The prognostic relevance of estimates of proliferative activity in early breast cancer.
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Antibodies, Monoclonal↗
Biomedical subjects
C Whiteside
Publications and source records attributed to C Whiteside.
Insulin, insulin-like growth factor-I, and platelet-derived growth factor activate extracellular signal-regulated kinase by distinct pathways in muscle cells.
We have investigated the signaling pathways initiated by insulin, insulin-like growth factor-1 (IGF-I), and platelet-derived growth factor (PDGF) leading to activation of the extracellular signal-regulated kinase (ERK) in L6 myotubes. Insulin but not IGF-I or PDGF-induced ERK activation was abrogated by Ras inhibition, either by treatment with the farnesyl transferase inhibitor FTP III, or by actin disassembly by cytochalasin D, previously shown to inhibit Ras activation. The protein kinase C (PKC) inhibitor bisindolylmaleimide abolished PDGF but not IGF-I or insulin-induced ERK activation. ERK activation by insulin, IGF-I, or PDGF was unaffected by the phosphatidylinositol 3-kinase inhibitor wortmannin but was abolished by the MEK inhibitor PD98059. In contrast, activation of the pathway involving phosphatidylinositol 3-kinase (PI3k), protein kinase B, and glycogen synthase kinase 3 (GSK3) was mediated similarly by all three receptors, through a PI 3-kinase-dependent but Ras- and actin-independent pathway. We conclude that ERK activation is mediated by distinct pathways including: (i) a cytoskeleton- and Ras-dependent, PKC-independent, pathway utilized by insulin, (ii) a PKC-dependent, cytoskeleton- and Ras-independent pathway used by PDGF, and (iii) a cytoskeleton-, Ras-, and PKC-independent pathway utilized by IGF-I.
Actins↗
Differences in quality of life across renal replacement therapies: a meta-analytic comparison.
A meta-analysis compared emotional distress and psychological well-being across renal replacement therapies (RRTs) and examined whether differences could be explained by: (1) treatment modalities, (2) case mix, or (3) methodologic rigor. Standard meta-analytic procedures were used to evaluate published comparative studies. Successful renal transplantation was associated with: (1) lower distress (effect size, d = -0.43 SD) and greater well-being (d = 0. 62 SD) than in-center hemodialysis (CHD) and (2) lower distress (d = -0.29 SD) and greater well-being (d = 0.53 SD) than continuous ambulatory peritoneal dialysis (CAPD). CAPD was characterized by greater well-being (d = 0.18 SD) than CHD and CHD was associated with greater distress (d = 0.16 SD) than home hemodialysis. Although methodologic rigor and case-mix differences did not correlate with the magnitude of psychosocial differences across RRTs, 10 of the 12 comparisons (83%) were threatened by publication bias (ie, that nonsignificant comparisons may have been underrepresented in the published literature). Thus, although significant quality-of-life differences were evident across treatment groups, the types of patients representative of the various RRTs also differed significantly in terms of case-mix variables relevant to psychosocial well-being and emotional distress. Published findings indicating differential quality of life across RRTs may thus be attributable to: (1) valid differences in effective renal replacement, reduced medical complications, and lifestyles afforded by these treatment modalities; (2) case-mix differences in the patient samples selected to represent them in research comparisons; or (3) both of these alternative explanations.
Adolescent↗
Diabetes mellitus increases endothelin-1 gene transcription in rat kidney.
