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Biomedical subjects

G S Martin

Publications and source records attributed to G S Martin.

At least 73 records · Page 4Linked to original sources
PubMed1991

Disruption of the caudal component of the reciprocal apparatus in two horses.

Two horses became acutely lame following a fall during strenuous exercise and were diagnosed as having disruption of the caudal component of the reciprocal apparatus. Clinical signs consisted of lameness of the right pelvic limb, characterized by flexion of the hock and simultaneous extension of the stifle. Radiography revealed an avulsion fracture from the supracondylar tuberosity and fossa of the distal portion of the femur in one horse. Clinical and radiographic findings indicated avulsion of the lateral origin of the gastrocnemius and superficial digital flexor muscles. Treatment consisted of stall rest, limb immobilization, and phenylbutazone administration. One horse recovered to soundness and the other deteriorated and was euthanatized.

Animals↗
PubMed1991

Cell cycle tyrosine phosphorylation of p34cdc2 and a microtubule-associated protein kinase homolog in Xenopus oocytes and eggs.

We have examined the time course of protein tyrosine phosphorylation in the meiotic cell cycles of Xenopus laevis oocytes and the mitotic cell cycles of Xenopus eggs. We have identified two proteins that undergo marked changes in tyrosine phosphorylation during these processes: a 42-kDa protein related to mitogen-activated protein kinase or microtubule-associated protein-2 kinase (MAP kinase) and a 34-kDa protein identical or related to p34cdc2. p42 undergoes an abrupt increase in its tyrosine phosphorylation at the onset of meiosis 1 and remains tyrosine phosphorylated until 30 min after fertilization, at which point it is dephosphorylated. p42 also becomes tyrosine phosphorylated after microinjection of oocytes with partially purified M-phase-promoting factor, even in the presence of cycloheximide. These findings suggest that MAP kinase, previously implicated in the early responses of somatic cells to mitogens, is also activated at the onset of meiotic M phase and that MAP kinase can become tyrosine phosphorylated downstream from M-phase-promoting factor activation. We have also found that p34 goes through a cycle of tyrosine phosphorylation and dephosphorylation prior to meiosis 1 and mitosis 1 but is not detectable as a phosphotyrosyl protein during the 2nd through 12th mitotic cell cycles. It may be that the delay between assembly and activation of the cyclin-p34cdc2 complex that p34cdc2 tyrosine phosphorylation provides is not needed in cell cycles that lack G2 phases. Finally, an unidentified protein or group of proteins migrating at 100 to 116 kDa increase in tyrosine phosphorylation throughout maturation, are dephosphorylated or degraded within 10 min of fertilization, and appear to cycle between low-molecular-weight forms and high-molecular-weight forms during early embryogenesis.

Animals↗
PubMed1990

Quantitation of vitamin K in human milk.

A quantitative method was developed for the assay of vitamin K in human colostrum and milk. The procedure combines preparative and analytical chromatography on silica gel in a nitrogen atmosphere followed by reversed phase high performance liquid chromatography (HPLC). Two HPLC steps were used: gradient separation with ultraviolet (UV) detection followed by isocratic separation detected electrochemically. Due to co-migrating impurities, UV detection alone is insufficient for identification of vitamin K. Exogenous vitamin K was shown to equilibrate with endogenous vitamin K in the samples. A statistical method was incorporated to control for experimental variability. Vitamin K1 was analyzed in 16 pooled milk samples from 7 donors and in individual samples from 15 donors at 1 month post-partum. Vitamin K1 was present at 2.94 +/- 1.94 and 3.15 +/- 2.87 ng/mL in pools and in individuals, respectively. Menaquinones, the bacterial form of the vitamin, were not detected. The significance of experimental variation to studies of vitamin K in individuals is discussed.

Adult↗
PubMed1990

Src homology region 2 domains direct protein-protein interactions in signal transduction.

