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Biomedical subjects

J C Yu

Publications and source records attributed to J C Yu.

At least 73 records · Page 4Linked to original sources
PubMed1995

Differential requirement of a motif within the carboxyl-terminal domain of alpha-platelet-derived growth factor (alpha PDGF) receptor for PDGF focus forming activity chemotaxis, or growth.

To determine the molecular basis for the transforming function of platelet-derived growth factor (PDGF)-A in NIH/3T3 cells, we have constructed chimerae consisting of the extracellular domain of the human CSF-1R (fms) linked to the cytoplasmic domain of the alpha PDGF receptor (alpha R) containing a series of deletion or point mutations. The ability of fms/alpha R chimerae to mediate CSF-1-dependent anchorage-independent growth, focus formation, and chemotaxis of NIH/3T3 cells was then examined. Our results provide evidence that a domain encompassing amino acid residues 977-1024 of the alpha PDGFR is required for ligand-dependent focus formation, but not chemotaxis or anchorage-independent growth, and that tyrosine residues within this domain constitute the major binding site for phospholipase C gamma. Therefore, our findings suggest that: (i) the focus forming function of alpha PDGFR correlates well with the ability of the receptor to bind phospholipase C gamma, and (ii) the mechanism of focus formation mediated by alpha PDGFR may be distinguished from that required for chemotaxis or anchorage-independent growth.

3T3 Cells↗
PubMed1995

A divalent metal ion binding site in the kinase insert domain of the alpha-platelet-derived growth factor receptor regulates its association with SH2 domains.

To investigate the effects of metal ion binding to the alpha-PDGFR kinase insert domain, a PCR product representing amino acid residues 691-795 (104 amino acids) was bacterially expressed and purified. Secondary structure prediction and circular dichroism spectroscopy indicated this domain to be a mixed alpha + beta protein with a large coil/turn contribution. This 16 kDa, soluble, nonphosphorylated domain bound to 45Ca2+ and 65Zn2+ through a common shared site. Of the unlabeled divalent and trivalent metal ions tested, Ho3+ = Zn2+ > Ni2+ > Ca2+ = Mn2+ > Mg2+, Ba2+ in competing for 45Ca2+ binding to this domain. In the presence of Ca2+ ions, the conformation of the KI domain changed significantly, and this changed conformation was resistant to subtilisin proteolysis. However, in the presence of Zn2+ ions, the conformation of the KI domain changed only slightly. Nevertheless, Zn2+ ions were more effective in rendering the KI domain resistant to proteolysis as compared to that shown by Ca2+ ions. In vitro binding studies using purified baculovirus-expressed alpha-PDGFR showed a marked increase in binding the p85 N-SH2 domain in the presence of Ca2+ or Zn2+ ions (KD = 0.5 microM), suggesting that metal ion binding enhances association of the p85 N-SH2 domain with the receptor. To confirm this, association of the alpha-PDGFR with the p85 N-SH2 domain was tested in the presence of the KI domain. The nonphosphorylated KI domain was effective in competing with the alpha-PDGFR for the binding of the p85 N-SH2 domain. This effect was more pronounced in the presence of Ca2+ ions. Microinjection of this domain into Xenopus oocytes delayed maturation in the presence of insulin but not progesterone. This suggests that the KI domain has a correctly folded three-dimensional structure compatible with biological activity. Together these findings indicate that the recombinant alpha-PDGFR KI domain binds the p85 N-SH2 domain and this binding is modulated by the presence of a novel divalent metal ion binding site within its structure.

Amino Acid Sequence↗
PubMed1995

Critical review of microfixation in pediatric craniofacial surgery.

The migration or passive intracranial translocation of microplates and screws in the pediatric craniofacial patient has been reported. A retrospective review was undertaken to clarify the incidence of microplate translocation and identify potential clinical implications. Computed tomographic imaging demonstrated internalization of microfixation in 14 of 27 pediatric patients. Statistically significant factors for microplate translocation include longer plates (p < 0.05) and those placed in the temporal region (p < 0.001). Younger patients and those with syndromic craniofacial dysostosis also had a higher incidence of translocation. Specific complications relating to the translocation of microplates were not found in any patient. The direct effects of translocated microplates and screws on the underlying brain and dura remain unclear.

