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Biomedical subjects

G S Martin

Publications and source records attributed to G S Martin.

At least 37 records · Page 2Linked to original sources
PubMed1998

Ras-independent activation of Ral by a Ca(2+)-dependent pathway.

The RalA and RalB proteins comprise a distinct family of small GTPases [1]. Ral-specific guanine-nucleotide exchange factors such as RalGDS, Rlf and RGL interact with activated Ras and cooperate with Ras in the transformation of murine fibroblasts [2-5]. Thus, the interaction of RalGDS with Ras and the subsequent activation of Ral are thought to constitute a distinct Ras-dependent signaling pathway. The function of Ral is largely unknown. There is circumstantial evidence that Ral may have a function in regulating the cytoskeleton through its interaction with RIP1 (also known as RLIP or RalBP1), a GTPase-activating protein specific for the small GTPases Cdc42 and Rac [6-8]. Ral also binds to phospholipase D (PLD) and thus may play a role in signaling through phospholipids [9]. We have examined endogenous levels of activated, GTP-bound Ral (Ral-GTP) in Rat-2 fibroblasts stimulated with various mitogens. Lysophosphatidic acid (LPA) and epidermal growth factor (EGF), which activate both Ras-dependent and Ras-independent signaling pathways [10,11], rapidly activated Ral. Inhibition of Ras activation by dominant-negative Ras (RasS17N) or pertussis toxin had little effect on Ral-GTP levels, however. Ral was activated by the Ca2+ ionophore ionomycin, and activation by LPA or EGF could be blocked by a phospholipase C (PLC) inhibitor. The results presented here demonstrate a Ca(2+)-dependent mechanism for the activation of Ral.

Animals↗
PubMed1998

Structural basis for the interaction of Ras with RalGDS.

The Ras protein signals to a number of distinct pathways by interacting with diverse downstream effectors. Among the effectors of Ras are the Raf kinase and RalGDS, a guanine nucleotide dissociation stimulator specific for Ral. Despite the absence of significant sequence similarities, both effectors bind directly to Ras, but with different specificities. We report here the 2.1 A crystal structure of the complex between Ras and the Ras-interacting domain (RID) of RalGDS. This structure reveals that the beta-sheet of the RID joins the switch I region of Ras to form an extended beta-sheet with a topology similar to that found in the Rap-Raf complex. However, the side chain interactions at the joining junctions of the two interacting systems and the relative orientation of the two binding domains are distinctly different. Furthermore, in the case of the Ras-RID complex a second RID molecule also interacts with a different part of the Ras molecule, the switch II region. These findings account for the cross-talk between the Ras and Ral pathways and the specificity with which Ras distinguishes the two effectors.

Animals↗
PubMed1998

Cardiac troponin-I accurately predicts myocardial injury in renal failure.

BACKGROUND: Non-specific elevations of creatine kinase isoenzymes (CK-MB) and cardiac troponin-T may be seen in renal failure, confusing the diagnosis of myocardial infarction. Cardiac troponin-I (cTn-I) has been shown to be specific for myocardial damage in several disease states, but has not been prospectively evaluated in the setting of renal failure. METHODS: This prospective case series evaluated 56 patients with acute or chronic renal failure or end-stage renal disease to assess the sensitivity and specificity of cTn-I for detecting myocardial injury in this patient population. During a 6-month period, patients admitted with suspected myocardial injury by history, physical examination, and electrocardiography were evaluated. Cardiac troponin-I (cTn-I) measurements were assessed between 8 and 48 h after admission. Appropriate medical care and further cardiac testing (echocardiography, stress testing, or arteriography) was performed at the discretion of the primary physician. RESULTS: Myocardial injury was diagnosed in 18/56 (32%) patients by positive cTn-I levels, while only 7/56 (13%) patients had evidence of myocardial damage by CK-MB. Twenty-one of 56 (38%) patients had indeterminate CK-MB levels and 53% of these patients demonstrated myocardial ischaemia on follow-up testing. Sixteen patients had negative cardiac studies; all of these patients had negative cTn-I levels, while seven of these 16 (44%) patients had indeterminate CK-MB measurements. All of the patients with positive cTn-I levels had positive cardiac studies. Positive troponin levels were associated with increased in-hospital mortality. Sensitivity and specificity for CK-MB were 44 and 56% respectively, and 94 and 100% for cTn-I. CONCLUSION: These data support the use of cTn-I for diagnosing myocardial injury in patients with renal failure. Elevated cTn-I levels are associated with increased short-term mortality in renal failure patients. The accuracy of cTn-I could potentially limit unnecessary cardiac testing in renal failure patients, while the enhanced sensitivity contributes to risk stratification and aids in diagnosing true myocardial injury in this population susceptible to non-specific elevations in other muscle enzymes.

