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16113055 | Low pH induces co-ordinate regulation of gene expression in oesophageal cells. | The development of gastro-oesophageal reflux disease (GORD) is known to be a causative risk factor in the evolution of adenocarcinoma of the oesophagus. The ponent of this reflux is gastric acid. However, the impact of low pH on gene expression has not been extensively studied in oesophageal cells. This study utilizes a transcriptomic and bioinformatic approach to assess regulation of gene expression in response to low pH. In more detail, oesophageal adenocarcinoma cell lines were exposed to a range of pH environments. Affymetrix microarrays were used for gene-expression analysis and results were validated using cycle limitation and real-time RT-PCR analysis, as well as northern and western blotting. Comparative promoter transcription factor binding site (TFBS) analysis (MatInspector) of hierarchically clustered gene-expression data was employed to identify the elements which may co-ordinately regulate individual gene clusters. Initial experiments demonstrated maximal induction of EGR1 gene expression at pH 6.5. Subsequent array experimentation revealed significant induction of gene expression from such functional categories as DNA damage response (EGR1-4, ATF3) and cell-cycle control (GADD34, GADD45, p57). Changes in expression of EGR1, EGR3, ATF3, MKP-1, FOSB, CTGF and CYR61 were verified in separate experiments and in a variety of oesophageal cell lines. TFBS analysis of promoters identified transcription factors that may co-ordinately regulate gene-expression clusters, Cluster 1: Oct-1, AP4R; Cluster 2: NF-kB, EGRF; Cluster 3: IKRS, AP-1F. Low pH has the ability to induce genes and pathways which can provide an environment suitable for the progression of malignancy. Further functional analysis of the genes and clusters identified in this low pH study is likely to lead to new insights into the pathogenesis and therapeutics of GORD and oesophageal cancer. |
16113056 | Effects of indole-3-carbinol and phenethyl isothiocyanate on colon carcinogenesis induced by azoxymethane in rats. | Indole-3-carbinol (I3C) and phenethyl isothiocyanate (PEITC) are breakdown products of the glucosinolates glucobrassicin and gluconasturtiin, respectively, and are thought to reduce carcinogen activation by P450 enzymes. To assess the effects of pounds on colon cancer risk, rats were divided into five groups and fed the following diets: control diet (AIN-93G), or diets with PEITC or I3C added to the control diet: high-PEITC (3.37 mmols/kg diet-high level of PEITC), low-PEITC (0.67 mmols/kg-low level of PEITC), high-I3C (6.8 mmols/kg-high level of I3C) and low-I3C (1.36 mmols/kg-low level of I3C). Diets were fed for 2 weeks before and 10 weeks after administration of the colon carcinogen azoxymethane. Precancerous lesion (aberrant crypt foci, ACF) number in the distal colon was significantly lower in both high-I3C and low-I3C groups (6.9 +/- 0.8 and 5.9 +/- 0.59 per cm2, respectively) pared with the control group (10.4 +/- 0.9). No significant difference in ACF number was found between the PEITC group and the control group. ACF expressing sialomucin, thought to indicate ACF more likely to progress to tumors, were greater in the high-PEITC group (13 +/- 3) than the control (5.6 +/- 2). Mucin-depleted ACF, suggested to have the greatest tumorigenic potential, tended to be lower in the low-I3C group (P < pared with the control group. Mucosal apoptotic and cell proliferation labeling indices did not differ among groups, suggesting that reduction in the ACF number by I3C does not involve alterations in mucosal cell kinetics. No significant differences were found among the groups in hepatic cytochrome P450 2E1 (CYP2E1) activity, the first enzyme involved in activation of azoxymethane. However, there was increased activity of NADPH- and NADH reductases with high-I3C, which are the enzymes involved in the transfer of reducing equivalents to cytochrome P450. These results suggest that I3C lowers colon cancer risk through a mechanism not involving reduction of carcinogen activation by CYP2E1. |
16113057 | Promoter hypermethylation of tetraspanin members contributes to their silencing in myeloma cell lines. | Multiple myeloma (MM) cell interactions with their microenvironment modulate acquired drug resistance and disease progression. Indeed, reported aberrant gene methylation underscores the possible role of epigenetic events in MM's molecular profile. Membranal tetraspanins are often inversely correlated with cancer prognosis and metastasis, however mutations were unidentified hitherto. Their promoter characteristics and frequent down-regulation conform to transcriptional silencing by chromatin remodeling. We delineated the baseline expression of select tetraspanins in MM cell lines (RPMI 8226, U266, ARP1, ARK, CAG and EBV transformed ARH77) and fresh bone marrow samples (n = 9) for the first time and determined reduced expression of CD9, CD81 and absence of CD82. Thus, we aimed to assess their promoter methylation status. Indeed, we established CD9, CD81 and CD82 promoter methylation in MM cell lines employing methyl-specific-PCR of bisulfite modified G-DNA and PCR of G-DNA digested with methylation-sensitive restriction enzyme (Hin6I). Re-transcription of assayed genes in the cell lines following de-methylation [5-aza-2'-deoxycytidine (5-aza-dC)] confirmed the mechanistic significance of methylation to their regulation. Combined de-methylation and de-acetylation [Trichostatin A (TsA)] induced synergistic elevation of CD82 mRNA. We conclude that chromatin remodeling contributes to tetraspanin silencing in MM. |
16113062 | Functional hierarchy of coronary circulation: direct evidence of a structure-function relation. | The heart muscle is nourished by plex system of blood vessels that make up the coronary circulation. Here we show that the design of the coronary circulation has a functional hierarchy. A full anatomic model of the coronary arterial tree, containing millions of blood vessels down to the capillary vessels, was simulated based on previously measured porcine morphometric data. A network analysis of blood flow through every vessel segment was carried out based on the laws of fluid mechanics and appropriate boundary conditions. Our results show an abrupt change in cross-sectional area that demarcates the transition from epicardial (EPCA) to intramyocardial (IMCA) coronary arteries. Furthermore, a similar pattern of blood flow was observed with a corresponding transition from EPCA to IMCA. These results suggest functional differences between the two types of vessels. An additional abrupt change occurs in the IMCA in relation to flow velocity. The velocity is fairly uniform proximal to these vessels but drops significantly distal to those vessels toward the capillary branches. This finding suggests functional differences between large and small IMCA. Collectively, these observations suggest a novel functional hierarchy of the coronary vascular tree and provide direct evidence of a structure-function relation. |
16113064 | Systemic venous circulation. Waves propagating on a windkessel: relation of arterial and venous windkessels to systemic vascular resistance. | Compared with arterial hemodynamics, there has been relatively little study of venous hemodynamics. We propose that the venous system behaves just like the arterial system: waves propagate on a time-varying reservoir, the windkessel, which functions as the reverse of the arterial windkessel. During later diastole, pressure increases exponentially to approach an asymptotic value as inflow continues in the absence of outflow. Our study in eight open-chest dogs showed that windkessel-related arterial resistance was approximately 62% of total systemic vascular resistance, whereas windkessel-related venous resistance was only approximately 7%. Total pliance was found to be 21 times larger than pliance (n = 3). Inferior vena pliance (0.32 +/- 0.015 ml x mmHg(-1) x kg(-1); mean +/- SE) was approximately 14 times the pliance (0.023 +/- 0.002 ml x mmHg(-1) x kg(-1); n = 8). Despite greater pliance, the variation in venous windkessel volume pliance x windkessel pulse pressure; 7.8 +/- 1.1 ml) was only approximately 32% of the variation in aortic windkessel volume (24.3 +/- 2.9 ml) because of the larger arterial pressure variation. In addition, and contrary to previous understanding, waves generated by the right heart propagated upstream as far as the femoral vein, but excellent proportionality between the excess pressure and venous outflow suggests that no reflected waves returned to the right atrium. Thus the venous windkessel model not only successfully accounts for variations in the venous pressure and flow waveforms but also, bination with the arterial windkessel, provides a coherent view of the systemic circulation. |
16113063 | Phosphorylation and binding of AUF1 to the 3'-untranslated region of cardiomyocyte SERCA2a mRNA. | Experimental animals and patients with cardiac hypertrophy and heart failure display abnormally slowed myocardial relaxation, which is associated with downregulation of sarco(endo)plasmic reticulum calcium ATPase 2a (SERCA2a), the cardiomyocyte sarcoplasmic reticulum Ca2+ pump. We previously showed that SERCA2a downregulation can be simulated in cultured neonatal rat ventricular myocytes (NRVM) by treatment with the hypertrophic agonist phorbol myristate acetate (PMA) or by overexpression of the novel protein kinase C (PKC) isoenzymes PKCdelta and PKCepsilon. PKC activation, in turn, decreased SERCA2a promoter activity and destabilized the SERCA2a mRNA. Here we demonstrate by using an RSV beta-galactosidase reporter system that a 609-nt fragment of the SERCA2a mRNA 3'-untranslated region (UTR), containing five adenylate-uridylate (AU)-rich regions, may be responsible for destabilizing the message following PMA treatment. UV cross-linking analysis demonstrated that several proteins found in the NRVM cell extracts bind to the 609-nt fragment. In addition, protein binding was transiently increased in response to PMA stimulation. 3'-UTR mRNA pull-down assays and Western blot analysis indicated that the AU binding protein AUF1 interacted with the SERCA2a 3'-UTR. AUF1 binding activity was predominantly found in the nuclear fraction, and PMA-induced AUF1 binding was associated with increased threonine phosphorylation of AUF1. These data suggest that the phosphorylation, binding, and location of AUF1 affect the posttranscriptional regulation of the SERCA2a message in NRVM. |
16113065 | 14,15-Dihydroxyeicosatrienoic acid activates peroxisome proliferator-activated receptor-alpha. | Epoxyeicosatrienoic acids (EETs), lipid mediators synthesized from arachidonic acid by cytochrome P-450 epoxygenases, are converted by soluble epoxide hydrolase (SEH) to the corresponding dihydroxyeicosatrienoic acids (DHETs). Originally considered as inactive degradation products of EETs, DHETs have biological activity in some systems. Here we examined the capacity of EETs and DHETs to activate peroxisome proliferator-activated receptor-alpha (PPARalpha). We find that among the EET and DHET regioisomers, 14,15-DHET is the most potent PPARalpha activator in a COS-7 cell expression system. Incubation with 10 microM 14,15-DHET produced a 12-fold increase in PPARalpha-mediated luciferase activity, an increase similar to that produced by the PPARalpha agonist Wy-14643 (20 microM). Although 10 microM 14,15-EET produced a threefold increase in luciferase activity, this was abrogated by the SEH inhibitor dicyclohexylurea. 14-Hexyloxytetradec-5(Z)-enoic acid, a 14,15-EET analog that cannot be converted to a DHET, did not activate PPARalpha. However, PPARalpha was activated by 2-(14,15-epoxyeicosatrienoyl)glycerol, which was hydrolyzed and the released 14,15-EET converted to 14,15-DHET. COS-7 cells incorporated 14,15-[3H]DHET from the medium, and the cells also retained a small amount of the DHET formed during incubation with 14,15-[3H]EET. Binding studies indicated that 14,15-[3H]DHET binds to the ligand binding domain of PPARalpha with a Kd of 1.4 microM. Furthermore, 14,15-DHET increased the expression of carnitine palmitoyltransferase 1A, a PPARalpha-responsive gene, in transfected HepG2 cells. These findings suggest that 14,15-DHET, produced from 14,15-EET by the action of SEH, may function as an endogenous activator of PPARalpha. |
16113066 | Endothelin-1 expression in vascular adventitial fibroblasts. | Endothelial cells are a major source of endothelin (ET)-1, but the possibility that vascular adventitial fibroblasts generate ET-1 has not been explored. We hypothesized that aortic adventitial fibroblasts have the ability to produce ET-1, which may contribute to extracellular matrix synthesis. Vascular adventitial fibroblasts were isolated from mouse aorta and incubated with various concentrations of angiotensin II (ANG II). mRNA levels of preproET-1 and type I procollagen were detected with relative RT-PCR. ET-1 levels in culture medium were measured with ELISA. Protein levels of procollagen were detected with Western blotting. ANG II (10 and 100 nM, 1 microM) induced a time- and concentration-dependent increase in preproET-1 mRNA levels (P < 0.05). Induction of preproET-1 mRNA was panied by release of immunoreactive peptide ET-1 (P < 0.05). ANG II-evoked increases in preproET-1 mRNA expression and ET-1 release were blocked by losartan (100 microM), an AT1 receptor antagonist, but not PD-123319 (100 microM), an AT2 receptor antagonist. To further confirm our findings, we cloned and then sequenced vascular fibroblast preproET-1 bidirectionally with T7 and M13 reverse sequencing primers. Their nucleotide sequences were identical to preproET-1 cDNA from mouse vascular endothelial cells (accession no. AB081657). Moreover, ANG II-induced type I procollagen mRNA and protein expression were inhibited by BQ-123 (10 microM), an ET(A) receptor inhibitor, but not BQ-788 (10 microM), an ET(B) receptor inhibitor, suggesting a significant role of adventitial ET-1 in regulation of extracellular matrix synthesis. The results demonstrate that vascular adventitial fibroblasts are able to synthesize and release ET-1 in response to ANG II. |
16113068 | Effect of targeted deletions of beta1- and beta2-adrenergic-receptor subtypes on heart rate variability. | Beta-adrenergic receptors (beta-ARs) play a major role in regulating heart rate (HR) and contractility in the intact cardiovascular system. Three subtypes (beta1, beta2, and beta3) are expressed in heart tissue, and the role of each subtype in regulating cardiac function has previously been determined by using both pharmacological and gene-targeting approaches. However, previous studies have only examined the role of beta-ARs in the macrolevel regulation of HR. We employed three knockout (KO) mouse lines, beta1-KO, beta2-KO, and beta1/beta2 double KO (DL-KO), to examine the role that beta-AR subtypes play in HR variability (HRV) and in the sympathetic and parasympathetic inputs into HR control. Fast Fourier transformation (FFT) in frequency domain methods of ECG spectral analysis was used to resolve HRV into high- and low-frequency (HF and LF) powers. Resting HR (in beats/min) was decreased in beta1-KO [488 (SD 27)] and DL-KO [495 (SD 12)] pared with wild-type [WT; 638 (SD 30)] or beta2-KO [656 (SD 51)] (P < 0.0005) mice. Mice lacking beta1-ARs (beta1-KO and DL-KO) had increased HRV (as illustrated by the standard deviation of normal R-R intervals) and increased normalized HF and LF pared with mice with intact beta1-ARs (WT and beta2-KO). These results demonstrate the differential role of beta-AR subtypes in regulating autonomic signaling. |