BACKGROUND: Mesangial cell hypertrophy and increased extracellular matrix (ECM) contribute to mesangial expansion in early progressive diabetic nephropathy. Previous studies suggest that the growth factor endothelin-1 (ET-1) is not only up-regulated in diabetes, but may mediate the effects of hyperglycemia on mesangial cell hypertrophy and ECM synthesis. In models of diabetes mellitus, the mechanisms underlying increased ET-1 peptide and mRNA remain unknown. Therefore, our purpose is to determine whether ET-1 gene activity increases in kidneys of streptozotocin (SZT)-treated rats. METHODS: Male Sprague-Dawley rats were injected with either SZT or vehicle. Parameters including glucose, body weight, 24-hour urine volume, urinary protein, and urinary ET-1 excretion were recorded. All rats were sacrificed at 12 weeks postinjection. Prepro-ET-1 mRNA from whole kidneys was determined using both RNase protection and reverse transcription-polymerase chain reaction (RT-PCR). The abundance of ET-1 peptide in primary cultured mesangial cells was detected by indirect immunofluorescence following treatment with 5.6, 11.2, or 22.5 mmol/L D-glucose for 24 hours. Cellular ET-1 mRNA was measured using RT-PCR in control cells at time 0 and also following exposure to increasing concentrations of glucose for 24 hours. Rat mesangial cells were transfected with a luciferase reporter construct containing the rat ET-1 promoter (pET1. Luc), and relative ET-1 promoter activity was measured after a 24-hour exposure to 5.6 and 22.5 mmol/L of D- or L-glucose. RESULTS: After 12 weeks of hyperglycemia, diabetic rats gained less weight (344 +/- 23.9 vs. 548.75 +/- 15.08 g), had increased urinary volume (158.6 +/- 24.32 vs. 8.38 +/- 1.56 mL/day), and had marked proteinuria (101.7 +/- 12.2 vs. 14.1 +/- 2.8 mg/day) compared with controls. Total urinary ET-1 peptide increased 26.4-fold in diabetic versus control rats (17.5083 +/- 5.405 vs. 0.6635 +/- 0.343 ng/day). ET-1 mRNA extracted from whole rat kidneys was increased 2.1-fold in diabetic versus control animals. Primary cultured rat mesangial cells demonstrated a significant increase in immunofluorescence labeling of ET-1 peptide and ET-1 mRNA in response to increasing concentrations of glucose. Furthermore, transfected mesangial cells exposed to 22.5 mmol/L D-glucose showed a 1.6-fold increase in ET-1 promoter activity relative to those treated with 5.6 mmol/L glucose. CONCLUSION: Glucose increases ET-1 gene expression in the kidney of the SZT-treated rat model of diabetes mellitus. Furthermore, high glucose induces ET-1 expression in primary cultured rat mesangial cells and directly enhances ET-1 promoter activity. The greater relative increase in peptide compared with transcription suggests the potential participation of other mechanisms such as increased mRNA stability, protein stability, and/or enhanced translational efficiency.
Animals↗
BMP-2/ALK3 and HGF signal in parallel to regulate renal collecting duct morphogenesis.
Bone morphogenetic protein (BMP)-2 and hepatocyte growth factor (HGF) exert antagonistic effects on renal collecting duct formation during embryogenesis. A current model proposes HGF inhibits BMP-2 signaling at the level of Smad1 in a common target cell. Here, we show that BMP-2 and HGF control collecting duct formation via parallel pathways. We examined the interactions between BMP-2 and HGF in the mIMCD-3 model of collecting duct morphogenesis. During tubule formation, HGF rescued the inhibitory effects of BMP-2 and of a constitutive active form of the BMP-2 receptor, ALK3, stably expressed in mIMCD-3 cells. To determine whether the effect of HGF occurs through known mediators which act downstream of the BMP-2/ALK3 complex, we examined the effect of HGF on BMP-2-induced Smad1 phosphorylation, Smad1/Smad4 complex formation, and Smad1 nuclear translocation. Neither HGF nor other receptor tyrosine kinase ligands (EGF, FGF-4) induced phosphorylation of endogenous Smad1 in mIMCD-3 cells or in Mv1Lu, MC3T3-E1 or P19 cells. Furthermore, none of these ligands blocked induction of the BMP-responsive promoter, Tlx2. Thus, HGF overcomes the inhibitory effects of BMP-2 on collecting duct morphogenesis without interrupting any of the known signaling events in the BMP-2 dependent Smad1 signaling pathway. We conclude that BMP-2/ALK3 and HGF function to control parallel pathways downstream of their respective cell surface receptors. Integration of these signals likely occurs at the level of transcriptional or post-transcriptional events.
Activin Receptors↗
Protein kinase A is a negative regulator of renal branching morphogenesis and modulates inhibitory and stimulatory bone morphogenetic proteins.
