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Biomedical subjects

J Lawler

Publications and source records attributed to J Lawler.

16 recordsLinked to original sources
PubMed1992

Molecular requirements for the interaction of thrombospondin with thrombin-activated human platelets: modulation of platelet aggregation.

We have investigated the molecular requirements for thrombospondin (TSP) to bind to the platelet surface and to support the subsequent secretion-dependent platelet aggregation. For this, we used two distinct murine monoclonal antibodies (MoAbs), designated MAI and MAII, raised against human platelet TSP, and three polyclonal antibodies, designated R3, R6, and R5, directed against fusion proteins containing the type 1 (Gly 385-Ile 522), type 2 (Pro 559-Ile 669), and type 3 (Asp 784-Val 932) repeating sequences, respectively. Among them, R5 and R6, but not R3, inhibited thrombin-induced aggregation of washed platelets and the concomitant secretion of serotonin. These antibodies, however, did not inhibit the expression of TSP on thrombin-activated platelets, as measured by the binding of a radiolabeled MoAb to TSP, suggesting that they may inhibit platelet aggregation by interfering with a physiologic event subsequent to TSP binding. In contrast, MoAb MAII, which reacts with an epitope located within the heparin-binding domain of TSP, inhibited both TSP surface expression and platelet aggregation/secretion induced by thrombin. In addition, this MoAb inhibited in a dose-dependent manner (IC50 approximately 0.5 mumol/L) the interaction of 125I-TSP with immobilized fibrinogen and platelet glycoprotein IV, both potential physiologic receptors for TSP on thrombin-activated platelets. These results indicate that the interaction of TSP with the surface of activated platelets can be modulated at the level of a specific epitope located within the amino terminal heparin-binding domain of the molecule. Thus, selective inhibition of the platelet/TSP interaction may represent an alternative approach to the inhibition of platelet aggregation.

Antibodies↗
PubMed1992

Expression and mutagenesis of thrombospondin.

Thrombospondin is a 420,000-dalton adhesive glycoprotein that is composed of three subunits of equivalent molecular weight. When the cDNA for the complete coding region of the human endothelial cell thrombospondin subunit is expressed in mouse NIH 3T3 cells, a 420,000-dalton protein is synthesized and secreted. The expressed protein comigrates with human platelet thrombospondin both in the presence and in the absence of a reducing agent. The expressed protein binds to a monoclonal anti-thrombospondin antibody, heparin, and calcium. In addition to the 420,000-dalton protein, the transfected cell lines also express a variable amount of a 140,000-dalton polypeptide. When the culture supernatants that are produced by cells that are expressing thrombospondin are applied to heparin-Sepharose, the 420,000-dalton and the 140,000-dalton proteins are bound to the column and are eluted with buffer containing 0.55 and 0.3 M NaCl, respectively. The 140,000-dalton protein only binds to heparin-Sepharose in the presence of calcium. Deletion of the region of homology with procollagen results in defective assembly of the trimer. Deletion of the type 1 or type 2 repeats results in decreased stability of the subunit with the predominant polypeptides that are expressed having molecular weights of 127,000 and 130,000, respectively. These polypeptides retain low-affinity heparin-binding activity. High-affinity heparin binding is markedly diminished by mutations in either of two sequence motifs that include clusters of lysines and arginines.(ABSTRACT TRUNCATED AT 250 WORDS)

3T3 Cells↗
PubMed1992

Colocalization of thrombospondin and syndecan during murine development.

Thrombospondin is an adhesive glycoprotein that is thought to play a role in tissue genesis and repair. We have used a monoclonal anti-thrombospondin antibody, designated 5G11, to localize thrombospondin in paraformaldehyde fixed, paraffin-embedded sections of developing mouse embryos. Thrombospondin expression is observed in uterine smooth muscle, endometrial glands, the decidua, and trophoblastic giant cells during the initial phase of post-implantation development in the embryo. Cardiac myocytes and neuroepithelial cells show positive staining for thrombospondin at day 8.5 of gestation, and this expression continues throughout the development of the myocardium and central nervous system. Strong staining for thrombospondin is seen in developing bone and in the liver. Thrombospondin is also observed in developing smooth muscle and skeletal muscle, as well as in a variety of epithelia, including the epidermis, small intestinal epithelium, lens epithelium, renal tubular epithelium, and the epithelium of the developing tooth. Comparison of thrombospondin staining with that of two known cell surface receptors for thrombospondin, syndecan and the vitronectin receptor, reveals remarkable colocalization of thrombospondin and syndecan in all tissues, but almost no coexpression with the vitronectin receptor. Coexpression of thrombospondin and syndecan may play an important role in cell-cell or cell-matrix interactions during development.

