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Biomedical subjects

G Carpenter

Publications and source records attributed to G Carpenter.

At least 55 records · Page 3Linked to original sources
PubMed1997

Phospholipase C-gamma1: regulation of enzyme function and role in growth factor-dependent signal transduction.

Phospholipase C(gamma)1 (PLC-gamma1), a tyrosine kinase substrate, is a multi-domain molecule that modulates the intracellular levels of the second messenger molecules: Ca2+ and diacylglycerol. Although a wide variety of growth factor receptor tyrosine kinases phosphorylate and activate PLC-gamma1, the biological role and necessity of this signal transduction element in mitogenesis has remained unclear. Recent results, however, point to a more essential role than was suggested by initial studies. Also, biochemical studies have indicated a putative means for the intramolecular repression of PLC-gamma1 activity and provide a means for interpreting activation signals through a derepression mechanism.

Animals↗
PubMed1997

Patient-validated content of epilepsy-specific quality-of-life measurement.

PURPOSE: To study the effects of epilepsy from the patients' perspective and assist determination of content validity of health-related quality-of-life (HRQOL) measures. METHODS: We asked 81 consecutive patients with moderately severe epilepsy to list in order of importance their concerns of living with recurrent seizures. To minimize investigator bias, patients completed the procedure in a private setting without staff involvement. RESULTS: Twenty-four distinct domains were generated by the patients. Concerns about driving (64%), independence (54%), employment (51%), social embarrassment (36%), medication dependence (33%), mood/stress (32%), and safety (31%) each were listed by > 30% of patients. Driving was listed as the most important concern by 28% of patients, followed by employment (21%), independence (9%), safety (6%), antiepileptic-drug side effects (5%), seizure unpredictability (5%), and seizure aversion (5%). CONCLUSIONS: The effect of epilepsy on HRQOL is not vague or obscure from the patients' perspective but is defined by a limited number of domains. Independence is an important concern that may not be evaluated adequately by currently available HRQOL instruments.

Activities of Daily Living↗
PubMed1996

Selective cleavage of the heregulin receptor ErbB-4 by protein kinase C activation.

The 180-kDa transmembrane tyrosine kinase ErbB-4 is a receptor for the growth factor heregulin. 125I-Heregulin binding to NIH 3T3 cells overexpressing the ErbB-4 receptor is rapidly decreased by 12-O-tetradecanoylphorbol-13-acetate (TPA) pretreatment. Immunologic analysis demonstrates that TPA treatment of cells induces the proteolytic cleavage of ErbB-4, producing an 80-kDa cytoplasmic domain fragment, which contains a low level of phosphotyrosine, and a 120-kDa ectodomain fragment, which is released into the extracellular medium. Cleavage of ErbB-4 was also enhanced by other protein kinase C activators, i.e. platelet-derived growth factor, ionomycin, and synthetic diacylglycerol, while protein kinase C inhibition or down-regulation suppressed the TPA stimulation of ErbB-4 degradation. TPA did not induce the degradation of related receptors (ErbB-1, ErbB-2, and ErbB-3) in the EGF receptor family. The phorbol ester-induced cleavage of ErbB-4 occurs within or close to the ectodomain, as the 80-kDa cytoplasmic domain fragment is recognized by antibody to the ErbB-4 carboxyl terminus and is membrane-associated. Coprecipitation experiments show that, while the 80-kDa ErbB-4 fragment is associated with the SH2-containing molecules PLC-gamma1 and Shc, TPA did not induce the phosphorylation of these substrates in intact cells. In addition, kinase assays in vitro indicate that the 80-kDa fragment is not an active tyrosine kinase. These results show that protein kinase C negatively regulates heregulin signaling through the ErbB-4 receptor by the activation of a selective proteolytic mechanism.

3T3 Cells↗
PubMed1996

Enhanced phospholipase C-gamma1 activity produced by association of independently expressed X and Y domain polypeptides.