Protein kinase A (PKA) regulates morphogenetic responses to bone morphogenetic proteins (BMPs) during embryogenesis. However, the mechanisms by which PKA regulates BMP function are unknown. During kidney development, BMP-2 and high doses of BMP-7 inhibit branching morphogenesis, whereas low doses of BMP-7 are stimulatory (Piscione, T. D., Yager, T. D., Gupta, I. R., Grinfeld, B., Pei, Y., Attisano, L., Wrana, J. L., and Rosenblum, N. D. (1997) Am. J. Physiol. 273, F961-F975). We examined the interactions between PKA and these BMPs in embryonic kidney explants and in the mouse inner medullary collecting duct-3 model of collecting duct morphogenesis. H-89, an inhibitor of PKA, stimulated branching morphogenesis and enhanced the stimulatory effect of low doses of BMP-7 on tubule formation. Furthermore, H-89 rescued the inhibition of tubulogenesis by BMP-2 (or high doses of BMP-7) by attenuating BMP-2-induced collecting duct apoptosis. In contrast, 8-bromo-cAMP, an activator of PKA, inhibited tubule formation and attenuated the stimulatory effects of low doses of BMP-7. To determine mechanisms underlying the interdependence of BMP signaling and PKA activity, we examined the effect of PKA on the known signaling events in the BMP-2-dependent Smad1 signaling pathway and the effect of BMP-2 on PKA activity. PKA did not induce endogenous Smad1 phosphorylation, Smad1-Smad4 complex formation, or Smad1 nuclear translocation. In contrast, BMP-2 increased endogenous PKA activity and induced phosphorylation of the PKA effector, cAMP-response element-binding protein, in a PKA-dependent manner. We conclude that BMP-2 induces activation of PKA and that PKA regulates the effects of BMPs on collecting duct morphogenesis without activating the known signaling events in the BMP-2-dependent Smad1 signaling pathway.
Animals↗
High glucose alters the response of mesangial cell protein kinase C isoforms to endothelin-1.
BACKGROUND: High glucose causes glomerular mesangial growth and increased matrix synthesis contributing to diabetic glomerulopathy. Our purpose was to determine if high glucose alters endothelin-1 (ET-1) or platelet-derived growth factor-B activation of mesangial cell diacylglycerol-sensitive protein kinase C (PKC) isoforms and subsequent stimulation of mitogen-activated protein kinase (MAPK; p42, p44). METHODS: Rat mesangial cells in primary culture were growth arrested for 48 hours in glucose 5.6 mM (NG) or 30 mM (HG). PKC-alpha, PKC-delta, and PKC-epsilon translocation from the cytosol-to-membrane and cytosol-to-particulate (cytoskeleton, nucleus) cellular fractions were measured by immunoblot using isoform-specific monoclonal antibodies. PKC isoforms were visualized also by confocal immunofluorescence microscopy. MAPK activation was measured by immunoblot using phospho-MAPK antibody and by detection of Elk-1 fusion protein phosphorylation following phospho-MAPK immunoprecipitation. RESULTS: In NG, ET-1 stimulated cytosol-to-membrane translocation of PKC-delta and PKC-epsilon but not PKC-alpha. In HG, the pattern of ET-1-stimulated PKC-delta and PKC-epsilon changed to a cytosol-to-particulate distribution, which was confirmed by confocal immunofluorescence imaging. Platelet-derived growth factor-B did not cause translocation of PKC-alpha, PKC-delta, or PKC-epsilon in either NG or HG. In HG, both basal and ET-1-stimulated MAPK activities were increased significantly. In HG, down-regulation of PKC isoforms with phorbol ester prevented the increased stimulation of MAPK by ET-1. CONCLUSION: In HG, the enhanced activation of mesangial cell MAPK by ET-1 is PKC dependent and associated with altered translocation of PKC-delta and PKC-epsilon. Enhanced mesangial cell signaling responsiveness to vasoactive peptides in HG may constitute an important mechanism contributing to diabetic nephropathy.
Animals↗
Effect of high glucose on mesangial cell protein kinase C-delta and -epsilon is polyol pathway-dependent.