Animals↗
PubMed1992

High intensity exercise training-induced metabolic alterations in respiratory muscles.

Limited information exists concerning the effects of high intensity interval exercise training (HIET) on metabolic alterations in both inspiratory and expiratory muscles. To test the hypothesis that HIET will improve the oxidative capacity of the diaphragm and major expiratory muscles, we examined Krebs cycle and beta oxidation enzyme activities in the diaphragm and three groups of expiratory (abdominal) muscles in rats subjected to 12 weeks (5 days.wk-1) of treadmill exercise. Two groups of female Sprague-Dawley rats (age ca 120 days) were studied: (1) HIET group (n = 10; animals performed 6 x ca 5-min running intervals.day-1 at ca 90-95% VO2max); (2) sedentary control group (n = 7). When compared to controls, HIET resulted in significantly elevated (P less than 0.05) activities of 3-hydroxy-acyl-Co-A dehydrogenase (HADH) and citrate synthase (CS) in the costal diaphragm, rectus abdominus, external obliques, and the plantaris muscles. In contrast, training did not increase (P greater than 0.05) the activities of CS or HADH in the crural diaphragm or the internal obliques/transversus abdominus muscles. By comparison, the training-induced increases in oxidative capacity (e.g., CS activity) in the costal diaphragm, rectus abdominus, and external obliques were relatively small (ca 23, 10, 12%, respectively) when contrasted to the exercise-induced increase in CS activity in the plantaris muscle (ca 47%). We conclude that HIET results in small but significant improvements in the oxidative and beta oxidation capacities of the costal diaphragm and at least two abdominal expiratory muscles.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1992

Modulation of tyrosine hydroxylase gene expression in the rat adrenal gland by exercise: effects of age.

Both aging and exercise are associated with alterations in circulating levels of catecholamines. To determine the interactions of age and exercise on tyrosine hydroxylase (TH) activity and TH mRNA, Fischer-344 female rats aged 5 months (young) and 25 months (old) were trained by treadmill running for 10 weeks. The elevation in maximum oxygen consumption in both groups was equivalent following exercise, indicating that training had occurred. In control rats, both TH activity and TH mRNA were greater in the older groups when compared with the younger animals. In young rats, exercise decreased TH activity by 25% and TH mRNA by 27%. In older rats, exercise was not associated with a decrease in TH activity and TH mRNA. Choline acetyltransferase activity (ChAT) was decreased and glutamic acid decarboxylase activity (GAD) was increased by exercise in young rats. The decrease in ChAT activity and increase in GAD activity suggest that trans-synaptic mechanisms play a role in the exercise-induced alteration of TH gene expression. Neither ChAT nor GAD was altered by exercise in older groups. Our data suggest that the previously reported diminution in catecholamines associated with exercise may be due to a decrease in TH mRNA and a resulting decrease in TH activity. There was no effect of exercise in the old rats, supporting previous observations that the plasticity of the sympathoadrenal system diminishes with age.

Acetylcholine↗
PubMed1992

Exercise training-induced alterations in skeletal muscle oxidative and antioxidant enzyme activity in senescent rats.

Limited data exist concerning exercise training-induced alterations in skeletal muscle oxidative and antioxidant enzyme activity in senescent animals. Therefore, the purpose of this study was twofold: 1) to examine the exercise training-induced changes in oxidative and antioxidant enzyme activity in skeletal muscle of old rats; and 2) to critically analyze the relationship between oxidative and antioxidant enzyme activities in skeletal muscle in both trained and untrained senescent rats. Female Fischer-344 rats (approximately 24-mo-old) were divided into 1) exercised trained (ET; n = 10) and 2) sedentary (S; n = 6) groups. The ET rats performed a 10-week training program of treadmill exercise (approximately 60 min, 5 days/wk). Training significantly (p less than 0.05) improved VO2max (delta 22.8%) in the ET rats above their age-matched controls. Further, the ET group had significantly elevated (p less than 0.05) activities of succinate dehydrogenase (SDH) in the soleus and red gastrocnemius (RG) muscles as well as greater (p less than 0.05) 3-hydroxyacyl-CoA dehydrogenase (HADH) activity in the RG when compared to the S group. However, training did not alter (p greater than 0.05) HADH activity within the white gastrocnemius (WG) or soleus muscles. Activity of the antioxidant enzyme, glutathione peroxidase (GPX) was higher (p less than 0.05) in the soleus and RG in ET rats when compared to the S rats; in contrast, training did not alter (p less than 0.05) GPX activity in the WG. Finally, the correlation coefficients between SDH and GPX activities (combined ET and S groups) for the RG, WG, and soleus muscles were r = .73, .17 and .36, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1992