16113067 | Asymmetrical dimethylarginine plasma clearance persists after acute total nephrectomy in rats. | Elevated plasma concentrations of symmetrical dimethylarginine (SDMA) and asymmetrical dimethylarginine (ADMA) are repeatedly associated with kidney failure. Both ADMA and SDMA can be excreted in urine. We tested whether renal excretion is necessary for acute, short-term maintenance of plasma ADMA and SDMA. Sprague-Dawley rats underwent sham operation, bilateral nephrectomy (NPX), ureteral ligation, or ureteral section under isoflurane anesthesia. Tail-snip blood samples (250 microl) were taken before and at 6- or 12-h intervals for 72 h after operation. Plasma clearance was assessed in intact and NPX rats. High-performance liquid chromatography determined SDMA and ADMA concentrations. Sodium, potassium, creatinine, blood urea nitrogen (BUN), and body weight were also measured. Forty-eight hours after NPX, SDMA increased 25 times (0.23 +/- 0.03 to 5.68 +/- 0.30 microM), whereas ADMA decreased (1.17 +/- 0.08 to 0.73 +/- 0.08 microM) by 38%. Creatinine and BUN increased, paralleling SDMA. Sham-operated animals showed no significant changes. Increased SDMA confirms continuous systemic production of SDMA and its obligatory renal excretion, much like creatinine. In contrast, decreased plasma ADMA suggests that acute total NPX either reduced systemic ADMA formation and/or systemic hydrolysis of ADMA increased 48-h post-NPX. However, plasma clearance of ADMA appeared unchanged 48 h after NPX. We conclude that renal excretory function is needed for SDMA elimination but not needed for acute, short-term ADMA elimination in that systemic hydrolysis is fully capable of clearing plasma ADMA. |
16113069 | Cardioprotective effects of estradiol include the activation of large-conductance Ca(2+)-activated K(+) channels in cardiac mitochondria. | The ponents of the large-conductance Ca(2+)-activated K(+) channels that are functionally expressed in mitochondria (mitoK(Ca)) in cardiac myocytes have not been identified. Our experimental results show that the transcript corresponding to the large-conductance Ca(2+)-activated K(+) channel beta1-subunit (BK-beta1) is substantially expressed in mammalian heart. A yeast two-hybrid assay showed the BK-beta1 protein can interact with a mitochondrial protein, cytochrome c oxidase subunit I (Cco1). Results from immunocytochemical experiments also demonstrated that BK-beta1 interacted with Cco1 and colocalized in rat cardiac mitochondria. Furthermore, 17beta-estradiol, which enhances the activity of the BK channel alpha-subunit only in the presence of the beta1-subunit, significantly increased flavoprotein oxidation in rat ventricle myocytes and decreased the rate of cell death under simulated ischemia. Single-channel recordings from mitochondrial inner membrane indicated that the activity of mitoK(Ca), which had a conductance of approximately 270 pS, was enhanced by 17beta-estradiol and blocked by paxilline. bination, the present study revealed a new mechanism for the cardioprotective effects of 17beta-estradiol, which include the activation of mitoK(Ca) via the interaction with BK-beta1. BK-beta1 may be an important ponent that functionally couples with both Cco1 and mitoK(Ca) pore-forming alpha-subunit. |
16113070 | Central command blunts sensitivity of arterial baroreceptor-heart rate reflex at onset of voluntary static exercise. | We have reported that baroreflex bradycardia by stimulation of the aortic depressor nerve is blunted at the onset of voluntary static exercise in conscious cats. mand may contribute to the blunted bradycardia, because the most blunted bradycardia occurs immediately before exercise or when a forelimb is extended before force development. However, it remained unknown whether the blunted bradycardia is due to either reduced sensitivity of the baroreflex stimulus-response curve or resetting of the curve toward a higher blood pressure. To determine this, we examined the stimulus-response relationship between systolic (SAP) or mean arterial pressure (MAP) and heart rate (HR) at the onset of and during the later period of static exercise in seven cats (n = 348 trials) by changing arterial pressure with infusion of nitroprusside and phenylephrine or norepinephrine. The slope of the MAP-HR curve decreased at the onset of exercise to 48% of the preexercise value (2.9 +/- 0.4 beats x min(-1) x mmHg(-1)); the slope of the SAP-HR curve decreased to 59%. The threshold blood pressures of the stimulus-response curves, at which HR started to fall due to arterial baroreflex, were not affected. In contrast, the slopes of the stimulus-response curves during the later period of exercise returned near the preexercise levels, whereas the threshold blood pressures elevated 6-8 mmHg. The maximal plateau level of HR was not different before and during static exercise, denying an upward shift of the baroreflex stimulus-response curves. Thus mand is likely to attenuate sensitivity of the ponent of arterial baroreflex at the onset of voluntary static exercise without shifting the stimulus-response curve. |
16113071 | Angiogenic effects of long-term enhanced external counterpulsation in a dog model of myocardial infarction. | Enhanced external counterpulsation (EECP) is an effective noninvasive treatment of coronary artery disease. Its mechanism of action remains unknown. An acute coronary occlusion dog model was created to explore the angiogenic effect of EECP. After coronary occlusion, 12 dogs were randomly assigned to either EECP (n = 6) or control (n = 6). Immunohistochemical studies of alpha-actin and von Willebrand factor (vWF) were used to detect newly developed microvessels. Systemic and local vascular endothelial growth factor (VEGF) were identified by ELISA and reverse transcriptase PCR analysis. There was a significant increase in the density of microvessels per squared millimeter in the infarcted regions of the EECP pared with the control group (vWF, 15.2 +/- 6.3 vs. 4.9 +/- 2.1, P < 0.05; alpha-actin, 11.8 +/- 5.3 vs. 3.4 +/- 1.2, P < 0.05). The positive-stained area per squared micrometer also increased significantly (alpha-actin, 6.6 x 10(3) +/- 2.9 x 10(3) microm2 vs. 0.6 x 10(3) +/- 0.5 x 10(3) microm2, P < 0.05; vWF, 5.7 x 10(3) +/- 1.9 x 10(3) microm2 vs. 1.7 x 10(3) +/- 1.4 x 10(3) microm2, P < 0.05). Immunohistochemical staining and reverse transcriptase PCR analysis documented a significant increase in VEGF expression. These factors associated with angiogenesis corresponded to improved myocardial perfusion by 99mTc-sestamibi single-photon puted tomography. Angiogenesis may be a mechanism of action for the improved myocardial perfusion demonstrated after EECP therapy. |
16113073 | ANG II stimulates phospholipase D through PKCzeta activation in VSMC: implications in adhesion, spreading, and hypertrophy. | ANG II stimulates phospholipase D (PLD) activity and growth of vascular smooth muscle cells (VSMC). The atypical protein kinase C-zeta (PKCzeta) plays a central role in the regulation of cell survival and proliferation. This study was conducted to determine the relationship between ANG II-induced activation of PKCzeta and PLD and their implication in VSMC adhesion, spreading, and hypertrophy. ANG II stimulated PKCzeta activity with maximal activation at 30 s followed by a decline in its activity to 45% above basal at 5 min. Inhibition of PKCzeta activity with a myristoylated pseudosubstrate peptide or overexpression of a kinase-inactive form of PKCzeta decreased ANG II-induced PLD activity. Moreover, depletion of PKCzeta with selective antisense oligonucleotides also decreased ANG II-induced PLD activity. Interaction between PLD2 and PKCzeta in VSMC was detected by coimmunoprecipitation. ANG II-induced PLD activity was inhibited by the primary alcohol n-butanol but not the tertiary alcohol t-butanol. The functional significance of PKCzeta and PLD2 in VSMC adhesion, spreading, and hypertrophy was investigated. Inhibition of PKCzeta and PLD2 activity or expression attenuated VSMC adhesion to collagen I and ANG II-induced cell spreading and hypertrophy. These results demonstrate that ANG II-induced PLD activation is regulated by PKCzeta and suggest a crucial role of PKCzeta-dependent PLD2 in VSMC functions such as adhesion, spreading, and hypertrophy, which are associated with the pathogenesis of atherosclerosis and malignant hypertension. |
16113072 | Inositol trisphosphate receptor calcium release is required for cerebral artery smooth muscle cell proliferation. | Vascular damage signals smooth muscle cells to proliferate, often exacerbating existing pathologies. Although the role of changes in "global" Ca2+ in vascular smooth muscle (VSM) cell dedifferentiation has been studied, the role of specific Ca2+ signals in determining VSM phenotype remains relatively unexplored. Earlier work with cultured VSM cells suggests that inositol 1,4,5-trisphosphate receptor (IP3R) expression and sarcoplasmic reticulum (SR) Ca2+ release may be linked to VSM cell proliferation in native tissue. Thus we hypothesized that SR Ca2+ release through IP3Rs in the form of discrete transient signals is necessary for VSM cell proliferation. To investigate this hypothesis, we used mouse cerebral arteries to design an organ culture system that permitted examination of Ca2+ dynamics in native tissue. Explanted arteries were cultured in normal medium with 10% FBS, and appearance of individual VSM cells migrating from explanted arteries (outgrowth cells) was tracked daily. Initial exposure to 10% FBS increased Ca2+ waves in myocytes in the arteries that were blocked by the IP3R antagonist 2-aminoethoxydiphenylborate (2-APB). Inhibition of IP3R opening (via 100 microM 2-APB, 10 microM xestospongin C, or 25 microM U-73122) dramatically reduced outgrowth cell pared with untreated or ryanodine-treated (10 microM) arteries. Consistent with this finding, 2-APB inhibited cell proliferation, as measured by reduced proliferating cell nuclear antigen immunostaining within 48 h of culture but did not inhibit cell migration. These results indicate that activation of IP3R Ca2+ release is required for VSM cell proliferation in these arteries. |
16113074 | Overexpression of prolylcarboxypeptidase enhances plasma prekallikrein activation on Chinese hamster ovary cells. | Plasma prekallikrein plexes with its receptor, high-molecular-weight kininogen (HK), on human umbilical vein endothelial cells (HUVEC). When assembled on endothelial cells, PK is activated to plasma kallikrein independent of factor XIIa by the serine protease prolylcarboxypeptidase (PRCP, Km= 9 nM). PRCP was shown to be a PK activator when isolated from HUVEC (J Biol Chem 277: 17962-17969, 2002) and produced as a binant protein (Blood 103: 4554-4561, 2004). To additionally confirm that human PRCP is a physiological PK activator, PRCP was overexpressed in Chinese hamster ovary (CHO) cells. CHO cells were transfected with full-length PRCP under the control of a cytomegalovirus promoter, and CHO binant PRCP was expressed as a fusion protein with COOH-terminal enhanced green fluorescence protein (EGFP). The presence of binant PRCP in transfected CHO cells was detected by real-time RT-PCR, immunoblot, and immunoprecipitation. PRCP mRNA and PK activation were two- to threefold higher in transfected than in control CHO cells. The increase in PRCP-induced PK activation in the transfected CHO cells paralleled the increase in PRCP antigen expression, as determined by anti-PRCP and anti-green fluorescence protein antibodies. PK activation of the transfected cells was blocked by small interfering RNA to PRCP. Anti-PRCP antibody and Z-Pro-Pro-aldehyde dimethyl acetate also blocked PK activation (IC50= 0.01 and 7.0 mM, respectively). Localization of PRCP in intact cells observed via confocal microscopy and flow cytometry also confirmed overexpression of PRCP on the external membrane. These investigations independently confirm that PRCP is expressed on cell membranes and that PRCP expression increases PK activation. |
16113076 | Increased ventricular repolarization heterogeneity in patients with ventricular arrhythmia vulnerability and cardiomyopathy: a human in vivo study. | Increased repolarization heterogeneity can provide the substrate for reentrant ventricular arrhythmias in animal models of cardiomyopathy. We hypothesized that ventricular repolarization heterogeneity is also greater in patients with cardiomyopathy and ventricular arrhythmia vulnerability (inducible ventricular tachycardia or positive microvolt T wave alternans, pared with a similar patient population without ventricular arrhythmia vulnerability (no VT/TWA). Endocardial and epicardial repolarization heterogeneity was measured in patients with (n = 12) and without (n = 10) VT/TWA by using transvenous 26-electrode catheters placed along the anteroseptal right ventricular endocardium and left ventricular epicardium. Local activation times (AT), activation-recovery intervals (ARI), and repolarization times (RT) were measured from unipolar electrograms. Endocardial RT dispersion along the apicobasal ventricle was greater (P < 0.005) in patients with VT/TWA than in those without VT/TWA because of greater ARI dispersion (P < 0.005). AT dispersion was similar between the two groups. Epicardial RT dispersion along the apicobasal ventricle was greater (P < 0.05) in patients with VT/TWA than in those without VT/TWA because of greater ARI dispersion (P < 0.05). AT dispersion was similar between the two groups. A plot of AT as a function of ARI revealed an inverse linear relationship for no VT/TWA such that progressively later activation was associated with progressively shorter ARI. The AT-ARI relationship was nonlinear in VT/TWA. In conclusion, patients with cardiomyopathy and VT/TWA have greater endocardial and epicardial repolarization heterogeneity than those without VT/TWA without associated conduction slowing. The steep repolarization gradients in VT/TWA may provide the substrate for functional conduction block and reentrant ventricular arrhythmias. |