The X and Y domains of phospholipase C (PLC)-gamma1, which are conserved in all mammalian phosphoinositide-specific PLC isoforms and are proposed to interact to form the catalytic site, have been expressed as individual hexahistidine-tagged fusion proteins in the baculovirus system. Following coinfection of insect cells with recombinant viruses, association of X and Y polypeptides was demonstrated in coprecipitation assays. When enzyme activity was examined, neither domain possessed catalytic activity when expressed alone; however, coexpression of the X and Y polypeptides produced a functional enzyme. This reconstituted phospholipase activity remained completely dependent on the presence of free Ca2+. The specific activity of the X:Y complex was significantly greater (20- to 100-fold) than that of holoPLC-gamma1 and was only moderately influenced by varying the concentration of substrate. The enzyme activities of holoPLC-gamma1 and the X:Y complex exhibited distinct pH optima. For holoPLC-gamma1 maximal activity was detected at pH 5.0, while activity of the X:Y complex was maximal at pH 7.2.

Amino Acid Sequence↗
PubMed1996

Craniosynostosis, Philadelphia type: a new autosomal dominant syndrome with sagittal craniosynostosis and syndactyly of the fingers and toes.

The acrocephalosyndactyly syndromes (ACS) are a group of clinically similar disorders that share the manifestations of craniosynostosis and a variety of hand and foot anomalies. Here we report on a 5-generation kindred segregating sagittal craniosynostosis and syndactyly of the fingers and the toes in an autosomal dominant manner. The anomalies seen in this kindred comprise a syndrome distinct from other craniosynostosis syndromes. For this novel syndrome, we propose the name craniosynostosis, Philadelphia type.

Acrocephalosyndactylia↗
PubMed1996

All ErbB receptors other than the epidermal growth factor receptor are endocytosis impaired.

Four transmembrane tyrosine kinases constitute the ErbB receptor family: the epidermal growth factor (EGF) receptor, ErbB-2, ErbB-3, and ErbB-4. We have measured the endocytic capacities of all four members of the EGF receptor family, including ErbB-3 and ErbB-4, which have not been described previously. EGF-responsive chimeric receptors containing the EGF receptor extracellular domain and different ErbB cytoplasmic domains (EGFR/ErbB) have been employed. The capacity of these growth factor-receptor complexes to mediate 125I-EGF internalization, receptor down-regulation, receptor degradation, and receptor co-immunoprecipitation with AP-2 was assayed. In contrast to the EGF receptor, all EGFR/ErbB receptors show impaired ligand-induced rapid internalization, down-regulation, degradation, and AP-2 association. Also, we have analyzed the heregulin-responsive wild-type ErbB-4 receptor, which does not mediate the rapid internalization of 125I-heregulin, demonstrates no heregulin-regulated receptor degradation, and fails to form association complexes with AP-2. Despite the substantial differences in ligand-induced receptor trafficking between the EGF and ErbB-4 receptors, EGF and heregulin have equivalent capacities to stimulate DNA synthesis in quiescent cells. These results show that the ligand-dependent down-regulation mechanism of the EGF receptor, surprisingly, is not a property of any other known ErbB receptor family member. Since endocytosis is thought to be an attenuation mechanism for growth factor-receptor complexes, these data imply that substantial differences in attenuation mechanisms exist within one family of structurally related receptors.

3T3 Cells↗
PubMed1995

Electrophoretic variation in adenylate kinase of Neisseria meningitidis is due to inter- and intraspecies recombination.