Acute Kidney Injury↗
PubMed1998

Intra-articular pressure, elastance and range of motion in healthy and injured racehorse metacarpophalangeal joints.

The objective of this study was to determine if intraarticular pressure, elastance of the soft tissue forming the dorsal pouch, and range of motion in flexion measurements are significantly different in Thoroughbred metacarpophalangeal joints with clinical evidence of idiopathic synovitis, primary synovitis, synovitis/capsulitis, or osteoarthritis relative to clinically normal joints. Forty-two metacarpophalangeal joints, in 25 active or retired Thoroughbred racehorses, were categorised by palpation and visual inspection of the palmar pouch into one of 4 increasing grades of distention. Intra-articular pressures were then measured using 2 pressure transducers attached to 22-gauge needles from both the dorsal and palmar pouches simultaneously while the horses stood squarely under mild sedation. After obtaining baseline pressure measurements, a third needle was inserted into the dorsal pouch, and 0.5 ml increments of saline solution were added every 10 s to perform a pressure/volume (elastance) study of the dorsal pouch. The elastance study for each joint ended when leakage into the palmar pouch was detected by the pressure transducer placed in that region. A lateral radiographic view was taken of each metacarpophalangeal joint in maximal flexion. The maximum angle of flexion was measured from the radiograph, and this angle was subtracted from 180 degrees to acquire the range of motion in flexion. In this study, all Thoroughbreds with clinical evidence of lameness and/or sensitivity to flexion, referable to the metacarpophalangeal joint region, had fluid distention of the palmar pouch (grade 2 or 3 distention). The 16 metacarpophalangeal joints with no clinical abnormalities had a mean palmar pouch pressure of -1.25 mmHg. Joints afflicted with synovitis/capsulitis had the highest intraarticular pressures (mean +51.00 mmHg); however, joints with idiopathic synovitis (mean +15.71 mmHg), primary synovitis (mean +28.33 mmHg) and osteoarthritis (mean +26.20 mmHg) also had significantly elevated intraarticular pressures relative to the clinically normal group. Thoroughbred metacarpophalangeal joints diagnosed with synovitis/capsulitis, or osteoarthritis, had significantly increased elastance (stiffness) of the soft tissue forming the dorsal pouch relative to the normal group and, probably, as a result significantly decreased range of motion in flexion. The presence of primary synovitis alone did not have a significant immediate effect on elastance of the dorsal pouch and range of motion in flexion. The 16 Thoroughbred metacarpophalangeal joints assessed as having no clinical abnormalities had a mean range of motion in flexion of 60.81 degrees. The mean range of motion in flexion of Thoroughbred metacarpophalangeal joints with a clinical diagnosis of primary synovitis was 53.67 degrees; idiopathic synovitis 52.14 degrees; synovitis/capsulitis 44.20 degrees; and those with radiographic evidence of moderate to marked osteoarthritis 30.80 degrees. This study demonstrated that, as the severity of the clinical evidence of metacarpophalangeal joint injury/disease increased, the range of motion in flexion decreased.

Analysis of Variance↗
PubMed1997

Validation of a regression model for standardizing lifetime racing performances of thoroughbreds.

OBJECTIVE: To determine the relationship between prediction errors of a regression model of racing finish times and earnings or finish position; the relationship between standardized finish times, determined by use of this model, and earnings or finish position; and whether this model was valid when applied to data for horses that underwent surgical treatment. DESIGN: Survey. SAMPLE POPULATION: Records of 6,700 healthy Thoroughbreds racing in Louisiana and of 31 Thoroughbreds with idiopathic left laryngeal hemiplegia that underwent surgical treatment. PROCEDURE: Predicted and standardized finish times were calculated by use of the regression model for healthy horses, and the relationships between prediction error (actual--predicted finish time) and standardized finish times, and earnings and finish position, were examined. Then, the regression model was applied to data for horses with hemiplegia to determine whether the model was valid when used to calculate predicted and standardized finish times for lifetime performance data. RESULTS: Prediction error and standardized finish times were negatively correlated with earnings and positively correlated with finish position and, thus, appeared to be reliable measures of racing performance. The regression model was found to be valid when applied to lifetime performance records of horses with laryngeal hemiplegia. CLINICAL IMPLICATIONS: Prediction error and standardized finish times are measures of racing performance that can be used to compare performances among Thoroughbred racehorses across a variety of circumstances that would otherwise confound comparison.

Animals↗
PubMed1997

Casein kinase II catalyzes tyrosine phosphorylation of the yeast nucleolar immunophilin Fpr3.