16113075 | Critical mass hypothesis revisited: role of dynamical wave stability in spontaneous termination of cardiac fibrillation. | The tendency of atrial or ventricular fibrillation to terminate spontaneously in finite-sized tissue is known as the critical mass hypothesis. Previous studies have shown that dynamical instabilities play an important role in creating new wave breaks that maintain cardiac fibrillation, but its role in self-termination, in relation to tissue size and geometry, is not well understood. This study puter simulations of two- and three-dimensional tissue models to investigate qualitatively how, in relation to tissue size and geometry, dynamical instability affects the spontaneous termination of cardiac fibrillation. The major findings are as follows: 1) Dynamical instability promotes wave breaks, maintaining fibrillation, but it also causes the waves to extinguish, facilitating spontaneous termination of fibrillation. The latter effect predominates as dynamical instability increases, so that fibrillation is more likely to self-terminate in a finite-sized tissue. 2) In two-dimensional tissue, the average duration of fibrillation increases exponentially as tissue area increases. In three-dimensional tissue, the average duration of fibrillation decreases initially as tissue thickness increases as a result of thickness-induced instability but then increases after a critical thickness is reached. Therefore, in addition to tissue mass and geometry, dynamical instability is an important factor influencing the maintenance of cardiac fibrillation. |
16113077 | Regulation of hydraulic conductivity in response to sustained changes in pressure. | The present study addresses the effect of a sustained change in pressure on microvascular permeability assessed by hydraulic conductivity (Lp) measurements from microvessels of the rat mesentery. With a microperfusion technique, transvascular filtration (normalized to surface area; Jv/S) and Lp were measured in small arterioles (baseline Lp= 0.26 x 10(-7) cm.s(-1).cmH2O(-1)) and venules (baseline Lp= 2.88 x 10(-7) cm.s(-1).cmH2O(-1)). The main finding of this study is that step increases in microvascular pressure led to time-dependent alterations of L(p). Immediately after a twofold step increase in pressure, Jv/S increased in proportion to the pressure change. This observation is consistent with Starling's law that predicts filtration proportional to the overall pressure gradient when Lp is constant. However, when Jv/S measurements continued for 60-90 min past the step in pressure, there was an initial decrease in Jv/S for 30 min ("sealing effect") followed by a substantial increase in Jv/S out to 90 min. The sustained increase in Jv/S suggests an increase in Lp of 36 +/- 7% for small arterioles and 42 +/- 5% for small venules (P < 0.05 for both). In addition, the increase in Lp in response to an increase in pressure was attenuated significantly by nitric oxide synthase inhibition. These results indicate that a pressure-induced mechanical stimulus (possibly Jv) activates a NO-dependent biochemical response that leads to an increase in hydraulic conductivity. |
16113078 | Myocardial infarction and heart failure in the db/db diabetic mouse. | Clinical studies have reported that the incidence and severity of myocardial infarction is significantly greater in pared with nondiabetics after correction for all other risk factors. The majority of studies investigating the pathophysiology of myocardial ischemia-reperfusion injury have focused on otherwise healthy animals. At present, there is a paucity of experimental investigations on the pathophysiology of heart failure in diabetic animals. We hypothesized that the severity of myocardial reperfusion injury and the development of congestive heart failure would be markedly enhanced in the db/db diabetic mouse. Accordingly, we studied the effects of varying durations of in vivo myocardial ischemia and reperfusion on the incidence of heart failure in db/db diabetic mice. Nondiabetic and db/db diabetic mice (10 wk of age) were subjected to 30, 45, or 60 min of left coronary artery occlusion and 28 days of reperfusion. Survival at 24 h of reperfusion was 100% in nondiabetic mice subjected to 30 min of myocardial ischemia and 88% in nondiabetic mice subjected to 45 min of myocardial ischemia. In contrast, survival was 53% in db/db diabetic mice subjected to 30 min of myocardial ischemia and 44% in db/db mice after 45 min of myocardial ischemia. Prolonged survival in nondiabetic mice was not significantly attenuated pared during the 28-day follow-up period with all groups experiencing >90% survival. Prolonged survival was significantly decreased in db/db mice after both 30 and 45 min of myocardial pared with sham controls. Furthermore, we observed a significant degree or left ventricular dilatation, cardiac hypertrophy, and cardiac contractile dysfunction in db/db mice subjected to 45 min of myocardial ischemia and 28 days reperfusion. In nondiabetic mice subjected to 45 min of myocardial ischemia, we failed to observe any changes in left ventricular dimensions or fractional shortening. These studies provide a feasible experimental model system for the investigation of heart failure secondary to acute myocardial infarction in the db/db diabetic mouse. |
16113079 | Renouncing electroneutrality is not free of charge: switching on electrogenicity in a Na+-coupled phosphate cotransporter. | Renal type IIa Na+-coupled inorganic phosphate (Pi) cotransporters (NaPi-IIa) mediate divalent Pi transport in an electrogenic manner, whereas the renal type IIc isoform (NaPi-IIc) is electroneutral, yet it shows high sequence identity with NaPi-IIa. Dual uptake (32Pi/22Na) assays confirmed that NaPi-IIc displayed Na+-coupled Pi cotransport with a 2:1 (Na+:Pi) pared with 3:1 established for NaPi-IIa. This finding suggested that the electrogenicity of NaPi-IIa arises from the interaction of an additional Na+ pared with NaPi-IIc. To identify the molecular elements responsible for the functional difference between isoforms, we used chimera and amino acid replacement approaches. Transport activity of chimeras constructed with NaPi-IIa and NaPi-IIc indicated that residues within the first six transmembrane domains were essential for the electrogenicity of NaPi-IIa. parison between electrogenic and electroneutral isoforms revealed differences in the charge and polarity of residues clustered in three areas, one of which included part of the predicted third transmembrane domain. Here, substitution of three residues with their NaPi-IIa equivalents in NaPi-IIc (S189A, S191A, and G195D) resulted in a transporter that displayed a 1:1 charge/Pi coupling, a 3:1 Na+:Pi stoichiometry, and transient currents that resembled pre-steady-state relaxations. The mutant's weaker voltage dependency and 10-fold lower apparent Pi pared with NaPi-IIa indicated that other residues important for the NaPi-IIa kinetic fingerprint exist. Our findings demonstrate that, through a minimal number of side chain substitutions, we can effect a switch from electroneutral to electrogenic cotransporter function, itant with the appearance of a cosubstrate interaction site. |
16113081 | Variation in IL-1beta gene expression is a major determinant of genetic differences in arthritis aggressivity in mice. | In humans and in animal models, susceptibility to arthritis is plex genetic control, reflecting influences on the immunological processes that initiate autoimmunity and on subsequent inflammatory mechanisms in the joints. The effector phases are conveniently modeled by the K/BxN serum transfer system, a robust model well suited for genetic analysis where arthritis is initiated by pathogenic Ig. Here, we mapped the genetic loci distinguishing the high-responder BALB/c vs. low-responder SJL strains. putational modeling of potential breeding schemes, we adapted a stepwise selective breeding strategy, with a whole-genome scan performed on a limited number of animals. Several genomic regions proved significantly associated with high sensitivity to arthritis. One of these regions, on distal chr2, was centered on the interleukin 1 gene family. Quantitation of transcripts of the Il1a and Il1b candidate genes revealed a 10-fold greater induction of Il1b mRNA in BALB/c than in SJL splenocytes after injection of LPS, whereas Il1a showed much less difference. The differential activity of the Il1b gene was associated with a particular sequence haplotype of noncoding polymorphisms. The BALB/c haplotype was found in 75% of wild-derived strains but was rare among conventional inbred strains (4/33 tested, one of which is DBA/1, the prototype arthritis-susceptible strain) and was associated with vigorous Il1b responses in a panel of inbred strains. Inbred strains carrying this allele were far more responsive to serum-transferred arthritis, confirming its broad importance in controlling arthritis severity. |
16113080 | Reelin and glutamic acid decarboxylase67 promoter remodeling in an epigenetic methionine-induced mouse model of schizophrenia. | Reduction of prefrontal cortex glutamic acid decarboxylase (GAD67) and reelin (mRNAs and proteins) expression is the most consistent finding reported by several studies of postmortem schizophrenia (SZ) brains. Converging evidence suggests that the reduced GAD67 and reelin expression in cortical GABAergic interneurons of SZ brains is the consequence of an epigenetic hypermethylation of RELN and GAD67 promoters very likely mediated by the overexpression of DNA methyltransferase 1 in cortical GABAergic interneurons. Studies of the molecular mechanisms (DNA methylation plus related chromatin remodeling factors) that cause the down-regulation of reelin and GAD67 in SZ brains have important implications not only to understand the disease pathogenesis but also to improve present pharmacological interventions to treat SZ. The mouse treated with l-methionine models some of the molecular neuropathologies detected in SZ, including the hypermethylation of RELN promoter CpG islands and the down-regulation of reelin and GAD67 expression. We now report that in these mice, RELN and GAD67 promoters express an increased recruitment of methyl-CpG binding domain proteins. In these mice the histone deacetylase inhibitor valproate, which increases acetylated histone content in cortical GABAergic interneurons, also prevents MET-induced RELN promoter hypermethylation and reduces the methyl-CpG binding domain protein binding to RELN and GAD67 promoters. These findings suggest that DNA hypermethylation and the associated chromatin remodeling may be critically important in mediating the epigenetic down-regulation of reelin and GAD67 expression detected in cortical GABAergic interneurons of SZ patients. |
16113082 | Reduced PU.1 expression causes myeloid progenitor expansion and increased leukemia penetrance in mice expressing PML-RARalpha. | PU.1 is a member of the ETS family of transcription factors that is known to be important for hematopoietic development. Recently, haploinsufficiency for PU.1 has been shown to cause a shift in myelomonocytic progenitor fate toward the myeloid lineage. We have previously shown that transgenic mice expressing PML-RARalpha (PR) and RARalpha-PML frequently develop acute promyelocytic leukemia (APL) in association with a large (>20 Mb) interstitial deletion of chromosome 2 that includes PU.1. To directly assess the relevance of levels of expression of PU.1 for leukemia progression, we bred hCG-PR mice with PU.1+/- mice and assessed their phenotype. Young, nonleukemic hCG-PR x PU.1+/- mice developed splenomegaly because of the abnormal expansion of myeloid cells in their spleens. hCG-PR x PU.1+/- mice developed a typical APL syndrome after a long latent period, but the penetrance of disease was pared with 7% in hCG-PR x PU.1+/+ mice (P < 0.0001). The residual PU.1 allele in hCG-PR x PU.1+/- APL cells was expressed, plete exonic resequencing revealed no detectable mutations in nine of nine samples. However, PR expression in U937 myelomonocytic cells and primary murine myeloid bone marrow cells caused a reduction in PU.1 mRNA levels. Therefore, the loss of one copy of PU.1 through a deletional mechanism, plus down-regulation of the residual allele caused by PR expression, may synergize to expand the pool of myeloid progenitors that are susceptible to transformation, increasing the penetrance of APL. |
16113083 | Orientation discrimination of single-stranded DNA inside the alpha-hemolysin membrane channel. | We characterize the voltage-driven motion and the free motion of single-stranded DNA (ssDNA) molecules captured inside the approximately 1.5-nm alpha-hemolysin pore, and show that the DNA-channel interactions depend strongly on the orientation of the ssDNA molecules with respect to the pore. Remarkably, the voltage-free diffusion of the 3'-threaded DNA (in the trans to cis direction) is two times slower than the corresponding 5'-threaded DNA having the same poly(dA) sequence. Moreover, the ion currents flowing through the blocked pore with either a 3'-threaded DNA or 5' DNA differ by approximately 30%. All-atom molecular dynamics simulations of our system reveal a microscopic mechanism for the asymmetric behavior. In a confining pore, the ssDNA straightens and its bases tilt toward the 5' end, assuming an asymmetric conformation. As a result, the bases of a 5'-threaded DNA experience larger effective friction and forced reorientation that favors co-passing of ions. Our results imply that the translocation process through a narrow pore is plicated than previously believed and involves base tilting and stretching of ssDNA molecules inside the confining pore. |
16113084 | Focal adhesions as mechanosensors: a physical mechanism. | Focal adhesions (FA) are large, plexes that provide a mechanical link between the cytoskeletal contractile machinery and the extracellular matrix. FA exhibit mechanosensitive properties; they self-assemble and elongate upon application of pulling forces and dissociate when these forces are decreased. We propose a thermodynamic model for the mechanosensitivity of FA, according to which a molecular aggregate, subjected to pulling forces, tends to grow in the direction of force application by incorporating additional molecules. We demonstrate that this principle is consistent with the phenomenology of FA dynamics by considering a one-dimensional protein aggregate subjected to pulling forces and anchored to the substrate. Depending on the force level, force distribution along the aggregate, and the character of its anchoring to the substrate, the aggregate is predicted to exhibit distinct modes of assembly that are largely consistent with the experimentally observed FA behavior. We define here specific conditions that can lead to the different regimes of FA assembly, including growth, steady state, and disassembly. |
16113087 | Growth factor signalling and response to endocrine therapy: the Royal Marsden Experience. | De novo resistance to endocrine therapy is a near-universal feature of oestrogen receptor (ER)- negative breast cancer. Although many ER-positive breast cancers also show no response to tamoxifen or aromatase inhibitors on objective clinical grounds the large majority show reduced proliferation indicating that some oestrogen dependence is present in almost all ER-positive breast cancer. In neoadjuvant studies HER2 positivity is associated with poor response rates to tamoxifen but not aromatase inhibitors, consistent with preclinical models. Acquired resistance to tamoxifen is associated with decreases in ER positivity but most recurrent lesions remain ER-positive. A small proportion of these show increased HER2 expression and in these patients increased phospho-p38 may contribute to the tamoxifen-resistant phenotype. There is an unfortunate paucity of clinical and biological data on acquired resistance to aromatase inhibitors. |