Bone Plates↗
PubMed1995

Secretion of endothelin-1 and endothelin-3 by human cultured vascular smooth muscle cells.

1. It is generally accepted that endothelial cells secrete endothelin (ET) to the underlying media which mediates the contractile effects of ET. However, there is some evidence that animal vascular smooth muscle cells (VSMCs) also secrete ET. We cultured VSMCs from human vessels representative of a number of different vascular beds to determine whether human VSMCs endogenously secrete ET. 2. VSMCs explanted from adult arterial vessels secrete picomolar quantities of immunoreactive mature ET: coronary artery 226.6 +/- 58.8 pM/10(6) cells (n = 7), thoracic aorta 169.5 +/- 105.4 pM/10(6) cells (n = 3), left internal mammary artery 102.4 +/- 23.1 pM/10(6) cells (n = 3) and saphenous vein 69.4 +/- 19.9 pM/10(6) cells (n = 3), as well as from umbilical vein (HUVSMCs) 38.3 +/- 4.3 pM/10(6) cells (n = 3). Secretion of immunoreactive big ET-1 was also detected: coronary artery 249.1 +/- 59.4 pM/10(6) cells (n = 7), thoracic aorta 120.0 +/- 13.4 pM/10(6) cells (n = 3), left internal mammary artery 170.0 +/- 68.2 pM/10(6) cells (n = 3), saphenous vein 105.1 +/- 30.7 pM/10(6) cells (n = 3) and from umbilical vein 146.3 +/- 7.4 pM/10(6) cells (n = 3). Comparable, intracellular levels of immunoreactive big ET-1 and mature ET were also detected in cultured VSMCs. 3. Since enzyme-dispersed VSMCs are thought to be more differentiated and more closely resemble their in vivo counterparts, and these enzyme-dispersed VSMCs from human umbilical vein (HUVSMCs) also secreted the greatest levels of immunoreactive peptides, they were characterized further. Reverse transcription-polymerase chain reaction assay demonstrated that HUVSMCs express ET-1 mRNA. High performance liquid chromatography coupled to radioimmunoassay revealed that HUVSMCs secrete ET-1 and ET-3, in addition to big ET-1. However, levels of ET are not altered by 100 AM phosphoramidon,an inhibitor of metalloproteases or by 100 microM pepstatin A, an aspartyl protease inhibitor.4. In concordance, KD and Bmax values for [125I]-ET-l saturation binding are not altered in HUVSMC cultures incubated for 24 h with 100 microM phosphoramidon (431 +/- 218 PM and 31.1 +/- 12.7 fmol mg-1;mean =/- s.e.mean, n = 3) or 100 microM pepstatin A (381 +/- 169PM and 19.9 +/- 7.8 fmol mg-1, n = 3) as compared to controls (355 +/- 99 pM and 33.3 +/- 9.3 fmol mg-1; n = 3). This observation indicates the absence of an autocrine 'unmasking' effect for ET receptors.5. HUVSMCs synthesize and secrete immunoreactive ET-1, ET-3 and big ET-1, and possess intracellular levels of immunoreactive mature ET and big ET-1. There is some evidence of common cellular mechanisms between growth factors and vasoconstrictor peptides, suggesting a close relationship between contraction and proliferation. Since the development of various vascular pathologies such as atherosclerosis, hypertension and after vessel injury has been attributed to alterations in the normal growth pattern of VSMCs, the role of ET in these diseases may be of significance.

Antimalarials↗
PubMed1995

Endothelin ETA receptor expression in human cerebrovascular smooth muscle cells.