In diabetes mellitus, enhanced activity of mesangial cell protein kinase C (PKC) may contribute to nephropathy. The purpose of this study was to determine whether high glucose alters mesangial cell diacylglycerol-sensitive PKC-alpha, -beta2, -delta, and -epsilon content, cellular distribution, and activity through polyol pathway activation. Primary cultured rat mesangial cells (passage 10) were growth-arrested in 0.5% fetal bovine serum and cultured in 5.6 mM glucose (NG) or 30 mM glucose (HG) for 48 h, with or without the aldose reductase inhibitor tolrestat or ARI-509. PKC isoform content in total cell lysates, or cytosol, membrane (Triton X-soluble), and particulate (sodium dodecyl sulfate-soluble) fractions was analyzed by immunoblotting, and band density in HG was expressed as a percentage of corresponding NG values. In HG at 48 h, increased total PKC-alpha (222 +/- 17% of NG, P < 0.001), -beta2 (209 +/- 12%, P < 0.001), and -epsilon (195 +/- 19%, P < 0.001) were observed. L-Glucose had no effect on total PKC isoform content. HG caused increased membrane- and particulate-associated PKC-alpha (257 +/- 87 and 327 +/- 66%, respectively, P < 0.05), membrane-associated PKC-delta (143 +/- 10%, P < 0.05), and membrane-associated PKC-epsilon (186 +/- 11%, P < 0.001), with no change in cytosol contents. The HG effects were not mimicked by L-glucose. In NG or HG, PKC-beta2 was not detected in the cytosol fraction, and membrane and particulate association were unchanged with phorbol ester stimulation. Confocal immunofluorescence imaging revealed that in HG, PKC-alpha, -delta, and -epsilon translocate to the nucleus and plasma membrane. Total PKC activity measured by in situ 32P-phosphorylation of the epidermal growth factor receptor substrate increased from 18 +/- 1 pmol/min per mg cell protein in NG to 33 +/- 3 pmol/min per mg cell protein in HG (P < 0.002 versus NG). In NG, tolrestat and ARI-509 exposure caused increased PKC activity, enhanced accumulation of total PKC-alpha and -beta2, with no change in total or fractional recovery of PKC-delta or -epsilon. In HG, tolrestat and ARI-509 prevented the increase in total PKC-epsilon and membrane-associated PKC-delta and -epsilon. It is concluded that within 48 h of HG, enhanced mesangial cell PKC activity is associated with accumulation and cellular redistribution of diacylglycerol-sensitive PKC isoforms, and that increased PKC-epsilon content and membrane-associated PKC-delta and -epsilon are dependent on polyol pathway activation.
Animals↗
Influences of educational interventions and adverse news about calcium-channel blockers on first-line prescribing of antihypertensive drugs to elderly people in British Columbia.
BACKGROUND: The way in which dissemination of evidence changes medical practice needs to be better understood. Controversy about calcium-channel blockers (CCB) in the past 3 years has provided a natural experiment, enabling assessment of the impact of media stories, a national warning letter, a teleconference, small group workshops, and newsletters on first-line prescribing of antihypertensive drugs. METHODS: We included all physicians (4403) in British Columbia who prescribed a thiazide diuretic, beta-blocker, inhibitor of angiotensin-converting enzyme (ACE), or CCB as the first antihypertensive agent for 36,507 residents aged 66 years and over, with no previous or concurrent sign of underlying cardiovascular disease. We used a database covering all prescriptions to elderly people to measure the change in proportion of newly treated patients who received each class of drug as first-line therapy. We used a matched cohort design for assessment of the teleconference and workshops, a randomised community design for the newsletters, and time-series analysis for the media impacts. FINDINGS: The proportion of patients who received a CCB as first-line therapy declined gradually from 22% in early 1994 to 15% in late 1996. This proportion was not affected by two waves of adverse news about CCBs in 1995, but fell by 5% for 5 months and by 3% for 1 month after two waves in 1996. The proportion of patients who received either a CCB or an ACE inhibitor as first-line therapy, contrary to guidelines, was still 42% overall in 1996. The workshops and newsletters were followed by shifts from first-line CCB to first-line thiazide prescribing. INTERPRETATION: Changes in prescribing practices occur gradually with the accumulation of small impacts from educational interventions and lay media attention.