16113085 | Allosteric modulation of the cannabinoid CB1 receptor. | We investigated the pharmacology of three pounds, Org 27569 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid [2-(4-piperidin-1-yl-phenyl)-ethyl]-amide), Org 27759 (3-ethyl-5-fluoro-1H-indole-2-carboxylic acid [2-94-dimethylamino-phenyl)-ethyl]-amide), and Org 29647 (5-chloro-3-ethyl-1H-indole-2-carboxylic acid (1-benzyl-pyrrolidin-3-yl)-amide, 2-enedioic acid salt), at the cannabinoid CB1 receptor. In equilibrium binding assays, the pounds significantly increased the binding of the CB1 receptor agonist [3H]CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol], indicative of a positively cooperative allosteric effect. The pounds caused a significant, but plete, decrease in the specific binding of the CB1 receptor inverse agonist [3H]SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride], indicative of a limited negative binding cooperativity. Analysis of the data according to an allosteric plex model revealed that the estimated affinity of each pound was not significantly different when the radioligand was [3H]CP 55,940 or [3H]SR 141716A. However, the estimated cooperatively factor for the interaction between modulator and radioligand was greater than 1 when determined against [3H]CP 55,940 and less than 1 when determined against [3H]SR 141716A. [3H]CP 55,940 dissociation kinetic studies also validated the allosteric nature of the pounds, because they all significantly decreased radioligand dissociation. These data suggest that the pounds bind allosterically to the CB1 receptor and elicit a conformational change that increases agonist affinity for the orthosteric binding site. In contrast to the binding assays, however, the pounds behaved as insurmountable antagonists of receptor function; in the reporter gene assay, the guanosine 5'-O-(3-[35S]thio)triphosphate binding assay and the mouse vas deferens assay they elicited a significant reduction in the Emax value for CB1 receptor agonists. The data presented clearly demonstrate, for the first time, that the cannabinoid CB1 receptor contains an allosteric binding site that can be recognized by synthetic small molecule ligands. |
16113086 | Consensus statement. Workshop on therapeutic resistance in breast cancer: impact of growth factor signalling pathways and implications for future treatment. | Anti-hormones (notably tamoxifen), chemotherapy and modern radiotherapeutic approaches are invaluable in the management of breast cancer, and collectively have contributed substantially to the improved survival in this disease. Moreover, there is promise that these successes will continue with the emergence of other endocrine agents (for example, aromatase inhibitors and pure anti-oestrogens). However, de novo and acquired prises a significant problem with all treatment approaches examined to date. This Workshop aimed to evaluate the contribution made by growth factor signalling pathways in the various resistant states, primarily focusing on resistance to anti-hormonal strategies and spanning experimental models and, where possible, clinical breast cancer data. The successes and limitations of therapeutic targeting of these pathways with various signal transduction inhibitors (STIs) were evaluated in model systems and from emerging clinical trials (including epidermal growth factor receptor inhibitors such as gefitinib). It was concluded that growth factor signalling is an important contributor in the development of endocrine resistance in breast cancer and that use of STIs provides a promising therapeutic strategy for this disease. However, the cancer cell is clearly able to harness alternative growth factor signalling pathways for growth and cell survival in the presence of STI monotherapy and, as a consequence, the efficacy of STIs is likely to be limited by the acquisition of resistance. A number of strategies were proposed from studies in model systems that appeared to enhance anti-tumour actions of existing STI monotherapy, notably bination therapies targeting multiple pathways. With the increased availability of diverse STIs and improved drug delivery, there is much hope that the plex therapeutic strategies proposed may ultimately be achievable in clinical practice. |
16113088 | Growth factor signalling in clinical breast cancer and its impact on response to conventional therapies: the Edinburgh experience. | Neoadjuvant endocrine treatment in which therapy is given while the primary tumour is still in the breast provides a highly useful model system by which to identify mechanisms associated with de novo resistance and signs of early acquired resistance. Most importantly, the model is clinically relevant. It has been confirmed that the absence of tumour oestrogen receptors confers resistance to endocrine therapy. Early changes in tumour cell proliferation following neoadjuvant treatment with the third-generation aromatase inhibitor, letrozole, do not predict accurately for subsequent clinical response. Additionally, changes in proliferation seen at later times can be the consequence of response and may be associated with early resistance. High expression of c-erbB2 does not reduce tumour responses to neoadjuvant treatment with aromatase inhibitors, but is associated with high tumour proliferation before and during treatment. It remains to be determined whether these characteristics confer subsequent resistance to treatment and early relapse in the adjuvant setting. |
16113089 | Growth factor signalling in clinical breast cancer and its impact on response to conventional therapies: a review of chemotherapy. | Adjuvant chemotherapy has been shown to provide survival benefits in patients with breast cancer, but some patients still relapse despite this. There is therefore a need for molecular markers present within the primary tumour that can predict for chemotherapy sensitivity or resistance. Until now, no single marker has emerged into routine clinical practice, but several candidate pathways are being extensively investigated. This paper summarises the current status of growth factor singalling and p53 function in this context. The data on human epidermal growth factor receptor-2, topoisomerase II and p53 expression in a variety of breast cancer treatment settings are discussed. |
16113090 | Overview of tyrosine kinase inhibitors in clinical breast cancer. | Studies of cell models and profiling of clinical breast cancer material to reveal the mechanisms of resistance to anti-oestrogen therapy, and to tamoxifen in particular, have reported that this phenomenon can be associated with increased expression and signalling through erbB Type 1 growth factor receptors, notably the epidermal growth factor receptor (EGFR) and HER2. Further molecular studies have revealed an intricate interlinking between such growth factor receptor pathways and oestrogen receptor (ER) signalling. Inhibition of receptor tyrosine kinase activity involved in the EGFR signalling cascade forms the basis for the use of EGFR specific tyrosine kinase inhibitors exemplified by gefitinib (ZD1839, Iressa) and erlotinib (OSI-774, Tarceva). Such agents have proved promising in pre-clinical studies and are currently in clinical trials in breast cancer, where gefitinib has been studied more extensively to date. Here, we present an overview of the current development of gefitinib in clinical breast cancer. This includes results from our clinical breast cancer trial 1839IL/0057 that demonstrate the efficacy of gefitinib within ER-positive, tamoxifen-resistant patients with locally advanced/metastatic disease, where parallel decreases in EGFR signal transduction and the Ki67 (MIB1) proliferation marker can be detected as predicted from model system studies. We also consider trials bination treatment with gefitinib and anti-hormonal strategies that will begin to address the clinically important question of whether gefitinib can delay/prevent onset of anti-hormone resistance. |
16113091 | Clinical trials of intracellular signal transductions inhibitors for breast cancer--a strategy to overcome endocrine resistance. | Acquired resistance to endocrine therapy in breast cancer is associated with an increase in peptide growth factor signaling that results in cross-talk activation of the estrogen receptor (ER). Small molecule signal transduction inhibitors (STIs) can ponents of these intracellular pathways, and may prove effective in anticancer therapy. However, early phase II clinical trials with various STIs as monotherapy in advanced breast cancer have shown only a modest level of efficacy for these intracellular inhibitors. Preclinical data suggest binations of tamoxifen with STIs may provide significantly greater growth inhibition than either therapy alone, and, furthermore, may delay the emergence of endocrine resistance. There are now several trials assessing the efficacy binations of small molecule tyrosine kinase inhibitors (TKIs), such as gefitinib and lapatinib, with either tamoxifen or aromatase inhibitors both in the second-line, endocrine-resistant and first-line, hormone-sensitive setting. Similar trials continue with both farnesyltransferase inhibitors (FTIs) and mTOR antagonists, where there are strong preclinical data to suggest additive or synergistic effects for either of these agents bination with endocrine therapies. Biomarker studies in the presurgical setting are also being utilized as an alternative approach to investigate bined endocrine/STI therapy is an effective clinical strategy. This article reviews some of the preclinical evidence supporting this strategy, together with the current status of clinical trials in this area. |
16113092 | Intrinsic and acquired resistance to EGFR inhibitors in human cancer therapy. | The epidermal growth factor receptor (EGFR) autocrine pathway plays a crucial role in human cancer since it contributes to a number of highly relevant processes in tumor development and progression, including cell proliferation, regulation of apoptotic cell death, angiogenesis and metastatic spread. Among a variety of approaches used to target EGFR signaling, EGFR blocking monoclonal antibodies and small molecular weight EGFR tyrosine pounds have been successfully developed. The results of a large body of preclinical studies and clinical trials suggest that targeting the EGFR could represent a significant contribution to cancer therapy. Both types of agent exert a significant antiproliferative activity when used alone or bination with conventional antitumor treatments, such as chemotherapy or radiation therapy. Although the advanced clinical development of EGFR blocking drugs demonstrates their efficacy in some human metastatic diseases, such as lung, head and neck and colorectal cancers, the issue of constitutive resistance in a large number of patients and the development of acquired resistance in the responders remains an unexplored subject of investigation. Recent evidence suggests the role of specific activating mutations within the tyrosine kinase domain of EGFR to explain the dramatic responses to small molecule tyrosine kinase inhibitors in a subgroup of lung cancer patients. However, the intrinsic molecular mechanisms of resistance to these drugs are still unclear. This review will focus on the preclinical findings on therapeutic resistance to EGFR targeting agents. |
16113093 | The epidermal growth factor receptor family. | The epidermal growth factor receptor family consists of four receptor genes and at least 11 ligands, several of which are produced in different protein forms. They create an interacting system that has the ability to receive and process information that results in multiple outputs. The family has an important role in directing and coordinating many normal processes, including growth and development, normal tissue turnover and wound healing. Its members are also aberrantly activated by overexpression or mutation in mon human tumour types and as such have been the target for anticancer drug development. |
16113094 | Development of strategies for the use of anti-growth factor treatments. | Aberrant signalling through the epidermal growth factor receptor (EGFR) is associated with increased cancer cell proliferation, reduced apoptosis, invasion and angiogenesis. Over-expression of the EGFR is seen in a variety of tumours and is a rational target for antitumour strategies. Among the classes of agent targeting the EGFR are small-molecule inhibitors, which include gefitinib (IRESSA), which acts by preventing EGFR phosphorylation and downstream signal transduction. De novo and acquired resistance, however, have been reported to gefitinib and here we describe evidence which indicates that the type II receptor tyrosine kinases (RTKs) insulin-like growth factor-I receptor (IGF-IR) and/or insulin receptor (InsR) play important roles in the mediation of responses to gefitinib in the de novo- and acquired-resistance phenotypes in several cancer types. bination strategies that additionally target the IGF-IR/InsR can enhance the antitumour effects of gefitinib. |
16113095 | Inhibitors of growth factor signalling. | The therapeutic utility of trastuzumab ('Herceptin') in breast cancer patients with tumours that overexpress erbB2 established the principle that targeted inhibition of specific signal transduction pathways can provide a new approach to cancer treatment. The ErbB family of protein tyrosine kinases, in particular the epidermal growth factor receptor (EGFR), monly overexpressed in many solid human tumours and EGFR was the initial target for a drug discovery programme seeking small molecule inhibitors of the EGFR tyrosine kinase (TK) enzyme activity. The description of the anilinoquinazoline class of potent and selective TK inhibitors led to several candidate drugs from this chemical class, for example gefitinib ('Iressa') and erlotinib ('Tarceva'), which are being evaluated in breast cancer patients. Rapid advances in cancer molecular genetics have identified numerous potential drug targets associated with abnormal control of cell division either downstream of the ErbBs, for example Ras and MEK, or in erbB-associated signalling networks, like Src kinase, which affect the tumour cell motility and invasiveness. Candidate drugs for several of these targets are currently being evaluated; for example, the prenylation inhibitor AZD3409, a mimetic of the CAAX box of K-Ras, inhibits protein farnesyl and geranylgeranyl tranferases and a novel, selective, orally active Src kinase inhibitor AZD0530 have entered Phase I clinical trials and may have utility in breast cancer therapy. |