In the yeast Saccharomyces cerevisiae, the nucleolar immunophilin, Fpr3, is phosphorylated at tyrosine and dephosphorylated by the phosphotyrosine-specific phosphoprotein phosphatase, Ptp1. In Ptp1-deficient cells, Fpr3 contains phospho-Tyr at a single site (Tyr184), but also contains phospho-Ser and phospho-Thr. Ser186 (adjacent to Tyr184) is situated within a canonical site for phosphorylation by casein kinase II (CKII). Yeast cell lysates contain an activity that binds to Fpr3 in vitro and phosphorylates Fpr3 at Ser, Thr, and Tyr; this activity was found to be dependent on expression of functional yeast CKII. Moreover, purified Fpr3 was phosphorylated on Tyr184 in vitro by either purified yeast CKII or purified, bacterially-expressed human CKII. Likewise, phosphorylation of Fpr3 at tyrosine in vivo was markedly enhanced in yeast cells overexpressing a heterologous (Drosophila) CKII, but was undetectable in yeast cells carrying only a temperature-sensitive allele of the endogenous CKII, even when the cells were grown at a permissive temperature. Phosphorylation of Fpr3 at Tyr184 by CKII in vitro lagged behind phosphorylation of Fpr3 at Ser, and was accelerated by pre-phosphorylation of Fpr3 at Ser using CKII. Furthermore, synthetic peptides corresponding to the sequence surrounding Tyr184 that contained P-Ser (or Glu) at position 186 were much more efficient substrates for CKII phosphorylation of Tyr184 than a synthetic peptide containing Ala at position 186. These findings indicate that CKII phosphorylates Fpr3 at tyrosine and serine both in vivo and in vitro and thus possesses dual specificity. These results also indicate that Tyr184 is phosphorylated by CKII via a two-step process, in which phosphorylation at the +2 position provides a negatively-charged specificity determinant that allows subsequent phosphorylation of Tyr184.

Amino Acid Isomerases↗
PubMed1997

Ras-independent transformation by v-Src.

Signaling by a variety of receptor and nonreceptor tyrosine kinases is mediated by Ras, a membrane-associated GTPase. Expression of v-Src, a transforming nonreceptor tyrosine kinase, results in Ras activation, and inhibition of Ras function in NIH 3T3 cells suppresses transformation by v-Src, indicating that in these cells Ras-dependent signaling pathways are required for v-Src to exert its biological effects. However, we show here that Ras was not activated in Rat-2 fibroblasts transformed by wild-type v-Src, or in chicken embryo fibroblasts transformed by SRX5, a v-Src mutant with a linker insertion at the major site of autophosphorylation. Expression of a dominant-negative mutant of Ras completely inhibited the ability of v-Src to activate the mitogen-activated protein kinase ERK2, which is downstream of Ras. However, dominant-negative Ras did not suppress transformation by v-Src as judged by a variety of criteria. Thus, v-Src can transform at least some cell types in the absence of Ras activation or Ras-stimulated ERK2 activity, and in these cells activation of Ras-independent signaling pathways must therefore be sufficient for transformation.

Animals↗
PubMed1997

Myocardial bridge as a cause of thrombus formation and myocardial infarction in a young athlete.

We report here a young male athlete who had an acute myocardial infarction after strenuous exercise. Coronary angiography clearly demonstrated thrombus formation within a prominent myocardial bridge. Periarterial resection surgery was performed, which resulted in resolution of the angiographic "milking" effect of muscle bridge. Our observation, along with previous reports, suggests that myocardial bridges should be added to the list of known causes of sudden death or myocardial infarction in young persons after strenuous exercise. Thrombus formation within a myocardial bridge may be a potential mechanism whereby this anatomic curiosity may cause a tragic clinical event.

Adult↗
PubMed1997

The role of the Src homology-2 domain in the lethal effect of Src expression in the yeast Saccharomyces cerevisiae.

Expression of the retroviral transforming gene v-src arrests the proliferation of the yeast Saccharomyces cerevisiae. A functional Src SH2 (Src homology 2) domain is required for this arrest. To examine the mechanism by which Src blocks yeast cell proliferation, and to determine the role of the Src SH2 domain in the growth arrest, src variants were expressed in yeast under the control of the galactose-inducible GAL1 promoter. Following galactose induction of Src expression, phosphotyrosyl-proteins were isolated by immunoprecipitation with beads coupled to either anti-phosphotyrosine antibody or to a recombinant fusion protein containing the Src SH2 domain. A group of SH2-binding phosphotyrosyl proteins was detected in cells expressing toxic forms of Src, but were not detected in cells expressing non-toxic variants. This group of phosphotyrosyl-proteins represents a minor subset of the proteins phosphorylated by v-Src. The lethality of v-Src and the phosphorylation of SH2-binding proteins were co-ordinately affected by alterations in phosphotyrosine-phosphatase activity. These observations indicate that the lethality of Src is correlated with the phosphorylation of proteins that bind to the Src SH2 domain. The role of the SH2 domain in determining the lethal effects of Src in yeast may be similar to its role in targeting Src to substrates necessary for its biological effects in vertebrate cells.