1. Endothelin (ET) has been implicated in cerebrovasospasm for example, following subarachnoid haemorrhage, and blocking the interaction of ET with its receptors on cerebral vessels, may be of therapeutic benefit. The aim of our study was to characterize endothelin receptor sub-types on medial smooth muscle cells of human cerebral vessels. Cultures of vascular smooth muscle cells were explanted from human cerebral resistance vessels and characterized as human brain smooth muscle cells (HBSMCs). 2. Over a 48 h incubation period, HBSMC cultures secreted comparable levels of immunoreactive (IR) big endothelin-1 (big ET-1) and IR endothelin (ET): 12.7 +/- 10.3 and 8.3 +/- 5.6 pmol/10(6) cells, respectively (mean +/- s.e. mean from three different individuals), into the culture medium. 3. Total RNA was extracted from cultures of human brain smooth muscle cells. Reverse-transcriptase polymerase chain reaction (RI-PCR) assays and subsequent product separation by agarose gel electrophoresis revealed single bands corresponding to the expected product sizes encoding cDNA for ETA (299 base pairs) and ETB (428 base pairs) (n = 3 different cultures). 4. Autoradiography demonstrated the presence of specific binding sites for [125I]-ET-1 which labels all ET receptors, and [125I]-PD151242, an ETA subtype-selective antagonist which exclusively labels ETA receptors, but no specific-binding was detected using ETB subtype-selective [125I]-BQ3020 (n = 3 different cultures, in duplicate). 5. In saturation binding assays, [123I]-ET-1 bound with high affinity: KD = 0.8 +/- 0.1 nM and Bmax = 690 +/- 108 fmol mg-1. A one-site fit was preferred and Hill slopes were close to unity over the concentration range (10(-12) to 10(-8) M). [125I]-PD151242 also bound with similar affinity: KD = 0.4 +/- 0.1 nM and Bmax = 388 +/- 68 fmol mg-1 (mean +/- s.e. mean, n = 3 different cultures). Again, a one-site fit was preferred and Hill slopes were close to unity over the concentration range. Unlabelled PD151242 competed for the binding of [125I]-ET-1 monophasically and analysis of the competition curves indicated that a one-site fit was preferred over a two-site model, implying that the cultures express mainly ETA receptors. 6. Although messenger RNA encoding both ETA and ETB receptors was detected, autoradiographical analysis, as well as binding studies indicate that human cultured brain smooth muscle cells express only ETA receptor protein. Antagonism of this sub-type may be necessary to block the actions of ET-1 in the human cerebral resistance vessels in the vasospasm observed subsequent to subarachnoid haemorrhage.

Aged↗
PubMed1995

Studies on cryopreservation of Toxoplasma gondii and its antigenicity in mice.

Two strains (RH and GC, the latter of which is a Taiwan isolate of porcine origin) of Toxoplasma gondii were kept at -20 degrees C, -60 degrees C, and in liquid nitrogen (-196 degrees C) to follow the time course change in viability and virulence of the parasites by direct count and animal inoculation methods. Changes in antibody titers in some of the mice inoculated with the thawed organisms were assayed by the indirect immunofluorescent antibody test. Viability and virulence of T. gondii were best preserved by storage in liquid nitrogen. Tachyzoites kept in liquid nitrogen for eight years still can lead to the death of the injected mice in 2-3 weeks. Virulence of the tachyzoites could be maintained for eight weeks at most at -20 degrees C and -60 degrees C. Dimethylsulfoxide (DMSO) seemed to be a better cryoprotectant for T. gondii than glycerol, but the DMSO-preserved organisms resulted in fewer tachyzoite-containing peritoneal exudates in inoculated mice than the glycerol-preserved organisms. The local isolate (GC strain) tachyzoites tolerated cryopreservation less well than the RH strain parasites. Low antibody titers (at most 1:64) were produced in mice that survived more than 16 days after inoculation with thawed tachyzoites.

Animals↗
PubMed1995

Regulation of endothelin receptor expression in vascular smooth-muscle cells.

Endothelin-1 (ET-1) has been implicated in atherosclerosis, hypertension, and restenosis, all of which involve abnormal vascular smooth muscle cell function and/or proliferation. We have previously established that human umbilical vein smooth-muscle cells (HUVSMCs) can secrete ET, (1) whereas A10 cells do not. Therefore, we investigated the effect of exogenously added ET-1 on receptor density (Bmax) of A10 cells. Compared to controls, A10s exposed to ET-1 for 48 h showed a significant decrease (79%) in receptor density, with no change in affinity. In contrast, incubation of A10s with the ETA-selective antagonist FR139317 (at a concentration blocking 99% of ET receptors) for 48 h caused a significant increase (245%) in Bmax and a significant decrease in affinity. These changes persisted after coincubation with both ET-1 and FR139317, indicating that the antagonist was able to block the effects of exogenous ET-1. In concordance, incubation of HUVSMCs with FR139317 for 24 h caused a significant increase in receptor density (> 1,000%), although there was no change in levels of immunoreactive (IR) ET or big ET-1. However, after a shorter incubation (1 h), there was no change in Bmax but IR ET was significantly elevated by 878%, whereas IR big ET-1 was depressed by 86% compared to controls. Incubation of HUVSMCs with FR139317 did not affect receptor affinity. Our observations suggest that whereas the application of exogenous ET to A10s causes downregulation of ET receptor expression, the ETA antagonist FR139317 causes upregulation of ET receptor expression in VSMCs regardless of their ability to secrete ET. In VSMCs capable of secreting ET, acute antagonism of the ETA receptor causes a significant increase in IR-ET and a decrease of IR-big ET-1 levels.