In prokaryotic and eukaryotic organisms, the electrophoretic variation in housekeeping enzymes from natural populations is assumed to have arisen by the accumulation of stochastic predominantly neutral mutations. In the naturally transformable bacterium Neisseria meningitidis, we show that variation in the electrophoretic mobility of adenylate kinase is due to inter- and intraspecies recombination rather than mutation. The nucleotide sequences of the adenylate kinase gene (adk) from isolates that express the predominant slow electrophoretic variant were rather uniform, differing in sequence at an average of 1.1% of nucleotide sites. The adk sequences of rare isolates expressing the fast migrating variant were identical to each other but had a striking mosaic structure when compared to the adk genes from strains expressing the predominant variant. Thus the sequence from the fast variants was identical to those of typical slow variants in the first 158 bp of the gene but differed by 8.4% in the rest of the gene (nt 159-636). The fast electrophoretic variant appears to have arisen by the replacement of most of the meningococcal gene with the corresponding region from the adk gene of a closely related Neisseria species. The adk genes expressing the electrophoretic variant with intermediate mobility were perfect, or almost perfect, recombinants between the adk genes expressing the fast and slow variants. Recombination may, therefore, play a major role in the generation of electrophoretically detectable variation in housekeeping enzymes of some bacterial species.

Adenylate Kinase↗
PubMed1995

Demonstration of functionally different interactions between phospholipase C-gamma and the two types of platelet-derived growth factor receptors.

Phosphorylated tyrosine residues in receptor tyrosine kinases serve as binding sites for signal transduction molecules. We have identified two autophosphorylation sites, Tyr-988 and Tyr-1018, in the platelet-derived growth factor (PDGF) alpha-receptor carboxyl-terminal tail, which are involved in binding of phospholipase C-gamma (PLC-gamma). The capacities of the Y988F and Y1018F mutant PDGF alpha-receptors, expressed in porcine aortic endothelial cells, to bind PLC-gamma are 60 and 5% of that of the wild-type receptor, respectively. Phosphorylated but not unphosphorylated peptides containing Tyr-1018 are able to compete with the intact receptor for binding to immobilized PLC-gamma SH2 domains; a phosphorylated Tyr-988 peptide competes 10 times less efficiently. The complex between PLC-gamma and the PDGF alpha-receptor is more stable than that of PLC-gamma and the PDGF beta-receptor. However, PDGF stimulation results in a smaller fraction of tyrosine-phosphorylated PLC-gamma and a smaller accumulation of inositol trisphosphate in cells expressing the alpha-receptor as compared with cells expressing the beta-receptor. We conclude that phosphorylated Tyr-988 and Tyr-1018 in the PDGF alpha-receptor carboxyl-terminal tail bind PLC-gamma, but this association leads to only a relatively low level of tyrosine phosphorylation and activation of PLC-gamma.

Amino Acid Sequence↗
PubMed1995

Stoichiometric interaction of the epidermal growth factor receptor with the clathrin-associated protein complex AP-2.

Plasma membrane clathrin-associated protein complexes (AP-2) have been shown to co-immunoprecipitate with the epidermal growth factor (EGF) receptor (Sorkin A., and Carpenter, G. (1993) Science 261, 612-615). Hence, we analyzed the stoichiometry of the EGF receptor interaction with AP-2 using a new antibody that efficiently immunoprecipitates native AP-2. EGF receptor AP-2 complexes were isolated from 35S-labeled cells treated with EGF by EGF receptor affinity chromatography followed by precipitation with the antibody to AP-2. Quantitation of the relative molar concentrations of the proteins found in the complex revealed that 1 mol of AP-2 was associated with approximately 1.1 mol of EGF receptor. No other proteins were present in significant molar concentrations relative to AP-2, indicating that other proteins are not stoichiometrically involved in the interaction of EGF receptors and AP-2 in vivo. Co-immunoprecipitation experiments in cells expressing a mutant EGF receptor demonstrated that the cytoplasmic carboxyl-terminal 214 residues of the EGF receptor are essential for interaction with AP-2.

3T3 Cells↗
PubMed1995

Cell density-dependent regulation of PLC gamma 1 tyrosine phosphorylation and catalytic activity in an intestinal cell line (IEC-6).