Adrenergic beta-Antagonists↗
High glucose-induced mesangial cell altered contractility: role of the polyol pathway.
Glomerular mesangial cells cultured in high glucose conditions display impaired contractile responsiveness. It was postulated that glucose metabolism through the polyol pathway leads to altered mesangial cell contractility involving protein kinase C. Rat mesangial cells were growth-arrested for 24 h with 0.5% fetal bovine serum in either normal (5.6 mmol/l) or high (30 mmol/l) glucose concentrations or high glucose plus the aldose reductase inhibitor, ARI-509 (100 micromol/l). The reduction of cell planar surface area (contraction) in response to endothelin-1 (0.1 micromol/l), or to phorbol 12-myristate 13-acetate (50 pmol/l), was studied by videomicroscopy. In response to endothelin-1, mesangial cells in normal glucose contracted to 52+/-3% of initial planar area. In high glucose, the significantly (p < 0.05) smaller cell size and no contractile responsiveness to endothelin-1 were normalized with ARI-509. Membrane-associated diacylglycerol, measured by a kinase specific 32P-phosphorylation assay, in high glucose was unchanged after 3 h, but significantly increased (p < 0.05) after 24 h which was normalized with ARI-509. Protein kinase C activity, measured by in situ 32P-phosphorylation of the epidermal growth factor receptor substrate was: increased by 32% at 3 h of high glucose, unchanged by ARI-509; and decreased significantly (p < 0.05) at 24 h compared to cells in normal glucose, normalized by ARI-509. Total cellular protein kinase C-alpha, -delta and -epsilon, analysed by immunoblotting, were unchanged in high glucose at 24 h. Only protein kinase C-epsilon content was reduced by ARI-509 in both normal and high glucose. Therefore, high glucose-induced loss of mesangial cell contractility, diacylglycerol accumulation and altered protein kinase C activity are mediated through activation of the polyol-pathway, although no specific relationship between elevated diacylglycerol and protein kinase C activity was observed. In high glucose, altered protein kinase C function, or another mechanism related to the polyol pathway, contribute to loss of mesangial cell contractile responsiveness.
Aldehyde Reductase↗
Immunohistochemical study of testicular sex cord-stromal tumors, including staining with anti-inhibin antibody.
Inhibin is a peptide hormone produced by ovarian granulosa cells and testicular Sertoli cells. Ovarian granulosa cell and other sex cord-stromal tumors usually exhibit positive immunohistochemical staining with antiinhibin antibodies, and this may be valuable in differentiating these neoplasms from histologic mimics. In the present study, we investigated the immunohistochemical staining of testicular sex cord-stromal tumors using antiinhibin. Immunostaining with CAM5.2, vimentin, S-100 protein, desmin, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), and placental alkaline phosphatase (PLAP) also was performed because few studies have investigated in detail the immunophenotype of testicular sex cord-stromal tumors. Fifteen of 16 Leydig cell tumors exhibited strong positive staining with antiinhibin. A proportion of Leydig cell tumors also stained positively with CAM5.2 (7 of 16), vimentin (14 of 16), S-100 protein (10 of 16), desmin (2 of 16) and epithelial membrane antigen (4 of 16). Four of six testicular sex cord-stromal tumors with varying degrees of Sertoli or granulosa cell differentiation were positive with antiinhibin, as were two of three sex cord-stromal tumors that were unclassified. Some of these tumors were positive with CAM 5.2, vimentin, S-100 protein, desmin, and epithelial membrane antigen. All tumors were negative with carcinoembryonic antigen and placental alkaline phosphatase. The immunohistochemical findings show that, analogous to their ovarian counterparts, most testicular sex cord-stromal tumors are immunoreactive with antiinhibin. Immunohistochemistry using this antibody as part of a panel may be valuable in confirming a diagnosis of testicular sex cord-stromal tumor and in differentiating these neoplasms from others that may mimic them.
Antibodies, Monoclonal↗
Chelation of intracellular calcium prevents mesangial cell proliferative responsiveness.