Cytoplasmic proteins that regulate signal transduction or induce cellular transformation, including cytoplasmic protein-tyrosine kinases, p21ras GTPase-activating protein (GAP), phospholipase C gamma, and the v-crk oncoprotein, possess one or two copies of a conserved noncatalytic domain, Src homology region 2 (SH2). Here we provide direct evidence that SH2 domains can mediate the interactions of these diverse signaling proteins with a related set of phosphotyrosine ligands, including the epidermal growth factor (EGF) receptor. In src-transformed cells GAP forms heteromeric complexes, notably with a highly tyrosine phosphorylated 62-kDa protein (p62). The stable association between GAP and p62 can be specifically reconstituted in vitro by using a bacterial polypeptide containing only the N-terminal GAP SH2 domain. The efficient phosphorylation of p62 by the v-Src or v-Fps tyrosine kinases depends, in turn, on their SH2 domains and correlates with their transforming activity. In lysates of EGF-stimulated cells, the N-terminal GAP SH2 domain binds to both the EGF receptor and p62. Fusion proteins containing GAP or v-Crk SH2 domains complex with similar phosphotyrosine proteins from src-transformed or EGF-stimulated cells but with different efficiencies. SH2 sequences, therefore, form autonomous domains that direct signaling proteins, such as GAP, to bind specific phosphotyrosine-containing polypeptides. By promoting the formation of these complexes, SH2 domains are ideally suited to regulate the activation of intracellular signaling pathways by growth factors.

Animals↗
PubMed1990

Behavioural change in injecting drug users: evaluation of an HIV/AIDS education programme.

The results obtained from the training and follow-up of 189 IDUs who participated in a programme consisting of an audiovisual presentation, pre-/post-testing and individual counselling are presented. Syringe sharing decreased from 35% at initial contact to 12% after 6 months. Sexual behaviour proved more resistant to change. However, condom use in at-risk situations increased from 49% to 70%. IDUs under continuous methadone treatment were less likely to engage high risk drug injecting practices than the other IDUs. Results indicate that an educational programme addressed toward risk reduction may determine relevant behavioural change among IDUs.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Novel yeast protein kinase (YPK1 gene product) is a 40-kilodalton phosphotyrosyl protein associated with protein-tyrosine kinase activity.

Extracts of bakers' yeast (Saccharomyces cerevisiae) contain protein-tyrosine kinase activity that can be detected with a synthetic Glu-Tyr copolymer as substrate (G. Schieven, J. Thorner, and G.S. Martin, Science 231:390-393, 1986). By using this assay in conjunction with ion-exchange and affinity chromatography, a soluble tyrosine kinase activity was purified over 8,000-fold from yeast extracts. The purified activity did not utilize typical substrates for mammalian protein-tyrosine kinases (enolase, casein, and histones). The level of tyrosine kinase activity at all steps of each preparation correlated with the content of a 40-kDa protein (p40). Upon incubation of the most highly purified fractions with Mn-ATP or Mg-ATP, p40 was the only protein phosphorylated on tyrosine. Immunoblotting of purified p40 or total yeast extracts with antiphosphotyrosine antibodies and phosphoamino acid analysis of 32P-labeled yeast proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the 40-kDa protein is normally phosphorylated at tyrosine in vivo. 32P-labeled p40 immunoprecipitated from extracts of metabolically labeled cells by affinity-purified anti-p40 antibodies contained both phosphoserine and phosphotyrosine. The gene encoding p40 (YPK1) was cloned from a yeast genomic library by using oligonucleotide probes designed on the basis of the sequence of purified peptides. As deduced from the nucleotide sequence of YPK1, p40 is homologous to known protein kinases, with features that resemble known protein-serine kinases more than known protein-tyrosine kinases. Thus, p40 is a protein kinase which is phosphorylated in vivo and in vitro at both tyrosine and serine residues; it may be a novel type of autophosphorylating tyrosine kinase, a bifunctional (serine/tyrosine-specific) protein kinase, or a serine kinase that is a substrate for an associated tyrosine kinase.