Azepines↗
PubMed1994

The alpha PDGFR tyrosine kinase mediates locomotion of two different cell types through chemotaxis and chemokinesis.

To determine the capability of alpha PDGFR to couple with chemotactic signaling, we established 32D cells expressing wild type alpha PDGFR (alpha RWT), a kinase-defective mutant of alpha PDGFR (alpha R627R), or wild type beta PDGFR (beta RWT). Using a modified Boyden chamber, we showed that PDGF induced significant cell migration of 32D cells expressing alpha RWT or beta RWT, but not of those expressing alpha R627R. Furthermore, the cell migration was largely reduced in each case when the same concentration of PDGF was present in both chambers, suggesting that cell migration observed in 32D expressing alpha RWT is mainly due to alpha PDGFR-mediated chemotaxis. Consistent with these results, PDGF-AA induced significant cell migration of NIH3T3 fibroblasts, which was markedly blocked by the presence of excess neutralizing polyclonal antibody to PDGF-AA. These results provide evidence that alpha PDGFR kinase activity is essential for mediating ligand-induced chemotactic and chemokinetic responses in two different cell types.

3T3 Cells↗
PubMed1994

Structural coincidence of alpha PDGFR epitopes binding to platelet-derived growth factor-AA and a potent neutralizing monoclonal antibody.

We have generated two groups of deletion mutants of the alpha PDGFR and one group of chimeras between alpha PDGFR and beta PDGFR to further investigate the structural requirements of the alpha PDGFR for binding to platelet-derived growth factor (PDGF)-AA and to a monoclonal antibody against alpha PDGFR (designated as mAb-alpha R1). The mAb-alpha R1 has recently been reported to block high affinity binding of PDGF-AA to alpha PDGFR. The first group of mutants were carboxyl-terminal deletion mutants encoding the first two immunoglobulin (Ig)-like domains (alpha R1-216), the first four Ig-like domains (alpha R1-415), or all five Ig-like domains (alpha R1-530) of the alpha PDGFR. Since these mutants lacked transmembrane domains, their expression in NIH/3T3 cells resulted in secretion of the truncated alpha PDGFRs. Using conditioned medium from NIH/3T3 transfectants, we showed that mAb-alpha R1 was able to immunoprecipitate each of these secreted form of alpha PDGFRs, suggesting that the epitope recognized by mAb-alpha R1 is located within Ig-like domains 1 and 2 of the alpha PDGFR. Furthermore, PDGF-AA exhibited detectable binding to alpha R1-415 or alpha R1-530 but failed to interact with alpha R1-216, suggesting that the first two Ig-like domains of the alpha PDGFR are not sufficient for PDGF-AA binding. The second group of alpha PDGFR mutants were internal deletion mutants lacking Ig-like loop 1 (alpha R delta 49-100), Ig-like loop 2 (alpha R delta 150-189), Ig-like loop 3 (alpha R delta 235-290), or part of Ig-like loops 4 and 5 (alpha R delta 375-450). The internal deletion mutants were transfected into 32D cells which lack both alpha PDGFR and beta PDGFR. PDGF-AA bound with high affinity to 32D cells expressing alpha R delta 375-450 but not to 32D cells expressing the other three internal deletion mutants, suggesting that the region required for PDGF-AA binding should be within the first three Ig-like domains of the alpha PDGFR. In addition, mAb-alpha R1 bound to 32D cells expressing alpha R delta 235-290 but failed to bind 32D cells transfected with alpha R delta 49-100 or alpha R delta 150-189.(ABSTRACT TRUNCATED AT 400 WORDS)

3T3 Cells↗
PubMed1994

Tyrosine phosphorylation of protein kinase C-delta in response to its activation.