16113096 | Polymer-drug conjugates: towards a novel approach for the treatment of endrocine-related cancer. | The last decade has seen successful clinical application of polymer-protein conjugates (e.g. Oncaspar, Neulasta) and promising results in clinical trials with polymer-anticancer drug conjugates. This, together with the realisation that nanomedicines may play an important future role in cancer diagnosis and treatment, has increased interest in this emerging field. More than 10 anticancer conjugates have now entered clinical development. Phase I/II clinical trials involving N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (PK1; FCE28068) showed a four- to fivefold reduction in anthracycline-related toxicity, and, despite cumulative doses up to 1680 mg/m2 (doxorubicin equivalent), no cardiotoxicity was observed. Antitumour activity in chemotherapy-resistant/refractory patients (including breast cancer) was also seen at doxorubicin doses of 80-320 mg/m2, consistent with tumour targeting by the enhanced permeability (EPR) effect. Hints, preclinical and clinical, that polymer anthracycline conjugation can bypass multidrug resistance (MDR) reinforce our hope that polymer drugs will prove useful in improving treatment of endocrine-related cancers. These promising early clinical results open the possibility of using the water-soluble polymers as platforms for delivery of a cocktail of pendant drugs. In particular, we have recently described the first conjugates bine endocrine therapy and chemotherapy. Their markedly enhanced in vitro activity encourages further development of such novel, bination therapies. This review briefly describes the current status of polymer therapeutics as anticancer agents, and discusses the opportunities for design of second-generation, bination therapy, including the cocktail of agents that will be needed to treat resistant metastatic cancer. |
16113097 | Growth factor signalling and resistance to selective oestrogen receptor modulators and pure anti-oestrogens: the use of anti-growth factor therapies to treat or delay endocrine resistance in breast cancer. | De novo insensitivity and acquired resistance to the selective oestrogen receptor modulator tamoxifen and the pure anti-oestrogen fulvestrant (faslodex) severely limit their effectiveness in breast cancer patients. This is a major clinical problem, since each year upward of 1 million women are dispensed anti-oestrogenic drugs. In order to investigate the phenomenon of anti-oestrogen resistance and to rapidly screen drugs that target the resistance mechanism(s), we have previously established several in vitro breast cancer models that have acquired resistance to anti-hormones. Such monly develop an ability to proliferate after approximately 3 months of exposure to 4-hydroxytamoxifen or fulvestrant, despite an initial endocrine-responsive (i.e. growth-suppressive) phase. The current paper explores the role that growth factor signalling plays in the transition of oestrogen receptor-positive endocrine-responsive breast cancer cells to anti-oestrogen resistance or insensitivity and how we might, in the future, most effectively use anti-growth factor therapies to treat or delay endocrine-resistant states. |
16113098 | The NFkappaB pathway and endocrine-resistant breast cancer. | Endocrine therapy with an estrogen receptor (ER)-targeted antiestrogen, such as tamoxifen, or estrogen ablation by aromatase inhibitors is clinically indicated for the management of all forms of ER-positive breast cancer. However, 30-50% of ER-positive breast cancer cases fail to benefit clinically from endocrine therapy alone, and recent molecular evidence suggests that 'crosstalk' pathways originating from activated receptor tyrosine kinases and/or other proliferative and survival signals may be contributing to this endocrine resistance. Molecular identification and validation of candidate ER crosstalking pathways will likely lead to clinically important prognostic markers and targets for the application of novel therapeutics bination with standard endocrine agents. This review focuses on a critical survival and proliferation pathway involving activation of nuclear factor-kappaB (NFkappaB), a family of ubiquitously expressed transcription factors that for nearly two decades have been known to be critical regulators of mammalian immune and inflammatory responses, and more recently have been associated with chemotherapy resistance. With the demonstration that activation of NFkappaB is absolutely required for normal mammary gland development, NFkappaB involvment in human breast cancers was initially explored and linked to the development of hormone-independent (ER-negative) breast cancer. Newer clinical evidence now implicates NFkappaB activation, particularly DNA-binding by the p50 subunit of NFkappaB, as a potential prognostic marker capable of identifying a high-risk subset of ER-positive, primary breast cancers destined for early relapse despite adjuvant endocrine therapy with tamoxifen. Furthermore, initial preclinical studies suggest that treatment strategies designed to prevent or interrupt activation of NFkappaB in cell-line models of these more aggressive, ER-positive breast cancers can restore their sensitivity to such standard endocrine agents as tamoxifen. |
16113099 | Downstream targets of growth factor and oestrogen signalling and endocrine resistance: the potential roles of c-Myc, cyclin D1 and cyclin E. | Antioestrogen therapy is a highly effective treatment for patients with oestrogen-receptor (ER)-positive breast cancer, emphasising the central role of oestrogen action in the development and progression of this disease. However, effective antioestrogen treatment is promised by acquired endocrine resistance, prompting the need for a greater understanding of the down-stream mediators of oestrogen action that may contribute to this effect. Recent studies have demonstrated a critical link between oestrogen's mitogenic effects and cell cycle progression, particularly at the G1 to S transition where key effectors of oestrogen action are c-Myc and cyclin D1, which converge on the activation of cyclin E-cdk2. ponents are rapidly upregulated in response to oestrogen, and can mimic its actions on cell cycle progression, including re-initiating cell proliferation in antioestrogen-arrested cells. Here we review the roles of c-Myc, cyclin D1 and cyclin E in oestrogen action and endocrine resistance, and identify their potential as markers of disease progression and endocrine responsiveness, and as novel therapeutic targets in endocrine-resistant breast cancer. |
16113100 | Long-term estradiol deprivation in breast cancer cells up-regulates growth factor signaling and enhances estrogen sensitivity. | Deprivation of estrogen causes breast tumors in women to adapt and develop enhanced sensitivity to this steroid. Accordingly, women relapsing after treatment with oophorectomy, which substantially lowers estradiol for a prolonged period, respond secondarily to aromatase inhibitors with tumor regression. We have utilized in vitro and in vivo model systems to examine the biologic processes whereby long-term estradiol deprivation (LTED) causes cells to adapt and develop hypersensitivity to estradiol. Several mechanisms are associated with this response, including up-regulation of estrogen receptor-alpha (ERalpha) and the MAP kinase, phosphoinositol 3 kinase (PI3-K) and mammalian target of rapamycin (mTOR) growth factor pathways. ERalpha is four- to tenfold up-regulated and co-opts a classical growth factor pathway using Shc, Grb-2 and Sos. This induces rapid non-genomic effects which are enhanced in LTED cells. The molecules involved in the non-genomic signaling process have been identified. Estradiol binds to cell membrane-associated ERalpha, which physically associates with the adaptor protein Shc, and induces its phosphorylation. In turn, Shc binds Grb-2 and Sos, which result in the rapid activation of MAP kinase. These non-genomic effects of estradiol produce biologic effects as evidenced by Elk-1 activation and by morphologic changes in cell membranes. Additional effects include activation of the PI3-K and mTOR pathways through estradiol-induced binding of ERalpha to the IGF-I and epidermal growth factor receptors. A major question is how ERalpha locates in the plasma membrane since it does not contain an inherent membrane localization signal. We have provided evidence that the IGF-I receptor serves as an anchor for ERalpha in the plasma membrane. Estradiol causes phosphorylation of the adaptor protein, Shc and the IGF-I receptor itself. Shc, after binding to ERalpha, serves as the 'bus' which carries ERalpha to Shc-binding sites on the activated IGF-I receptors. Use of small inhibitor (si) RNA methodology to knockdown Shc allows the conclusion that Shc is needed for ERalpha to localize in the plasma membrane. In order to abrogate growth factor-induced hypersensitivity, we have utilized a drug, farnesylthiosalicylic acid, which blocks the binding of GTP-Ras to its membrane acceptor protein, galectin 1, and reduces the activation of MAP kinase. We have also shown that this drug is a potent inhibitor of mTOR as an additional mechanism of inhibition of cell proliferation. The concept of 'adaptive hypersensitivity' and the mechanisms responsible for this phenomenon have important clinical implications. The efficacy of aromatase inhibitors in patients relapsing on tamoxifen could be explained by this mechanism and inhibitors of growth factor pathways should reverse the hypersensitivity phenomenon and result in prolongation of the efficacy of hormonal therapy for breast cancer. |
16113101 | Elevated ERK1/ERK2/estrogen receptor cross-talk enhances estrogen-mediated signaling during long-term estrogen deprivation. | The knowledge that steroids play a pivotal role in the development of breast cancer has been exploited clinically by the development of endocrine treatments. These have sought to perturb the steroid hormone environment of the tumour cells, predominately by withdrawal or antagonism of oestrogen. Unfortunately, the beneficial actions of existing endocrine treatments are attenuated by the ability of tumours to circumvent the need for steroid hormones, whilst in most cases, retaining the nuclear steroid receptors. The mechanisms involved in resistance to estrogen deprivation are of major clinical relevance for optimal treatment of breast cancer patients and the development of new therapeutic regimes. We have shown that long-term culture of MCF7 cells in medium depleted of oestrogen (LTED) results in hypersensitivity to oestradiol (E2) coinciding with elevated levels of both ERalpha phosphorylated on Ser(118) and ERK1/ERK2. Our data suggest elevated ERK1/ERK2 activity results wholly or in part from enhanced ERBB2 expression in the LTED cells. These cells showed greater sensitivity to the tyrosine kinase inhibitor ZD1839 in both ERalpha-mediated transcription and growth pared with the wt-MCF7. Similarly the MEK inhibitor U0126 decreased basal ERalpha-mediated transcription and proliferation in the LTED cells by 50% and reduced their sensitivity to the proliferative effects of E2 10-fold, whilst having no effect on the wild type (wt). plete suppression of ERK1/ERK2 activity in the LTED cells did not inhibit ERalpha Ser(118) phosphorylation suggesting that the cells remained ligand-dependent. This was further confirmed by the increased sensitivity of the LTED cells to the growth suppressive effects of ICI 182,780 and suggested that the LTED cells remained wholly or partially dependent on oestrogen receptor (ER)/oestrogen responsive elements directed growth. These findings suggest that treatments targeted at growth factor signalling pathways may be useful in patients acquiring resistance to oestrogen deprivation with aromatase inhibitors and that the pure anti-oestrogen ICI 182,780 may also be effective by blocking or destabilizing ER and hence disrupting cross-talk. |
16113102 | Acquired resistance to oestrogen deprivation: role for growth factor signalling kinases/oestrogen receptor cross-talk revealed in new MCF-7X model. | In vitro models of long-term oestrogen deprivation utilise increased oestrogen receptor (ER) and are oestrogen hypersensitive, with emerging evidence that growth factor signalling contributes and interacts with ER. However, such models monly derived in the presence of serum growth factors that may force the resistance mechanism. Our new in vitro model, MCF-7X, has thus been developed under conditions of both oestrogen and growth factor depletion. ER expression, serine 118 phosphorylation on this receptor and its transcriptional activity were modestly pared to the parental MCF-7 cells, although MCF-7X cells were not oestrogen hypersensitive. Faslodex (0.1 microM) partially decreased ER and its transcriptional activity, with associated decreases in serine 118 phosphorylation. Faslodex inhibited MCF-7X growth by 50% for 10 weeks. Classical growth factor receptors did not impact on MCF-7X growth and only a modest contribution for MAP kinase was revealed using PD98059 (25 microM; 35% inhibition for 3 weeks). However, the phosphatidylinositol-3-OH (PI3)-kinase inhibitor LY294002 (5 microM) inhibited MCF-7X growth by 65% for 10 weeks. In contrast to PD98059, LY294002 also partially-inhibited ER transcriptional activity and decreased serine 167 ER phosphorylation. Co-treatment with faslodex plus LY294002 to decrease activity of both serine 118 and 167 proved superior vs the single agents in decreasing ER transcriptional activity and MCF-7X growth (90% inhibition for 25 weeks). However, triple treatment including PD98059 was required to prevent resistance in MCF-7X, an event dependent on maximal depletion of serine 118 phosphorylation and ER transcriptional activity. Kinases clearly contribute in resistance to oestrogen deprivation, cross-talking with ER signalling via AF-1 phosphorylation. While inhibiting each pathway has potential to treat this bined therapy targeting all regulators of ER phosphorylation may be required to block subsequent emergence of resistance. |
16113103 | Overview of the impact of conventional systemic therapies on breast cancer. | Survival in women with breast cancer is improving in the western world, in part related to improved surgery, radiotherapy and adjuvant systemic therapy. Aromatase inhibitors are superior to tamoxifen in this clinical situation and several studies indicate that taxane-based chemotherapy is superior to non-taxane-based regimens. Herceptin is active alone in HER-2/neu +ve advanced breast cancer and four clinical trials are testing this agent in the adjuvant situation. It seems likely that herceptin will add to conventional therapies and thus will be the paradigm for the introduction of other biological therapies to improve cure rates. |
16113104 | Epidermal growth factor receptor/HER2/insulin-like growth factor receptor signalling and oestrogen receptor activity in clinical breast cancer. | Breast cancer models of acquired tamoxifen resistance, oestrogen receptor (ER)+ /ER- de novo resistance and gene transfer studies cumulatively demonstrate the increased importance of growth factor receptor signalling, notably the epidermal growth factor receptor (EGFR)/HER2, in tamoxifen resistance. Our recent in vitro studies also suggest that EGFR signalling productively cross-talks with insulin-like growth factor receptor (IGF-1R) and, where present, activates ER on key AF-1 serine residues to facilitate acquired tamoxifen-resistant growth. This paper presents our immunohistochemical evidence that EGFR/HER2 signalling (i.e. transforming growth factor (TGF)alpha, EGFR and HER2 expression; phosphorylation of EGFR, HER2 and ERK1/2 MAP kinase) is also prominent in clinical de novo resistant and modestly increased in acquired tamoxifen-resistant states, suggesting that anti-EGFR/HER2 strategies may prove valuable treatments. Primary breast cancer samples employed were obtained for (1) patients subsequently treated with tamoxifen for advanced disease where endocrine response and survival data were available and (2) ER+ elderly patients during tamoxifen response and relapse. We also present our clinical immunohistochemical findings that IGF-1R expression, its phosphorylation on tyrosine 1316, and also phosphorylation on serine 118 of ER are not only prominent in ER+ tamoxifen-responsive disease, but are also detectable in ER+ de novo and acquired tamoxifen-resistant breast cancer, where there is evidence of EGFR/ER cross-talk. Our data suggest that agents to deplete effectively ER or IGF-1R signalling may be of value in treating ER+ de novo/acquired tamoxifen resistance in addition to tamoxifen-responsive disease in vivo. IGF-1R inhibitors may also prove valuable in ER- patients, since considerable IGF-1R signalling activity was apparent within approximately 50% of such tumours. |