Amino Acid Sequence↗
PubMed1990

Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation.

We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.

Animals↗
PubMed1990

Intracellular localization of pp60c-src in human platelets.

We have studied the intracellular distribution of the proto-oncogene product pp60c-src in human platelets by immunofluorescence microscopy and by immunogold electron microscopy on frozen thin sections. Virtually all of the immunoreactive pp60c-src was found to be associated with the plasma membrane and with membranes of the surface-connected canalicular system. This localization suggests that pp60c-src may relay signals from an as yet unidentified cell surface protein to intracellular targets.

Antibodies, Monoclonal↗
PubMed1989

Thrombin stimulates the activities of multiple previously unidentified protein kinases in platelets.

We have used a renaturation method to search for previously unidentified protein kinases in human platelets. The method involves subjecting lysates to denaturing gel electrophoresis, transferring the proteins to blotting membranes, and treating the blotted proteins with guanidine. The guanidine is then removed to allow the proteins to renature, and the blots are overlaid with [gamma-32P]ATP. We have identified 14 electrophoretically distinct, serine/threonine-specific protein kinases. Eleven of the kinases clearly differ in molecular weight from all previously described platelet serine/threonine kinases. Ten of these novel kinases (PK220, PK200, PK170, PK150, PK64, PK60, PK56, PK52, PK48, and PK40) were found to possess markedly increased in vitro activity when isolated from thrombin-stimulated platelets, presumably as a result of thrombin-stimulated covalent modification. Treatment of intact platelets with the calcium ionophore ionomycin and phorbol 12-myristate 13-acetate also increased the in vitro activity of these kinases. The agonist-stimulated kinases could be divided into three classes: 1) one kinase whose activity was increased by in vivo phorbol ester treatment but not by ionomycin (PK150); 2) two kinases whose activity was increased by ionomycin but not phorbol ester (PK48 and PK40); 3) seven kinases whose activity was markedly increased by combinations of phorbol ester and ionomycin, but not by either agent alone (PK220, PK200, PK170, PK64, PK60, PK56, and PK52). This third mode of regulation is what would be expected of enzymes that mediate the biological effects of inositide-mobilizing stimuli.

Amino Acids↗
PubMed1989

Tyrosine-specific protein phosphorylation is regulated by glycoprotein IIb-IIIa in platelets.

We have previously shown that a number of platelet proteins become phosphorylated at tyrosine residues in response to platelet-activating agents. Here we present two lines of evidence implicating a platelet integrin, glycoprotein IIb-IIIa, in the regulation of a specific subset of these tyrosine phosphorylations. (i) Two peptides that inhibit the binding of fibrinogen and other ligands to gpIIb-IIIa, Arg-Gly-Asp-Ser and His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val, also inhibited the thrombin-induced tyrosine phosphorylation of this subset of proteins. The tetrapeptide Arg-Gly-Glu-Ser, which does not inhibit fibrinogen binding, did not inhibit thrombin-stimulated tyrosine phosphorylation. (ii) Platelets lacking gpIIb-IIIa (from a subject with Glanzmann thrombasthenia) did not undergo this subset of tyrosine phosphorylation in response to thrombin, although other serine, threonine, and tyrosine phosphorylations proceeded normally. These findings suggest a role for tyrosine-specific protein phosphorylation in integrin-mediated cell-matrix recognition.

Adenosine Diphosphate↗
PubMed1989

Nucleotidylation, not phosphorylation, is the major source of the phosphotyrosine detected in enteric bacteria.