Mesangial cell transformation into a proliferative phenotype, observed in many glomerular diseases, occurs in response to growth factors and cytokines. This study tests the hypothesis that intracellular calcium is necessary for stimulation of mesangial cell proliferative responsiveness to a variety of growth factors. Furthermore, these experiments tested whether nonspecific calcium entry via a calcium ionophore was sufficient to elicit the same response. Rat primary mesangial cells (passages 5 to 10) were growth-arrested for 48 h in 0.5% fetal bovine serum (FBS), then stimulated with 0.1 microM endothelin-1, 1.9 microM platelet-derived growth factor (PDGF)-BB, 0.5% FBS, or 0.1 microM ionomycin, with or without the intracellular calcium chelator 1,2-bis-(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid (BAPTA). Calcium signaling was measured in Fura-2-loaded cells on coverslips by dual-wavelength spectrofluorometry and in Fluo-3-loaded cells by confocal fluorescence laser microscopy. [3H]-Thymidine incorporation was measured after 12 to 24 h of stimulation with each test agent. Expression of c-fos mRNA was analyzed by Northern blot. All test agents stimulated a significant increase in cytosolic and nuclear calcium, which were both effectively inhibited with BAPTA. All agents stimulated a significant increase in [3H]-thymidine incorporation and enhanced c-fos mRNA expression (no detectable c-fos mRNA was observed in quiescent cells). BAPTA prevented the enhanced [3H]-thymidine incorporation stimulated by endothelin-1 and PDGF, and partial inhibition of FBS-stimulated incorporation with BAPTA was observed. BAPTA inhibited c-fos expression observed in response to these agents. Phorbol ester induction of c-fos mRNA in the absence of raised cytosolic or nuclear calcium was also suppressed by BAPTA. Cell viability as measured by thiazolyl blue and trypan blue was not altered by BAPTA. It is concluded that normal regulation of intracellular calcium is necessary for mesangial cell proliferative responsiveness.
Animals↗
Altered expression and subcellular localization of diacylglycerol-sensitive protein kinase C isoforms in diabetic rat glomerular cells.
Protein kinase C (PKC) is implicated in the pathogenesis of diabetic nephropathy. This study was designed to identify the expression of diacylglycerol (DAG)-sensitive PKC-alpha, -betaII, -delta, and -epsilon isoforms in normal and diabetic rat glomerular cells and to determine the effects of high glucose and insulin on PKC isoform cellular compartmentalization and PKC activity. Diabetic rats treated with or without insulin and normal rats were examined 2 and 4 weeks after streptozotocin/vehicle injection. Renal cortical tissue immunogold-labeled with anti-PKC-alpha, -betaII, -delta, or -epsilon antibody was visualized by electron microscopy. From isolated glomeruli, total cell lysate and cytosol and membrane fractions were immunoblotted with the same anti-PKC isoform antibodies. PKC activity in isolated glomeruli was measured by 32P-phosphorylation of the epidermal growth factor (EGF)-receptor substrate. Immunogold labeling revealed expression of the four PKC isoforms by glomerular visceral epithelial, endothelial, and mesangial cells of both normal and diabetic rats. Immunoblot analysis of the diabetic rat glomeruli at 2 weeks demonstrated a significant increase in membrane-associated PKC-alpha, -delta, and -epsilon and a significant decrease in membrane PKC-betaII content compared with normal, which were similar at 4 weeks. Insulin treatment normalized membrane PKC isoform contents and caused a significant decrease in the cytosol content of PKC-alpha, -betaII, and -delta and total cellular PKC-alpha compared with normal. Although PKC activity in the cells of diabetic rat glomeruli was increased by 20% compared with normal, the difference did not reach statistical significance. In insulin-treated diabetic rat glomeruli, PKC activity was significantly decreased compared with non-insulin-treated diabetic rat glomeruli. In conclusion, DAG-sensitive PKC-alpha, -betaII, -delta, and -epsilon isoforms are all found in the three major glomerular cell types in rats, and the expression, compartmentalization, and activity are modulated independently by high glucose and insulin.
Animals↗
Research data on alendronate: biased opinions?
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Alendronate↗
Mesangial cell actin disassembly in high glucose mediated by protein kinase C and the polyol pathway.