Retroviral vectors containing five different protein kinase C (PKC) isoenzymes (alpha, delta, epsilon, eta, zeta) were expressed in 32D hematopoietic cells and NIH-3T3 fibroblasts. In an effort to investigate signaling events regulated by PKC activation, we analyzed whether tyrosine phosphorylation of cellular proteins would occur after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of the various transfectants. While no detectable tyrosine-specific phosphorylation was observed after treatment of the majority of the transfectants, pronounced TPA-dependent tyrosine phosphorylation of an 82-kDa protein was detected in the 32D/PKC-delta and NIH-3T3/PKC-delta lines. Interestingly, the 82-kDa substrate proved to be PKC-delta itself. Tyrosine phosphorylation of purified PKC-delta by src family or receptor tyrosine kinases in vitro enhanced PKC-delta activity, suggesting that tyrosine phosphorylation of PKC-delta may positively affect its function.

3T3 Cells↗
PubMed1994

Comparison of calcium-dependent conformational changes in the N-terminal SH2 domains of p85 and GAP defines distinct properties for SH2 domains.

Src-homology region 2 (SH2) domains are stretches of about 100 amino acids which are found to be structurally conserved in a number of signaling molecules. These regions have been shown to bind with high affinity to phosphotyrosine residues within activated receptor tyrosine kinases. Here we report the bacterial expression and purification of individual N-terminal SH2 (NSH2) domains of phosphatidylinositol 3-kinase (PI-3K) binding subunit (p85) and Ras GTPase activating protein (GAP) in amounts suitable for structure-function studies. The p85NSH2 domain stains dark purple and absorbs around 620-640 nm with Stains-all, a dye known to bind to calcium binding proteins. This effect was not observed for the GAPNSH2 domain. Circular dichroism analysis of the N-terminal SH2 domain of these proteins shows that p85NSH2, but not GAPNSH2, undergoes a significant dose-dependent change in conformation in the presence of increasing calcium concentrations. Moreover, the conformational change of p85NSH2 induced by calcium could be replicated by addition of a phosphorylated hexapeptide (DYpMDMK) representing the alpha-PDGFR binding site for p85. Limited proteolysis studies showed a significant calcium-dependent increase in protection of p85NSH2 but not GAPNSH2 from degradation by subtilisin. Our results further indicate that holmium, a trivalent lanthanide ion, which has been previously shown to substitute for calcium, could also protect the p85NSH2 domain from proteolysis even at 10-fold lower concentrations. In vitro binding studies using purified preparations of activated alpha-PDGFR show that calcium did not affect the binding of GAPNSH2 domains to activated alpha-PDGFR.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Biological function of PDGF-induced PI-3 kinase activity: its role in alpha PDGF receptor-mediated mitogenic signaling.

The tyrosine phosphorylation sites in the human alpha PDGF receptor (alpha PDGFR) required for association with PI-3 kinase have been identified as tyrosines 731 and 742. Mutation of either tyrosine substantially reduced PDGF-induced PI-3 kinase activity but did not impair the receptor-mediated mitogenic response. We sought to determine whether PDGF-induced PI-3 kinase activity could be further ablated so as to exclude a low threshold requirement for PDGFR signal transduction. Thus, we mutated both tyrosine 731 and 742 and expressed the double mutant (Y731F/Y742F) in 32D hematopoietic cells. In such transfectants, PDGF induced no detectable receptor-associated or anti-P-Tyr recoverable PI-3 kinase activity. Under the same conditions, neither mobility shift of raf-1 nor tyrosine phosphorylation of either PLC gamma or MAP kinase was impaired. 32D transfectants expressing the double mutant showed wild-type alpha PDGFR levels of mitogenic and chemotactic responses to PDGF. To examine the effect of the double mutation in cells that normally respond to PDGF, we generated chimeras in which the cytoplasmic domains of wild-type alpha PDGFR, Y731F, and Y731F/Y742F were linked to the extracellular domain of colony-stimulating factor-1 (CSF-1) receptor (fms). After introduction of the chimeric receptors into mouse NIH/3T3 fibroblasts, the ability of CSF-1 to stimulate growth of these transfectants was examined. Our data show that all these chimeric receptors exhibited similar abilities to mediate CSF-1-stimulated cell growth. These findings lead us to conclude that PDGF-induced PI-3 kinase activity is not required for PDGF-stimulated mitogenic pathway in both NIH/3T3 fibroblasts and 32D hematopoietic cells.