Administration of epidermal growth factor (EGF) to rats has been shown to induce both mitogenic and nonmitogenic responses in the intestine. The mechanisms to describe a multiplicity of hormonal responses within a single tissue are unclear but likely involve selectivity among receptor substrates. A nontransformed rat jejunal crypt intestinal epithelial cell line (IEC-6) was studied to determine if the regulation of receptor tyrosine kinase substrates is affected by cell population physiology. EGF stimulated a rapid increase in inositol trisphosphate in confluent but not subconfluent cells. Similarly, treatment of confluent IEC-6 cells with EGF provoked a significant increase in the hydrolysis of PtdIns 4,5-P2 by immunoisolated PLC gamma 1. The tyrosine phosphorylation state of PLC gamma 1 and the association of PLC gamma 1 with the EGF receptor were increased by EGF in confluent cells only. In contrast, the autophosphorylation state of the EGF receptor and the tyrosine phosphorylation state of another SH2-containing EGF receptor substrate SHC were increased by EGF regardless of cell density. Western blot analysis revealed equal protein expression of PLC gamma 1 in confluent and subconfluent cells. EGF receptor protein expression and ligand binding capacity were slightly increased in confluent compared to subconfluent cells. EGF regulation of PLC gamma 1, therefore, is regulated by physiological factors dependent on cell density in IEC-6 cells.

Animals↗
PubMed1995

Baculovirus expression and purification of the second messenger enzyme phospholipase C-gamma 1, a tyrosine kinase substrate.

A rat cDNA encoding phospholipase C-gamma 1 (PLC-gamma 1) was expressed as a histidine-tagged fusion protein in insect cells utilizing the expression vector pBlueBacHis. The fusion protein was purified by Ni(2+)-agarose affinity chromatography to apparent homogeneity as defined by SDS-PAGE and Coomassie staining. Using a Triton X-100/PIP2 mixed micelle assay, (His6)-PLC-gamma 1 exhibited a calcium-dependent specific enzyme activity of 3 mumol/min/mg, a value similar to that reported for purified bovine brain PLC-gamma 1. Also similar to bovine brain PLC-gamma 1, (His6)-PLC-gamma 1 activity was stimulated by phosphatidic acid and inhibited by adenosine 5'monophosphate. (His6)-PLC-gamma 1 interacted directly with two known PLC-gamma 1 binding proteins, dynamin and the activated EGF receptor. Also, purified (His6)-PLC-gamma 1 was a tyrosine phosphorylation substrate for purified EGF receptor. These results suggest that (His6)-PLC-gamma 1 can be overexpressed as a functional enzyme in baculovirus-infected insect cells and purified by a one-step metal affinity chromatography procedure.

Animals↗
PubMed1995

Interspecies recombination in nature: a meningococcus that has acquired a gonococcal PIB porin.

A vaginal isolate of Neisseria has been reported to resemble Neisseria meningitidis in biochemical characteristics but to react with serological reagents that are specific to the PI porin from Neisseria gonorrhoeae. We have confirmed that this isolate has the biochemical attributes of a meningococcus and have shown that it clusters among meningococcal isolates on a dendrogram based on isoenzyme variation within housekeeping enzymes from populations of N. meningitidis and N. gonorrhoeae. Furthermore, the sequences of the fbp and adk genes were typical of those of N. meningitidis and were distinct from those of N. gonorrhoeae. However, the porB gene was very similar to the por genes of N. gonorrhoeae isolates that express the PIB class of outer-membrane porin (differing from one gonococcal por allele at only a single nucleotide site), and was clearly distinct from the porB genes of N. meningitidis. The isolate therefore appears to be a typical meningococcus, except that its porB gene has been replaced with the por gene from a gonococcus.

Adenylate Kinase↗
PubMed1995

Tannic acid's effect on bird antigen.