16113105 | Orientation behavior of retinal photoreceptors in alternating electric fields. | In alternating electric (AC) fields, particles experience polarizing effects that induce dipoles that orient elongated specimens either parallel or perpendicular to the field lines. In this work we studied the behavior of photoreceptor cells' rod outer segments (ROS) in AC fields of different frequencies. We showed that at low frequencies, ROS orient parallel to the field, whereas at higher frequencies they orient perpendicular to the field lines (in the frequency range from 100 Hz to 10 MHz). We found this behavior to be dependent on the physiological state of cells (due to modifications in their electrical properties). To simulate cell damage, the membrane conductivity was changed by treating the cell with gramicidin A, which resulted in a decrease of cytosol conductivity and, consequently, in a change of the orientation behavior of the treated cells. The change of cell orientation with cytosol conductivity is rather sharp, suggesting the potential of the method for accurate evaluation of the cell physiological status. We modeled the interaction between ROS and AC fields approximating the rod cell by a prolate spheroid with a very long axis. The partment of the ellipsoid was considered to be filled with an inhomogeneous medium consisting of alternating layers of membrane and cytoplasm as media modeling the disks. This theoretical model proved to be in good agreement with the experimental results and enabled the derivation (by fitting with the experimental results) of the membrane and cytosol parameters for normal and damaged cells. |
16113106 | Direct mechanical force measurements during the migration of Dictyostelium slugs using flexible substrata. | We use the flexible substrate method to study how and where mechanical forces are exerted during the migration of Dictyostelium slugs. This old and contentious issue has been left poorly understood so far. We are able to identify clearly separate friction forces in the tip and in the tail of the slug, traction forces mostly localized in the inner slug/surface contact area in the prespore region and large perpendicular forces directed in the outward direction at the outline of contact area. Surprisingly, the magnitude of friction and traction forces is decreasing with slug velocity indicating that these quantities are probably related to the dynamics of cell/substrate plexes. Contrary to what is always assumed in models and simulations, friction is not of fluid type (viscous drag) but rather close to solid friction. We suggest that the slime sheath confining laterally the cell mass of the slug experiences a tension that in turn is pulling out the elastic substrate in the direction tangential to the slug profile where sheath is anchored. In addition, we show in the appendix that the iterative method we developed is well adapted to study forces over large and continuous fields when the experimental error is sufficiently low and when the plane of recorded bead deformations is close enough to the elastomer surface, requirements fulfilled in this experimental study of Dictyostelium slugs. |
16113108 | pH dependence of amide chemical shifts in natively disordered polypeptides detects medium-range interactions with ionizable residues. | A growing number of natively disordered proteins undergo a folding/binding process that is essential for their biological function. An interesting question is whether these proteins have pletely solvated regions that drive the folding/binding process. Although the presence of predominantly hydrophobic buried regions can be easily ascertained by high-sensitivity differential scanning calorimetry analysis, the identification of those residues implicated in the burial requires NMR analysis. We have selected a partially solvated natively disordered fragment of Escherichia coli, thioredoxin, C37 (38-108), for full NMR spectral assignment. The secondary chemical shifts, temperature coefficients, and relaxation rates (R(1) and R(2)) of this fragment indicate the presence of a flexible backbone without a stable hydrogen bond network near neutral pH. (1)H-(15)N heteronuclear single quantum coherence analysis of the pH dependence of amide chemical shifts in fragment C37 within pH 2.0 and 7.0 suggests the presence of interactions between nonionizable residues and the carboxylate groups of four Asp and four Glu residues. The pH midpoints (pH(m)) of the amides in the ionizable residues (Asp or Glu) and, consequently, the shifts in the pH(m) (DeltapH(m)) of these residues with respect to model tetrapeptides, are sequence-dependent; and the nonionizable residues that show pH dependence cluster around the ionizable ones. The same pH dependence has been observed in two fragments: M37 (38-73) and C73 (74-108), ruling out the participation of long-range interactions. Our studies indicate the presence of a 15-residue pH-dependent segment with the highest density of ionizable sites in the disordered ensembles of fragments C37 and M37. The observed correlations between ionizable and nonionizable residues in this segment suggest the organization of the backbone and side chains through local and medium-range interactions up to nine residues apart, in contrast to only a few interactions in fragment C73. These results agree qualitatively with the predominantly hydrophobic buried surface detected only in fragments C37 and M37 by highly sensitive differential scanning calorimetry analysis. This work offers a sensitive and rapid new tool to obtain clues about local and nonlocal interactions between ionizable and nonionizable residues in the growing family of natively disordered small proteins with full NMR assignments. |
16113107 | Anomalous diffusion of proteins due to molecular crowding. | We have studied the diffusion of tracer proteins in highly concentrated random-coil polymer and globular protein solutions imitating the crowded conditions encountered in cellular environments. Using fluorescence correlation spectroscopy, we measured the anomalous diffusion exponent alpha characterizing the dependence of the mean-square displacement of the tracer proteins on time, r(2)(t) approximately t(alpha). We observed that the diffusion of proteins in dextran solutions with concentrations up to 400 g/l is subdiffusive (alpha < 1) even at low obstacle concentration. The anomalous diffusion exponent alpha decreases continuously with increasing obstacle concentration and molecular weight, but does not depend on buffer ionic strength, and neither does it depend strongly on solution temperature. At very high random-coil polymer concentrations, alpha reaches a limit value of alpha(l) approximately 3/4, which we take to be the signature of a coupling between the motions of the tracer proteins and the segments of the dextran chains. A similar, although less pronounced, subdiffusive behavior is observed for the diffusion of streptavidin in concentrated globular protein solutions. These observations indicate that protein diffusion in the cell cytoplasm and nucleus should be anomalous as well, with consequences for measurements of solute diffusion coefficients in cells and for the modeling of cellular processes relying on diffusion. |
16113111 | Variability in couplon size in rabbit ventricular myocytes. | Variation in couplon size is thought to be essential for graded Ca(2+) transients in cardiac myocytes. We examined this variation by investigating spark appearance in rabbit ventricular myocytes at various locations and at potentials from -20 to 0 mV. At 0 mV, sparks appeared at the beginning of the voltage step with a probability of unity. On the other hand, at -20 mV, sparks appeared later during the voltage step with a lower probability. The cumulative spark probabilities at various potentials were fitted with exponential functions of both time and voltage. Spark latency became longer as spark probability decreased at more negative potentials. At -20 mV, the cumulative spark probability and the mean spark latency were not only variable among locations but also inversely related. Under the assumption that a single opening of an L-type Ca(2+) channel triggers a spark, we suggest a simple mathematical explanation for the distribution of spark appearance. The variation in spark probability and latency with location suggests that the couplon size, and hence the number of L-type Ca(2+) channels in a couplon is variable. |
16113109 | Morphological behavior of lipid bilayers induced by melittin near the phase transition temperature. | Morphological changes of DMPC, DLPC, and DPPC bilayers containing melittin (lecithin/melittin molar ratio of 10:1) around the gel-to-liquid crystalline phase transition temperatures (Tc) were examined by a variety of biophysical methods. First, giant vesicles with the diameters of approximately 20 microm were observed by optical microscopy for melittin-DMPC bilayers at 27.9 degrees C. When the temperature was lowered to 24.9 degrees C (Tc = 23 degrees C for the neat DMPC bilayers), the surface of vesicles became blurred and dynamic pore formation was visible in the microscopic picture taken at different exposure times. Phase separation and association of melittin molecules in the bilayers were further detected by fluorescent microscopy and mass spectrometry, respectively. These vesicles pletely at 22.9 degrees C. It was thus found that the melittin-lecithin bilayers reversibly undergo their fusion and disruption near the respective Tcs. The fluctuation of lipids is, therefore, responsible for the membrane fusion above the Tc, and the association of melittin molecules causes membrane fragmentation below the Tc. Subsequent magnetic alignments were observed by solid-state (31)P NMR spectra for the melittin-lecithin vesicles at a temperature above the respective Tcs. On the other hand, additional large amplitude motion induced by melittin at a temperature near the Tc breaks down the magnetic alignment. |
16113110 | Probing vesicle dynamics in single hippocampal synapses. | We use fluorescence correlation spectroscopy and fluorescence recovery after photobleaching to study vesicle dynamics inside the synapses of cultured hippocampal neurons labeled with the fluorescent vesicle marker FM 1-43. These studies show that when the cell is electrically at rest, only a small population of vesicles is mobile, taking seconds to traverse the synapse. Applying the phosphatase inhibitor okadaic acid causes vesicles to diffuse freely, moving 30 times faster than vesicles in control synapses. These results suggest that vesicles move sluggishly due to binding to elements of the synaptic cytomatrix and that this binding is altered by phosphorylation. Motivated by these results, a model is constructed consisting of diffusing vesicles that bind reversibly to the cytomatrix. This stick-and-diffuse model accounts for the fluorescence correlation spectroscopy and fluorescence recovery after photobleaching data, and also predicts the well-known exponential refilling of the readily releasable pool. Our measurements suggest that the movement of vesicles to the active zone is the rate-limiting step in this process. |
16113112 | Interactions of liquid crystal-forming molecules with phospholipid bilayers studied by molecular dynamics simulations. | Recent experiments have shown that liquid crystals can be used to image mammalian cell membranes and to amplify structural reorganization in phospholipid-laden liquid crystal-aqueous interfaces. In this work, molecular dynamics simulations were employed to explore the interactions monly used liquid crystal-forming molecules and phospholipid bilayers. In particular, umbrella sampling was used to obtain the potential of mean force of 4-cyano-4'-pentylbiphenyl (5CB) and 4'-(3,4-difluor-phenyl)-4-pentyl-bicylohexyl (5CF) molecules partitioning into a dipalmitoylphosphatidylcholine bilayer. In addition, results of simulations are presented for systems consisting of a fully hydrated bilayer with 5CB or 5CF molecules at the lowest (4.5 mol %) and highest (20 mol %) concentrations used in recent laboratory experiments. It is found that mesogens preferentially partition from the aqueous phase into the membrane; the potential of mean force exhibits highly favorable free energy differences for partitioning (-18 k(B)T for 5CB and -26 k(B)T for 5CF). The location and orientation of mesogens associated with the most stable free energies in umbrella sampling simulations of dilute systems were found to be consistent with those observed in liquid-crystal-rich bilayers. It is found that the presence of mesogens in the bilayer enhances the order of lipid acyl tails, and changes the spatial and orientational arrangement of lipid headgroup atoms. These effects are more pronounced at higher liquid-crystal concentrations. paring the behavior of 5CB and 5CF, a stronger spatial correlation (i.e., possibly leading to aggregation) is observed between 5CB molecules within a bilayer than between 5CF molecules. Also, the range of molecular orientations and positions along the bilayer normal is larger for 5CB molecules. At the same time, 5CF molecules were found to bind more strongly to lipid headgroups, thereby slowing the lateral motion of lipid molecules. |
16113113 | Comparison of tRNA motions in the free and ribosomal bound structures. | A general method is presented that allows the separation of the rigid body motions from the nonrigid body motions of structural subunits when bound in plex. The application presented considers the motions of the tRNAs: free, bound to the ribosome and to a synthase. We observe that both the rigid body and nonrigid body motions of the structural subunits are highly controlled by the large ribosomal assembly and are important for the functional motions of the assembly. For the intact ribosome, its major parts, the 30S and the 50S subunits, are found to have counterrotational motions in the first few slowest modes, which are consistent with the experimentally observed ratchet motion. The tRNAs are found to have on average approximately 72-75% rigid body motions and principally translational motions within the first 100 slow modes of plex. Although the three tRNAs exhibit different apparent total motions, after the rigid body motions are removed, the remaining internal motions of all three tRNAs are essentially the same. The direction of the translational motions of the tRNAs are in the same direction as the requisite translocation step, especially in the first slowest mode. Surprisingly the small intrinsically flexible mRNA has all of its internal pletely inhibited and shows mainly a rigid-body translation in the slow modes of the plex. On the other hand, the required nonrigid body motions of the tRNA during translocation reveal that the anticodon-stem-loop, as well as the acceptor arm, of the tRNA enjoy a large mobility but act as rigid structural units. In summary, the ribosome exerts its control by enforcing rigidity in the functional parts of the tRNAs as well as in the mRNA. |