3T3 Cells↗
PubMed1994

Autotransfusion and reduction mammaplasty: a reappraisal in the 1990s.

Since the first report of autotransfusion and blood loss in major anesthetic procedures by Noone et al. in 1972, much has changed. The single most significant event is the development of the AIDS epidemic. With today's stringent and precisely defined transfusion criteria, the current practice of routinely using autologous units for reduction mammaplasties has caused considerable controversy. This study reviews the mean estimated blood loss during reduction mammaplasty and the use of autologous transfusion during a 46-month period at the Hospital of the University of Pennsylvania ending in November of 1991. The need for red blood cell transfusion is retrospectively analyzed. One-hundred female patients were entered into the study. The results demonstrate a decrease in blood loss from an average of 865 ml in the 1970s to 385 ml in the 1990s and suggest that routine employment of autologous transfusion is no longer necessary.

Adolescent↗
PubMed1994

Stimulation of the platelet-derived growth factor beta receptor signaling pathway activates protein kinase C-delta.

The murine myeloid progenitor cell line 32D was recently shown to undergo monocytic differentiation when protein kinase C-delta (PKC-delta) was overexpressed and activated by 12-O-tetradecanoylphorbol-13-acetate (TPA) (H. Mischak, J.H. Pierce, J. Goodnight, M.G. Kazanietz, P.M. Blumberg, and J.F. Mushinski, J. Biol. Chem. 268:20110-20115, 1993). Tyrosine phosphorylation of PKC-delta occurred when PKC-delta-transfected 32D cells were stimulated by TPA (W. Li, H. Mischak, J.-C. Yu, L.-M. Wang, J.F. Mushinski, M.A. Heidaran, and J.H. Pierce, J. Biol. Chem. 269:2349-2352, 1994). In order to elucidate the role played by PKC-delta in response to activation of a receptor tyrosine kinase, we transfected platelet-derived growth factor beta receptor (PDGF-beta R) alone (32D/PDGF-beta R) or together with PKC-delta (32D/PDGF-beta R/PKC-delta) into 32D cells. NIH 3T3 cells which endogenously express both PDGF-alpha R and PDGF-beta R were also transfected with PKC-delta (NIH 3T3/PKC-delta). Like TPA treatment, PDGF-BB stimulation caused striking phosphorylation of PKC-delta in vivo and translocation of some PKC-delta from the cytosol fraction to the membrane fraction in both cell systems. Some of the phosphorylation induced by PDGF-BB treatment was found to be on a tyrosine residue(s). Tyrosine-phosphorylated PKC-delta was observed only for the membrane fraction after stimulation with PDGF-BB or TPA. The enzymatic activity of PKC-delta in the membrane fraction also increased after stimulation with TPA or PDGF, providing a positive correlation between PKC-delta tyrosine phosphorylation and its activation. Overnight treatment of 32D/PDGF-beta R/PKC-delta cells with PDGF-BB induced monocytic differentiation as judged by an increase in expression of cell surface macrophage differentiation markers. PDGF-BB had much weaker effects on 32D/PDGF-beta R cell differentiation, suggesting that increased PKC-delta expression enhanced monocytic differentiation. These results indicate that PKC-delta is a downstream molecule in the PDGFR signaling pathway and may play a pivotal role in PDGF-beta R-mediated cell differentiation.

Animals↗
PubMed1993

Differences in substrate specificities of alpha and beta platelet-derived growth factor (PDGF) receptors. Correlation with their ability to mediate PDGF transforming functions.