BACKGROUND: Birds have been associated with many diseases including hypersensitivity pneumonitis and allergic diseases such as asthma and rhinitis. Bird antigen from homes of patients with hypersensitivity pneumonitis persists long after the bird is removed from the home. This may account for the persistence of symptoms, signs, and bird-specific IgG in patients with hypersensitivity pneumonitis. Tannic acid application has been effective in decreasing cat and mite allergen levels. No data have been available on tannic acid's effect on bird antigen. OBJECTIVE: It is the purpose of this study to determine whether tannic acid reduces bird antigen in the home. METHOD: Dust samples were collected from homes with bird antigen before and after application of tannic acid. Samples were assayed for bird antigen levels using a competitive inhibition ELISA. Pre- and post-bird antigen levels were compared using a paired t test to determine whether antigen was reduced significantly. RESULTS: There was not a statistical difference between bird antigen levels before and after application of tannic acid as compared by paired t test (P = .09). CONCLUSION: Tannic acid is not effective in decreasing bird antigen levels. In patients with hypersensitivity pneumonitis or allergic disease to birds, the bird should be removed from the home and environmental cleanup should be undertaken, but tannic acid application is not indicated.

Allergens↗
PubMed1994

Limited proteolysis of phospholipase C-gamma 1 indicates stable association of X and Y domains with enhanced catalytic activity.

Phospholipase C-gamma 1 (PLC-gamma 1) was treated with Staphylococcus aureus V8 protease (V8) and the digestion products were analysed with site-specific antibodies. V8 treatment generated three immunodetectable PLC-gamma 1 fragments of 120, 97, and 39 kDa. The 39 kDa fragment is derived from the C-terminus of PLC-gamma 1 and includes the conserved Y domain present in all PLC isoenzymes. The 120 and 97 kDa fragments are derived from the N-terminus of PLC-gamma 1, possess the conserved X domain common to all PLC isoenzymes, and the src-homology domains unique to PLC-gamma 1 and -gamma 2. It is likely that the 97 kDa fragment is a V8 product of the 120 kDa fragment. As the C-terminal 39 kDa fragment, and either of the N-terminal 120 or 97 kDa fragments, were precipitable with antibody specific to a sequence present in only the 39 kDa fragment, the data indicate co-precipitation of separate polypeptide chains that remain associated after V8 proteolysis. Importantly, V8 treatment increased the activity of PLC-gamma 1 and did not alter the calcium requirement. The influence of other modulators of PLC-gamma 1 activity, however, was lost following V8 treatment. These results suggest the stable association of the X and Y domains within PLC-gamma 1, and demonstrate that proteolysis in the region of PLC-gamma 1 that is subject to tyrosine phosphorylation can enhance catalytic activity.

Cell Line↗
PubMed1994

Individual epidermal growth factor receptor autophosphorylation sites do not stringently define association motifs for several SH2-containing proteins.

To determine whether individual autophosphorylation sites in the epidermal growth factor (EGF) receptor define specific interaction sites for the in vivo association of signal transduction proteins that contain src homology 2 (SH2) domains, the capacity of wild-type and mutant EGF receptors to associate with several SH2 domain-containing proteins has been assayed. Mutants included receptors with single autophosphorylation site mutations at each of five autophosphorylation sites and receptors in which multiple autophosphorylation sites were removed by point mutation or deletion of carboxyl-terminal residues. Receptor association, as measured by coimmunoprecipitation, has been determined for phospholipase C-gamma 1, the ras GTPase-activating protein, the p85 subunit of phosphatidylinositol 3-kinase, and the src homology and collagen protein. In contrast to data obtained with single autophosphorylation site mutants of other receptor tyrosine kinases, none of the EGF receptor single site mutants was dramatically impaired in its capacity to associate with any of these SH2-containing proteins. However, association was completely abrogated when all five autophosphorylation sites were mutated or removed by deletion. These results indicate that individual autophosphorylation sites in the EGF receptor are not stringently required for the recognition and association of different SH2-containing substrates. Thus, EGF receptor autophosphorylation sites seem to be flexible and/or compensatory in their capacity to mediate association with these four SH2-containing substrates.

3T3 Cells↗
PubMed1994

Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C.

Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.

Amino Acid Sequence↗