The majority of the phosphotyrosine recovered from partial acid hydrolysates of 32P-labeled Escherichia coli is derived from a single prominent protein. We show here by biochemical, genetic, and immunological criteria that this protein is actually glutamine synthetase adenylylated (not phosphorylated) at tyrosine. Furthermore, all of the phosphotyrosine detectable in partial acid hydrolysates of 32P-labeled Salmonella typhimurium was eliminated in a strain deficient in both glutamine synthetase and uridylyltransferase, an enzyme which uridylylates the regulatory protein PII at a tyrosine residue. These results suggest that protein-tyrosine phosphorylation represents a rare modification in eubacterial cells.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1989

Linker insertion-deletion mutagenesis of the v-src gene: isolation of host- and temperature-dependent mutants.

The host cell regulators and substrates of the Rous sarcoma virus transforming protein pp60v-src remain largely unknown. Viral mutants which induce a host-dependent phenotype may result from mutations which affect the interaction of pp60v-src with host cell components. To isolate such mutants and to examine the role of different regions of src in regulating pp60v-src function, we generated 46 linker insertion and 5 deletion mutations within src. The mutant src genes were expressed in chicken embryo fibroblasts and in rat-2 cells by using retrovirus expression vectors. Most linker insertions within the kinase domain (residues 260 to 512) inactivated kinase activity and transforming capacity, while most insertions in the N-terminal domain and at the extreme C terminus were tolerated. A number of mutations generated a host-dependent phenotype. Insertions after residues 225 and 227, within the N-terminal regulatory domain (SH2), produced a fusiform transformation in chicken embryo fibroblasts and abolished transformation in rat-2 cells; a similar phenotype also resulted from two deletions affecting SH2 (residues 149 to 174 and residues 77 to 225). Insertions immediately C terminal to Lys-295, which is involved in ATP binding, also produced a conditional phenotype. Insertions after residues 299 and 300 produced a temperature-sensitive phenotype, while insertions after residues 304 and 306 produced a host cell-dependent phenotype. An insertion which removed the major tyrosine autophosphorylation site (Tyr-416) greatly reduced transformation of rat-2 cells, a property not previously observed with other mutations at this site. We conclude that mutations at certain sites within src result in conditional phenotypes. These sites may represent regions important in interactions with host cell components.

Animals↗
PubMed1988

Nonenzymatic phosphorylation of tyrosine and serine by ATP is catalyzed by manganese but not magnesium.

Many cellular and retroviral protein-tyrosine kinases display either a requirement or a preference for manganese over magnesium for maximal activity. We have observed that peptides and proteins are non-enzymatically phosphorylated at tyrosine and serine by ATP when heated in the presence of MnCl2 at neutral pH. The extent of the reaction is negligible below 50 degrees C but increases rapidly at higher temperatures. The reaction proceeds in the presence of sodium dodecyl sulfate but is blocked by EDTA. No reaction is observed in the absence of Mn2+, even if Mg2+ is present. Manganese therefore acts as a catalyst for the non-enzymatic reaction, but magnesium does not. We propose that the preference for manganese shown by many protein tyrosine kinases is due at least in part to the intrinsic ability of Mn2+ to catalyze the transfer of phosphate from ATP to a phosphate acceptor such as tyrosine. The nonenzymatic phosphorylation reaction also offers a new synthetic pathway for the preparation of radiolabeled peptides containing phosphotyrosine and phosphoserine.

Adenosine Triphosphate↗
PubMed1988

Effect of third carpal slab fracture and repair on racing performance in Thoroughbred horses: 31 cases (1977-1984).