16113114 | Stepwise length changes in single invertebrate thick filaments. | Previous experiments on thick filaments of the anterior byssus retractor muscle of Mytilus and the telson-levator muscle of Limulus polyphemus have shown large, reversible length changes up to 23% and 66% of initial length, respectively, within the physiological tension range. Using nanofabricated cantilevers and newly developed high-resolution detection methods, we investigated the dynamics of isolated Mytilus anterior byssus retractor muscle thick filaments. Single thick filaments were suspended between the tips of two microbeams oriented perpendicular to the filament axis: a deflectable cantilever and a stationary beam. Axial stress was applied by translating the base of the deflectable nanolever away from the stationary beam, which bent the nanolever. Tips of flexible nanolevers and stationary beam were imaged onto a photodiode array to track their positions. Filament shortening and lengthening traces, obtained immediately after the motor had imposed stress on the filament, showed steps and pauses. Step sizes were 2.7 nm and integer multiples thereof. Steps of this same size paradigm have been seen both during contraction of single eres and during active interaction between single isolated actin and myosin filaments, raising the question whether all of these phenomena might be related. |
16113115 | Plasticity of acetylcholine receptor gating motions via rate-energy relationships. | Like other protein conformational changes, ion channel gating requires the protein to achieve a high-energy transition-state structure. It is not known whether ion channel gating takes place on a broad energy landscape on which many alternative transition state structures are accessible, or on a narrow energy landscape where only a few transition-state structures are possible. To address this question, we measured how rate-equilibrium free energy relationships (REFERs) for di-liganded and unliganded acetylcholine receptor gating vary as a function of the gating equilibrium constant. A large slope for the REFER plot indicates an openlike transition state, whereas a small slope indicates a closedlike transition state. Due to this relationship between REFERs and transition-state structure, the sensitivity of the REFER slope to mutation-induced energetic perturbations allows estimation of the breadth of the energy landscape underlying gating. The relatively large sensitivity of di-liganded REFER slopes to energetic perturbations suggests that the motions underlying di-liganded gating take place on a broad, shallow energy landscape where many alternative transition-state structures are accessible. |
16113116 | N-terminal-mediated homomultimerization of prestin, the outer hair cell motor protein. | The outer hair cell lateral membrane motor, prestin, drives the cell's mechanical response that underpins mammalian cochlear amplification. Little is known about the protein's structure-function relations. Here we provide evidence that prestin is a 10-transmembrane domain protein whose membrane topology differs from that of previous models. We also present evidence that both intracellular termini of prestin are required for normal voltage sensing, with short truncations of either terminal resulting in absent or modified activity despite quantitative findings of normal membrane targeting. Finally, we show with fluorescence resonance energy transfer that prestin-prestin interactions are dependent on an intact N-terminus, suggesting that this terminus is important for homo-oligomerization of prestin. These domains, which we have perturbed, likely contribute to allosteric modulation of prestin via interactions among prestin molecules or possibly between prestin and other proteins, as well. |
16113118 | Protein folding, stability, and solvation structure in osmolyte solutions. | An understanding of the impact of the crowded conditions in the cytoplasm on its biomolecules is of clear importance to biochemical, medical, and pharmaceutical science. Our previous work on the use of small pounds to crowd protein solutions indicates that a quantitative description of their nonideal behavior is possible and straightforward. Here, we show the structural origin of the nonideal solution behavior. We discuss the consequences of these findings regarding protein folding stability and solvation in crowded solutions through a structural analysis of the m-value or the change in free-energy difference of a macromolecule in solution with respect to the concentration of a ponent. |
16113117 | Cyclical interactions between two outer doublet microtubules in split flagellar axonemes. | The beating of cilia and flagella is based on the localized sliding between adjacent outer doublet microtubules; however, the mechanism that produces oscillatory bending is unclear. To elucidate this mechanism, we examined the behavior of frayed axonemes of Chlamydomonas by using high-speed video recording. A pair of doublet microtubules frequently displayed association and dissociation cycles in the presence of ATP. In many instances, the dissociation of two microtubules was not panied by noticeable bending, suggesting that the dynein-microtubule interaction is not necessarily regulated by the microtubule curvature. On rare occasions, association and dissociation occurred simultaneously in the same interacting pair, resulting in a tip-directed movement of a stretch of gap between the pair. Based on these observations, we propose a model for cyclical bend propagation in the axoneme. |
16113119 | Temperature dependence and thermodynamic properties of Ca2+ sparks in rat cardiomyocytes. | To elucidate the temperature dependence and underlying thermodynamic determinants of the elementary Ca2+ release from the sarcoplasmic reticulum, we characterized Ca2+ sparks originating from ryanodine receptors (RyRs) in rat cardiomyocytes over a wide range of temperature. From 35 degrees C to 10 degrees C, the normalized fluo-3 fluorescence of Ca2+ sparks decreased monotonically, but the Delta[Ca2+]i were relatively unchanged due to increased resting [Ca2+]i. The time-to-peak of Ca2+ sparks, which represents the RyR Ca2+ release duration, was prolonged by 37% from 35 degrees C to 10 degrees C. An Arrhenius plot of the data identified a jump of apparent activation energy from 5.2 to 14.6 kJ/mol at 24.8 degrees C, which presumably reflects a transition of sarcoplasmic reticulum lipids. Thermodynamic analysis of the decay kinetics showed that active transport plays little role in early recovery but a significant role in late recovery of local Ca2+ concentration. These results provided a basis for quantitative interpretation of intracellular Ca2+ signaling under various thermal conditions. The relative temperature insensitivity above the transitional 25 degrees C led to the notion that Ca2+ sparks measured at a "warm room" temperature are basically acceptable in elucidating mammalian heart function. |
16113120 | Pulsed interleaved excitation. | In this article, we demonstrate the new method of pulsed interleaved excitation (PIE), which can be used to extend the capabilities of multiple-color fluorescence imaging, fluorescence cross-correlation spectroscopy (FCCS), and single-pair fluorescence resonance energy transfer (spFRET) measurements. In PIE, multiple excitation sources are interleaved such that the fluorescence emission generated from one pulse plete before the next excitation pulse arrives. Hence, the excitation source for each detected photon is known. Typical repetition rates used for PIE are between approximately 1 and 50 MHz. PIE has many applications in various fluorescence methods. Using PIE, dual-color measurements can be performed with a single detector. In fluorescence imaging with multicolor detection, spectral cross talk can be removed, improving the contrast of the image. Using PIE with FCCS, we can eliminate spectral cross talk, making the method sensitive to weaker interactions. FCCS measurements plexes that undergo FRET can be analyzed quantitatively. Under specific conditions, the FRET efficiency can be determined directly from the amplitude of the measured correlation functions without any calibration factors. We also show the application of PIE to spFRET measurements, plexes that have low FRET efficiency can be distinguished from those that do not have an active acceptor. |
16113121 | Indentation and adhesive probing of a cell membrane with AFM: theoretical model and experiments. | In probing adhesion and cell mechanics by atomic force microscopy (AFM), the mechanical properties of the membrane have an important if neglected role. Here we theoretically model the contact of an AFM tip with a cell membrane, where direct motivation and data are derived from a prototypical ligand-receptor adhesion experiment. An AFM tip is functionalized with a prototypical ligand, SIRPalpha, and then used to probe its native receptor on red cells, CD47. The interactions prove specific and typical in force, and also show in detachment, a sawtooth-shaped disruption process that can extend over hundreds of nm. The theoretical model here that accounts for both membrane indentation as well as membrane extension in tip retraction incorporates membrane tension and elasticity as well as AFM tip geometry and stochastic disruption. Importantly, indentation depth proves initially proportional to membrane tension and does not follow the standard Hertz model. Computations of detachment confirm nonperiodic disruption with membrane extensions of hundreds of nm set by membrane tension. Membrane mechanical properties thus clearly influence AFM probing of cells, including single molecule adhesion experiments. |
16113129 | Measured versus reported parental height. | Parental height data are essential in the assessment of linear growth in children. A number of studies have documented inaccuracy in self-reported adult height. |
16113128 | Serious neurological disorders in children with chronic headache. | To determine the prevalence of serious neurological disorders among children with chronic headache. |
16113130 | Ethics support in clinical practice. | Ethical considerations have long been a part of clinical decision making. However, just as doctors and healthcare professionals are being increasingly challenged about the evidence base for their practice, there is increasing focus on the ethical dimension of clinical care. |
16113131 | Neuroradiological aspects of subdural haemorrhages. | To review the neuroimaging of a series of infants and young children admitted to hospital with subdural haemorrhage (SDH). |
16113133 | Acid suppression does not change respiratory symptoms in children with asthma and gastro-oesophageal reflux disease. | Epidemiological studies have shown an association between gastro-oesophageal reflux disease (GORD) and asthma, and oesophageal acid perfusion may cause bronchial constriction. However, no causative relation has been proven. |
16113132 | Subdural haematoma and effusion in infancy: an epidemiological study. | To determine incidence, aetiology, and clinical features of subdural haematoma and effusion (SDH/E) in infancy throughout the British Isles. |
16113135 | Prolonged QT interval in an infant of a fluoxetine treated mother. | Fluoxetine is an antidepressant drug, only recently discovered to be a QT interval prolonging agent. The first case is presented of an infant of a mother treated with fluoxetine during pregnancy who had a transiently prolonged QTc interval, concluded to be drug induced. Clinical and electrocardiographic monitoring of newborns of antidepressant treated mothers is needed. |
16113136 | Child survival: district hospitals and paediatricians. | In a previous article in this series, Zulfiquar Bhutta outlined many of the key sociopolitical issues, both national and international, that currently affect the delivery of health care to children in developing countries. The clear summary of our situation is that we are failing to provide even basic health care (both preventive and curative) that could reduce child mortality globally by more than half. Paediatricians, who have perhaps in the past felt they were at the forefront of articulating and promoting a global health agenda, should be challenged by these conclusions. The successful ratification of the United Nations Convention on the Rights of the Child that unequivocally target health was not a finishing line, a goal achieved, but rather a foundation for action. Therefore while researchers might have felt some satisfaction at successes in defining optimum treatment approaches, the pathways to delivering services were, and remain, far from clear. Progress is plicated by the diverse conditions and obstacles that may be encountered worldwide. |
16113134 | Prediction of total body water in infants and children. | In paediatric clinical practice treatment is often adjusted in relation to body size, for example the calculation of pharmacological and dialysis dosages. In addition to use of body weight, for some purposes total body water (TBW) and surface area are estimated from anthropometry using equations developed several decades previously. Whether such equations remain valid in contemporary populations is not known. |
16113149 | EUROCAT: 25 years of European surveillance of congenital anomalies. | The surveillance of congenital anomalies serves two main purposes: to facilitate the identification of teratogenic (malformation causing) exposures and to assess the impact of primary prevention and prenatal screening policy and practice at a population level. EUROCAT, the European network of population based registers for the epidemiological surveillance of congenital anomalies, now covers 1.2 million births per year, a quarter of births in Europe. The added value of European collaboration is particularly great for congenital ing from the opportunity to pool data, pare data between regions and countries, to give mon response to European public health questions, and to share expertise and resources, puting tools. EUROCAT provides essential epidemiological information on congenital anomalies in Europe, facilitates the early warning of teratogenic exposures, evaluates the effectiveness of primary prevention, assesses the impact of developments in prenatal screening, acts as an information and resource centre regarding clusters, provides a ready collaborative network and infrastructure for research, and acts as a catalyst for the setting up of registries throughout Europe. |