Fungal Proteins↗
PubMed1997

Three-dimensional structure of the Ras-interacting domain of RalGDS.

The Ras-interacting domains of the the protein-kinase Raf and the Ral guanine nucleotide dissociation stimulator, RalGDS, lack extensive sequence similarity, but their overall three-dimensional structures are very similar to each other. Mutational analysis indicated that three residues in the RalGDS domain are critical for its interaction with Ras.

Amino Acid Sequence↗
PubMed1997

The role of the Src homology domains in morphological transformation by v-src.

The Src homology (SH2 and SH3) domains of v-Src are required for transformation of Rat-2 cells and for wild-type (morphr) transformation of chicken embryo fibroblasts (CEFs). We report herein that the N-terminal domains of v-Src, when expressed in trans, cannot complement the transformation defect of a deletion mutant lacking the "unique," SH3, and SH2 regions. However, the same regions of Src can promote transformation when translocated to the C terminus of v-Src, although the transformation of CEFs is somewhat slower. We conclude that the SH3 and SH2 domains must be present in cis to the catalytic domain to promote transformation but that transformation is not dependent on the precise intramolecular location of these domains. In CEFSs and in Rat-2 cells, the expression of wild-type v-Src results in tyrosine phosphorylation of proteins that bind to the v-Src SH3 and SH2 domains in vitro; mutations in the SH2 or SH3 and SH2 domains prevent the phosphorylation of these proteins. These findings are most consistent with models in which the SH3 and SH2 domains of v-Src directly or indirectly target the catalytic domain to substrates involved in transformation. However, the N-terminal domains of v-Src can promote tyrosine phosphorylation of certain proteins, in particular p130Cas, even when expressed in the absence of the catalytic domain, indicating that the N-terminal domains of v-Src have effects that are independent of the catalytic domain.

Animals↗
PubMed1997

Prepartum protein restriction does not alter norepinephrine-induced thermogenesis or brown adipose tissue function in newborn calves.

We examined the effect of prepartum protein restriction on thermogenesis and several aspects of perirenal (brown) adipose tissue (BAT) in newborn calves. Lipid synthesis and morphology also were compared between BAT and sternum (white) adipose tissue. During the last 140 d of gestation, heifers were fed isocaloric diets containing adequate (10.4%) or restricted (average of 6.8%, dry matter basis) levels of protein. Body condition scores and weight gain during gestation were significantly lower in heifers fed the restricted-protein diet. However, newborn calf birth weight, calf BAT weight and composition, and calf thermoneutral metabolic rates were not affected by prepartum protein restriction. Similarly, visceral organ weights, except for lung plus trachea, were not affected (P > 0.10) by prepartum protein treatment. Peak metabolic rates were not affected (P > 0.10) by prepartum protein treatment and on average were twice the thermoneutral metabolic rates. Consistent with this, BAT of calves from heifers fed adequate- or restricted-protein diets did not differ in lipid synthesis, cellularity, or uncoupling protein mRNA:28S rRNA ratios. Although both perirenal and sternum adipocytes were mostly unilocular, perirenal adipocytes contained numerous large mitochondria with well-differentiated cristae; sternum adipocytes contained a small number of mitochondria with poorly developed cristae. Fatty acid biosynthesis from acetate was high in BAT (55-57 nmol acetate incorporated.100 mg-1.h-1) but barely detectable in sternum adipose tissue. Conversely, fatty acid biosynthesis from glucose was 80-110% higher in sternum adipose tissue than in BAT (4.5 vs 2.1-2.5 nmol glucose incorporated.100 mg-1.h-1). Thus maternal protein restriction severely affected heifers but had no effect on neonatal calf thermogenesis or BAT function.

Adipose Tissue, Brown↗
PubMed1997

Mck1, a member of the glycogen synthase kinase 3 family of protein kinases, is a negative regulator of pyruvate kinase in the yeast Saccharomyces cerevisiae.

An interaction between the Saccharomyces cerevisiae protein kinase Mck1 and pyruvate kinase (Pyk1) was detected by using the two-hybrid method. Purified Mck1 was able to phosphorylate purified Pyk1 on Ser in vitro. Pyruvate kinase activity was elevated in mck1 delta cells. Several of the phenotypes of mck1 delta mutants are similar to those observed in cells overexpressing PYK1. Co-overexpression of MCK1 suppressed all of the phenotypes associated with PYK1 overexpression. These results indicate that Mck1 negatively regulates pyruvate kinase activity, possibly by direct phosphorylation.

Calcium-Calmodulin-Dependent Protein Kinases↗