Between 1977 and 1984, 31 Thoroughbred horses (mean age, 2.8 years) were surgically treated for slab fractures of the third carpal bone. All fractures involved the articular surfaces of the intercarpal and the carpometacarpal joints in a frontal plane and had a cuboidal or slab shape. In 20 of the horses, the bone was fractured during racing and in 8 of the horses, the bone was fractured during race training. The right limb was affected more frequently than was the left limb (24 vs 7, P less than 0.05). Twenty-one (67.6%) horses raced at least once after recovery from the surgery. Data were available from 11 claiming horses that had raced at least twice before their injuries and 4 times after recovery. A claiming horse is one that competes where any horse entered is subject to being purchased for the designated amount of the claiming race; therefore, each race tends to automatically attract entrants of similar ability. In this group of 11 horses, claiming value decreased from a mean of $13,900 to a mean of $6,500 (P less than 0.05), and the mean finish position was 5.8 +/- 3.16 before injury and 5.8 +/- 3.30 after recovery. The mean claiming value for horses that had not raced before injury, but had raced after recovery (n = 5) was $8,150.

Animals↗
PubMed1988

A host-dependent temperature-sensitive mutant of Rous sarcoma virus: evidence for host factors affecting transformation.

We have characterized a host range mutant of Rous sarcoma virus in order to identify host cell factors involved in transformation. This mutant, tsLA33-1, which was isolated from a stock of the temperature-sensitive mutant tsLA33, is not temperature-sensitive for transformation of chicken embryo fibroblasts, as judged by its ability to induce morphological changes and agar colony formation at both 36 and 41.5 degrees. In Rat-3 cells, however, this mutant induced a temperature-dependent transformation: infected Rat-3 cells were transformed at 34 degrees but not at 39.5 degrees. Retransformants were isolated from tsLA33-1-infected Rat-3 cells by growth in agar suspension at 39.5 degrees. Virus rescued from these retransformants induced a temperature-dependent transformation when reintroduced into rat cells. The level of expression of pp60v-src at 39.5 degrees was unchanged in the retransformants. When the retransformants were treated with herbimycin, an antibiotic which induces turnover of certain protein-tyrosine kinases, they reverted to a normal phenotype, indicating that the transformed phenotype of the retransformants was dependent on continued expression of pp60v-src. The retransformants are therefore pseudorevertants in which a cellular alteration has occurred that allows transformation at 39.5 degrees by the mutant pp60v-src. Thus the temperature-dependence of transformation by tsLA33-1 is affected by the cellular environment, and is suppressed or complemented both in chicken cells and in the rat cell pseudorevertants. No clear correlation between levels of phosphorylation at tyrosine and transformation was observed. In Rat-3 cells the pp60v-src encoded by tsLA33-1 may be defective in its interaction with low abundance substrates that are critical for transformation; alternatively the nonpermissive cells may require a higher threshold dose of pp60v-src for transformation.

Animals↗
PubMed1988

pp60v-src tyrosine kinase is expressed and active in sarcoma-free avian embryos microinjected with Rous sarcoma virus.

Early embryonic avian tissue is resistant to transformation by Rous sarcoma virus. To determine the nature of this resistance, we examined the expression and properties of the Rous sarcoma virus transforming protein pp60v-src, in infected embryonic chicken limbs in ovo. Lysates from Rous sarcoma virus-infected limbs contained the viral structural protein p19gag, as detected by immunoblot analysis, and showed pp60v-src kinase activity in vitro. Immunoblot analysis of lysates with anti-phosphotyrosine antibodies revealed a number of phosphotyrosine-containing proteins present in lysates of Rous sarcoma virus-infected embryos but not in lysates of control, uninfected embryos. Anti-phosphotyrosine immunoreactivity was observed in frozen sections in the same cell types that expressed pp60v-src and p19gag. These studies demonstrate that pp60v-src is co-expressed with viral structural determinants in infected embryonic avian tissue. Furthermore, pp60v-src is active in ovo as a tyrosine-specific phosphotransferase, despite the apparent lack of sarcoma induction. The localization pattern of the major src gene substrate p36 (calpactin I) was compared with that of p19gag by double-label immunofluorescence and found to be generally nonoverlapping. These observations are consistent with the concept that the induction of tumors in ovo requires complementation between viral determinants and host factors. These host factors, which may be critical substrates of pp60v-src, are subject to developmental regulation in the avian embryo.

Animals↗