Recombinant expression of either the alpha or beta platelet-derived growth factor (PDGF) receptors in 32D hematopoietic cells allows efficient coupling of PDGF with mitogenic and chemotactic signaling pathways inherently expressed by those cells. PDGF-BB stimulation of 32D-alpha R or beta R cells results in anti-P-Tyr recovery of cellular proteins possessing similar as well as distinct phosphotyrosine signals. Comparison of the ability of each receptor to couple with known second messengers revealed that both receptors associated with and/or tyrosine phosphorylated phospholipase C-gamma (PLC gamma) and phosphatidylinositol 3-kinase (p85) with similar stoichiometry. However, the beta platelet-derived growth factor receptor (PDGFR) was significantly more efficient at in vivo tyrosine phosphorylation of GTPase-activating protein (GAP). Similar differences in binding affinity for GAP were observed in NIH/3T3 cells which express both receptors. To quantitate the affinities of each receptor for GAP or PLC gamma, we utilized baculovirus-expressed alpha and beta PDGFRs purified by anti-P-Tyr affinity chromatography. Exposure of immunoblots containing bacterially expressed GAP or PLC gamma to activated alpha or beta PDGF receptors led to a comparable high affinity binding of each receptor to PLC gamma, while the beta PDGFR showed a 5-fold higher binding affinity for GAP. In an effort to correlate differences in their substrate specificities with biological properties of the receptors, we compare their abilities to enhance PDGF-A transforming function in NIH/3T3 cells. Cotransfection of PDGF-A with the alpha PDGFR increased PDGF-A transforming activity by approximately 2-fold. However, cotransfection with a chimeric receptor with the catalytic domain of the beta PDGFR but possessing alpha PDGFR ligand binding properties resulted in 17-fold enhancement of PDGF-A transformation. These findings argue that differences in alpha and beta PDGF receptor substrate specificity in NIH/3T3 fibroblasts correlate with greater transforming activity mediated by the beta PDGFR.

3T3 Cells↗
PubMed1993

Thrombin promotes aortic endothelial cell spreading and microfilament formation in nonconfluent monolayer cultures.

Thrombin is present at sites of vascular injury. The objective of this study was to determine if thrombin may regulate endothelial repair. To address this our study was designed to determine the effect of thrombin on cell shape and microfilament distribution of porcine aortic endothelial cells in low-density cultures. Since these cells grow as islands of cells, low-density cultures serve as a model for conditions in which reendothelialization occurs from several foci following prominent patchy endothelial denudation. Thrombin incubation for 1, 2, and 4 hr at 0.5, 4, and 8 U/ml caused a significant increase in both the surface area of endothelial cells and the amount of microfilament bundles occupying the cell surface area. The increase in microfilament bundles was more than would be expected from the increased surface area. However, the pattern of distribution of microfilaments and of vinculin adhesion plaques within the cells was not altered by thrombin. Attempts at understanding the molecular mechanisms involved were undertaken. The role of protein kinase C was studied. Phorbol 12-myristate 13-acetate (PMA) caused cell spreading as well but no increase in microfilaments within the first 2 hr of incubation. Some cells showed mild disruption of microfilaments. Following 24 hr incubation with PMA, which reduces protein kinase C, the cells showed retraction and arborization along with thinning and disruption of microfilaments. Removal of PMA and addition of thrombin reversed these changes to normal within 2 hr while washout of PMA on its own did not alter cell shape or microfilaments. Thrombin also partially reversed the effects of H-7, a protein kinase C inhibitor, which on its own also caused cell retraction and disruption of microfilaments. Neomycin sulfate did not alter the thrombin effect suggesting that the participation of the phosphotidylinositol system is not directly required. In conclusion, thrombin promotes endothelial activities which are associated with long-term endothelial repair; however, these effects do not appear to be directly due to the involvement of conventional isoforms of protein kinase C or the phosphotidylinositol system.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1993

[Continuous monitoring for blood glucose after surgery of insulinoma and the use of insulin].

The changes of blood glucose in 60 patients after surgery of insulinoma were analysed. It was found that in more than half of the patients blood glucose was elevated to a standard level of total resection at 30' and in all patients at 24 hrs. More than 90% patients remained hyperglycemic for about 9 days. The higher the blood glucose level was elevated, the earlier the hyperglycemia developed. There was no relationship between the elevating speed of blood glucose and the severity of preoperative hypoglycemia, and the course of the disease. But there was positive relationship between the recovering time of hyperglycemia and the course of disease. Insulin is indicated in some patients during postoperative period to help safe recovery especially in a hyperglycemic state.

Adolescent↗