16113151 | How has research in the last five years changed my clinical practice? | Although the practice of neonatology is increasingly evidence based, much of our assessment and treatment of newborn infants is still founded on mon sense, guesswork, and myth. Research may put a particular issue beyond dispute, but it often raises new questions. This review looks at the benefits of research in one area: temperature control. |
16113150 | Feeding growth restricted preterm infants with abnormal antenatal Doppler results. | Absence or reversal of end diastolic flow (AREDF) in the umbilical artery is associated with poor e, and elective premature delivery mon. Feeding these infants is a challenge. They often have poor tolerance of enteral feeding, and necrotising enterocolitis may develop. This review explores current practice to see if there is evidence on which to base guidelines. The incidence of necrotising enterocolitis is increased in infants with fetal AREDF, especially plicated by fetal growth restriction. Abnormalities of splanchnic blood flow persist postnatally, with some recovery during the first week of life, providing justification for a delayed and careful introduction of enteral feeding. Such a policy exposes babies to the risks of parenteral nutrition, with no trials to date showing any benefit of delayed enteral nutrition. Trials are required to determine the optimum timing for introduction of enteral feeds in growth restricted infants with fetal AREDF. |
16113152 | Evaluation of the National Congenital Anomaly System in England and Wales. | To evaluate the National Congenital Anomaly System (NCAS). |
16113153 | Prevalence of congenital anomalies in five British regions, 1991-99. | To describe trends in total and live birth prevalence, regional differences in prevalence, and e of pregnancy of selected congenital anomalies. |
16113154 | Neuropsychological and educational problems at school age associated with neonatal encephalopathy. | Adverse cognitive and educational es are often ascribed to perinatal hypoxia without good evidence. |
16113155 | Severity of the ductal shunt: a comparison of different markers. | When the ductus arteriosus (DA) is patent, the ductal shunt is proportional to the ratio of left ventricular output (LVO) to systemic blood flow. Systemic blood flow can be estimated by measuring flow in the superior vena cava (SVC). |
16113156 | Families' views on ward rounds in neonatal units. | To discover parental preferences about visiting during ward rounds. |
16113157 | J Clifton Edgar (1859-1939) of New York and his obstetric text. | Edgar, a leading academic obstetrician/gynaecologist in the United States at the beginning of the 20th century, explored a number of interesting aspects of pregnancy and childbirth in his 1903 textbook on obstetrics. |
16113166 | Comparative biochemical analyses of venous blood and peritoneal fluid from horses with colic using a portable analyser and an in-house analyser. | Fifty-six horses with colic were examined over a period of three months. The concentrations of glucose, lactate, sodium, potassium and chloride, and the pH of samples of blood and peritoneal fluid, were determined with a portable clinical analyser and with an in-house analyser and the results pared. Compared with the in-house analyser, the portable analyser gave higher pH values for blood and peritoneal fluid with greater variability in the alkaline range, and lower pH values in the acidic range, lower concentrations of glucose in the range below 8.3 mmol/l, and lower concentrations of lactate in venous blood in the range below 5 mmol/l and in peritoneal fluid in the range below 2 mmol/l, with less variability. On average, the portable analyser underestimated the concentrations of lactate and glucose in peritoneal fluid parison with the in-house analyser. Its measurements of the concentrations of sodium and chloride in peritoneal fluid had a higher bias and were more variable than the measurements in venous blood, and its measurements of potassium in venous blood and peritoneal fluid had a smaller bias and less variability than the measurements made with the in-house analyser. |
16113167 | Review of the current involvement of homeopathy in veterinary practice and research. | Homeopathy has e the focus of increasing interest and use as plementary and alternative treatment for both human and animal disease. However, from the point of view of academic medicine, this type of therapy is controversial. The use of highly diluted remedies cannot be reconciled with the scientific theories on which the current understanding of disease and its treatment is based, and clinical research in the field is considered to be neither extensive enough nor of a high enough standard to determine whether homeopathic treatments are clinically effective. Animals have no choice in their treatment and are dependent on the judgements of their owners and their therapists. There is therefore a need for information about the effects and consequences of the use of alternative therapies. This paper discusses the use of homeopathy in the treatment of animal disease from the point of view of academic veterinary medicine, and the various approaches to research in this field, with an emphasis on the randomised clinical trial. It also discusses the role of the placebo response and the natural resolution of disease in the clinical evaluation of homeopathic treatment. |
16113168 | Risk factors for the infection of Swiss goat herds with small ruminant lentivirus: a case-control study. | In the mid-1980s, Switzerland started a programme to eradicate caprine arthritis-encephalitis - an infectious disease of goats caused by the small ruminant lentivirus (srlv). Since 1996, progress towards eradication has slowed down, owing to infections occurring on farms from which the infection had previously been eliminated. To investigate specific risk factors for these new infections and to improve the eradication programme, a case-control study was conducted. Cases consisted of farms that had been officially free of srlv for at least three consecutive years but on which at least one srlv-seropositive animal had been detected during the annual serological surveys of 2001 and 2002. On all the case and control farms where sheep were housed together with goats, a subset of sheep was screened for antibodies to srlv. Potential risk factors were analysed in a logistic regression model; the results indicated that close contact with srlv-seropositive sheep was highly correlated with seroconversion in srlv-seronegative goat herds (odds ratio=26.9), supporting the hypothesis that srlv can be transmitted between sheep and goats, and suggesting that the measures taken so far will not lead to plete eradication of srlv from Switzerland within the next few years. |
16113176 | Naloxone in opioid poisoning: walking the tightrope. | Acute opioid intoxication and overdose mon causes of presentation to emergency departments. Although naloxone, a pure opioid antagonist, has been available for many years, there is still confusion over the appropriate dose and route of administration. This article looks at the reasons for this uncertainty and undertakes a literature review from which a treatment algorithm is presented. |
16113178 | Simplifying thrombolysis decisions in patients with left bundle branch block. | To redesign and simplify an existing decision algorithm for the management of patients who present to the emergency department with chest pain and left bundle branch block (LBBB) based on the Sgarbossa criteria. pare its reliability with the current algorithm. |
16113180 | Odontoid lateral mass asymmetry: do we over-investigate? | This study aimed to evaluate the necessity for further radiological investigation in patients with suspected traumatic rotatory subluxation of the plex on plain radiography following acute cervical trauma and outline guidelines for assessment of patients with atlanto-axial asymmetry on plain radiography. |
16113179 | Rate of metoclopramide infusion affects the severity and incidence of akathisia. | To investigate the effect of the rate of metoclopramide infusion on akathisia incidence, severity, onset of symptoms, and duration in patients with headache, and/or nausea/vomiting in the emergency department (ED) setting. |
16113181 | To lead or not to lead? Prospective controlled study of emergency nurses' provision of advanced life support team leadership. | In many emergency departments advanced life support (ALS) trained nurses do not assume a lead role in advanced resuscitation. This study investigated whether emergency nurses with previous ALS training provided good team leadership in a simulated cardiac arrest situation. |
16113182 | Core-peripheral temperature gradient as a diagnostic test in dyspnoea. | To evaluate whether the core-peripheral temperature gradient could be used to distinguish between cardiac and respiratory causes of dyspnoea. |
16113183 | Detection of hypertension in the emergency department. | To assess whether an emergency department (ED) is a suitable location for the targeted screening of hypertension. |
16113185 | Pressurised metered dose inhalers with spacers versus nebulisers for beta-agonist delivery in acute asthma in children in the emergency department. | Wet nebulisers are widely used for beta-2 agonist delivery in Australasian emergency departments (ED). An increasing body of work suggests that pressurised metered dose inhalers with spacers (pMDI-S) are as effective. This study aims to investigate the effect on admission rates, total time in the ED, and total time in hospital after initiation of routine pMDI-S treatment for mild to moderate asthma in children presenting to the ED. |
16113186 | So much for percentage, but what about the weight? | The use of resuscitation formulae for burns is advocated for A&E departments. Much care is taken to calculate the percentage of the burn, but this is then multiplied by an approximate weight. How accurate is this figure and should it be more carefully measured? Forty two sets of case notes of patients with resuscitation sized burns were reviewed. In 32, a weight was documented in the A&E notes. This pared with the measured weight on arrival at the burn centre. In half the cases there was a greater than 5 kg difference and in nine patients over 10 kg of difference. The methods of patient weight assessment in 16 A&E departments were reviewed. The majority have only stand on scales. Three departments have sit on scales; however, if they were assessing the weight of a patient who is unable to sit they would need to ask the patient, relatives, or simply guess. Investment in weighing equipment should be encouraged if resuscitation formulae are to have any place in the A&E management of burn patients. |
16113188 | Best evidence topic report. Do non-steroidal anti-inflammatory drugs cause a delay in fracture healing? | A short cut review was carried out to establish whether there is any evidence that non-steroidal anti-inflammatory drugs (NSAIDs) might delay fracture healing. A total of 514 papers were found using the reported search, of which three represent the best evidence to answer the clinical question. The author, date and country of publication, patient group studied, study type, relevant es, results, and study weaknesses of these best papers are tabulated. At present, although there are theoretical concerns about the adverse effects of NSAIDs on fracture healing, there is not enough clinical evidence to deny patients with simple fractures their analgesic benefits. |
16113190 | Best evidence topic report. Rectal or intravenous non-steroidal anti-inflammatory drugs in acute renal colic. | A short cut review was carried out to establish whether rectal non-steroidal anti-inflammatory drugs (NSAIDs) are as effective as IV NSAIDs in the management of acute renal colic. Altogether 179 papers were found using the reported search, of which two represent the best evidence to answer the clinical question. The author, date and country of publication, patient group studied, study type, relevant es, results and study weaknesses of these best papers are tabulated. Rectal NSAIDs are an effective form of analgesia for patients with acute renal colic and have fewer side pared with intravenous NSAIDs. |
16113191 | Best evidence topic report. Nebulised furosemide in acute adult asthma. | A short cut review was carried out to establish whether the addition of nebulised furosemide to beta-agonist therapy improves es in acute asthma. Altogether 87 papers were found using the reported search, of which two presented the best evidence to answer the clinical question. A further relevant paper was found on scanning the references of these papers. The author, date and country of origin, patient group studied, study type, relevant e, results, and study weaknesses of the best papers are tabulated. There is currently insufficient evidence to support the routine addition of nebulised furosemide to standard beta agonist therapy in acute asthma in adults. |
16113192 | Best evidence topic report. Are antibiotics indicated following human bites? | A short cut review was carried out to establish whether antibiotics are indicated for human bites. Eighty nine papers were found using the reported search, of which two represent the best evidence to answer the clinical question. The author, date and country of publication, patient group studied, study type, relevant es, results, and study weaknesses of these best papers are tabulated. Prophylactic antibiotics should be given to all patients with human bites to the hands, feet, and skin overlying joints or cartilaginous structures, and to all patients with bites that penetrate deeper than the epidermal layer. |
16113194 | Emergencies in the air. | Medical emergencies in the air are topical. This study, undertaken by the Faculty of Prehospital Care at the Royal College of Surgeons of Edinburgh, analyses retrospectively consecutive medical emergencies occurring over a 6 month period on a single major international airline. Clinical problems are related to pre-existing problems (65%), new medical problems (28%), and traumatic injury (7%). The paper highlights the need for better pre-flight health advice and screening, a reminder to carry personal medication-especially for asthma-and the need to include ankle and leg exercises prior to getting out of the seat to reduce the incidence of syncope, which was responsible for 91% of new medical problems. |
16113195 | Prehospital management of lower limb fractures. | Lower limb fractures mon injuries in prehospital care. Untreated fractures can lead to hypovolaemic shock especially if open, and should be treated with effective haemorrhage control and splintage. A brief assessment for open fractures, deformity, and promise should be followed by effective analgesia, wound management, reduction (if needed), splintage, and packaging of the patient. Early appropriate management reduces the morbidity and mortality of lower limb fractures. |
16113197 | Hereditary motor and sensor neuropathy: a cause of acute stridor. | We present an acute stridor secondary to bilateral vocal cord paresis in a patient with demyelinating form (type I) of hereditary motor and sensory neuropathy (HMSN). Management problems are discussed and HMSN reviewed. |
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