High glucose alters mesangial cell cytoskeletal structure and function. We postulated that high glucose causes mesangial cell filamentous (F) actin disassembly through a protein kinase C (PKC) mechanism involving the polyol pathway. Rat mesangial cells (passage < 10, N = 60/group) were growth-arrested and then cultured in glucose 5.6 mM (NG), 15 mM (MG) or 30 mM (HG) for 48 hours, with or without the aldose reductase inhibitor Tolrestat 0.3 mM. F and globular (G) actin were labeled with rhodamine-phalloidin and FTTC-DNase-1, respectively. Both fluorescence probes were imaged simultaneously in each cell using dual-channel confocal laser microscopy. In HG, F-actin disassembly was observed and measured by a 40% decrease in F-/G-actin fluorescence intensity ratio (no change in NG + mannitol 24.4 mM). In separate experiments, cells were labeled with BODIPY FL-bisindolylmaleimide, specific for most PKC isoforms, and fluorescence intensity/cell was measured. In NG, exposure to phorbol 12-myristate 13-acetate (PMA) 0.1 microM for 15 minutes caused perinuclear and nuclear translocation of PKC, and F-actin disassembly identical to observations in HG alone. In HG, total PKC fluorescence increased by 50% and PMA exposure for 24 hours normalized both the total PKC and F-/G-actin fluorescence ratio. In NG and HG, exposure (15 min) to PMA 0.1 microM increased PKC activity three to four times, measured by in situ 32P-phosphorylation of EGF-receptor substrate. By immunofluorescence and confocal imaging, diacylglycerol-sensitive PKC-delta was localized to the cytosol in NG, and after 15 minutes exposure to PMA, translocated to the perinuclear region and plasma membrane. In HG. PKC-delta immunofluorescence was significantly increased/cell, distributed in a cytoskeletal pattern and the intensity was glucose-concentration dependent (30 > 15 > 5.6 mM). In HG, exposure to PMA for 24 hours returned the PKC-delta fluorescence to the intensity and cytosolic pattern observed in NG, and simultaneously prevented F-actin disassembly. Tolrestat significantly reduced the total PKC and PKC-delta fluorescence intensity and F-actin disassembly observed in HG. Immunoblot confirmed increased PKC-delta in HG, which was normalized by Tolrestat. The immunofluorescence pattern of diacylglycerol-insensitive PKC-delta was unchanged in HG, with PMA or Tolrestat. We conclude that mesangial cell F-actin disassembly in high glucose is likely mediated through diacylglycerol-sensitive PKC isoforms, including PKC-delta and involves the polyol pathway.
Actins↗
Women in biomedical research--addressing the challenges.
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Academic Medical Centers↗
Identifying the need for curriculum change. When a rural training program needs reform.
OBJECTIVE: To identify what changes should be made in the University of British Columbia's rural family practice training program curriculum to help graduates be better prepared to practice. DESIGN: Two cross-sectional surveys via mailed questionnaires: one designed to measure physicians' self-reported preparedness for practice and the other to measure the importance of various rural family medicine components. SETTING: Rural training program graduates and preceptors representing rural communities in British Columbia. PARTICIPANTS: Thirty-nine graduates of the rural training program between 1982 and 1991 and 14 community-based rural training program preceptors representing eight communities throughout the province participated in this study. MAIN OUTCOME MEASURES: Percentage of graduates of the rural program who reported themselves to be underprepared on each family practice item and preceptors' mean scores for the attributed importance to rural practice of each item on this questionnaire. RESULTS: A list of curriculum areas most in need of reform was created. This list included trauma, counseling skills, radiology, vacuum extraction, fracture care, exercising community leadership, cost-effective use of diagnostic tests, using community health resources, obtaining hospital privileges, ophthalmology, dermatology, otolaryngology, personal and professional growth, relationships with other physicians, and personnel issues. CONCLUSIONS: Using both the level of graduates' self-reported underpreparedness and the attributed importance of elements of rural practice, as indicated by the preceptor survey, we developed a list of the areas of the rural training program curriculum most in need of reform.
Adult↗
UBC program meets rural medical needs.
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British Columbia↗