Aging and respiratory muscle metabolic plasticity: effects of endurance training.

We examined the oxidative and antioxidant enzyme activities in respiratory and locomotor muscles in response to endurance training in young and aging rats. Young adult (4-mo-old) and old (24-mo-old) female Fischer 344 rats were divided into four groups: 1) young trained (n = 12), 2) young untrained (n = 12), 3) old trained (n = 10), and 4) old untrained (n = 6). Both young and old endurance-trained animals performed the same training protocol during 10 wk of continuous treadmill exercise (60 min/day, 5 days/wk). Compared with young untrained animals, the young trained group had significantly elevated (P less than 0.05) activities of 3-hydroxyacyl-CoA dehydrogenase (HADH), glutathione peroxidase (GPX), and citrate synthase (CS) in both the costal diaphragm and the plantaris muscle. In contrast, training had no influence (P greater than 0.05) on the activity of lactate dehydrogenase within the costal diaphragm in young animals. In the aging animals, training did not alter (P greater than 0.05) activities of CS, HADH, GPX, or lactate dehydrogenase in the costal diaphragm but significantly (P less than 0.05) increased CS, HADH, and GPX activities in the plantaris muscle. Furthermore, training resulted in higher activities of CS and HADH in the intercostal muscles in the old trained than in the old untrained animals. Finally, activities of CS, HADH, and GPX were significantly (P less than 0.05) lower in the plantaris in the old untrained than in the young untrained animals; however, CS, HADH, and GPX activities were greater (P less than 0.05) in the costal diaphragm in the old sedentary than in the young untrained animals.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1992

Alterations in diaphragmatic oxidative and antioxidant enzymes in the senescent Fischer 344 rat.

We investigated age-related changes in antioxidant, glycolytic, beta-oxidation, and tricarboxylic acid cycle enzyme activity in the diaphragm and plantaris muscle of female Fischer 344 rats. Tissue samples from the costal and crural diaphragm and plantaris muscle were obtained from 30 animals in the following age groups: 1) 6 mo old (n = 10), 2) 26 mo old (n = 10), and 3) 30 mo old (n = 10). Aging had no effect (P greater than 0.05) on the activities of citrate synthase (CS) and 3-hydroxyacyl-CoA dehydrogenase (HADH) in the costal or crural diaphragm. Similarly, no age-related differences existed (P greater than 0.05) in the crural diaphragm in lactate dehydrogenase (LDH) or glutathione peroxidase (GPX) activity. In contrast, the activities of LDH and GPX were significantly (P less than 0.05) higher in the costal diaphragm in the 30- than in the 6-mo old animals. In addition, the ratio of LDH to CS activity increased (P less than 0.05) as a function of age in the costal diaphragm. Conversely, the ratio of CS to GPX activity in the costal diaphragm was lower (P less than 0.05) in the 30- than in the 6-mo old animals. No significant (P greater than 0.05) age-related differences existed in LDH-to-CS or CS-to-GPX activity ratios in the crural diaphragm. Finally, aging resulted in a significant decrease (P less than 0.05) in the activities of LDH, CS, and HADH in the plantaris muscle. These data demonstrate that, unlike many hindlimb locomotor muscles, the oxidative capacity of the Fischer 344 rat diaphragm does not decrease in old age.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1992

Endurance training-induced increases in expiratory muscle oxidative capacity.

Recent evidence demonstrates that endurance exercise training improves the oxidative capacity of the major mammalian inspiratory muscle (e.g., costal diaphragm). In contrast, no data exist concerning the effects of exercise training on abdominal expiratory muscles. We tested the hypothesis that 12 wk of endurance exercise training would significantly increase the activity of selected beta oxidation and Krebs cycle enzymes of abdominal expiratory muscles of the rat. To test this hypothesis two groups of female Sprague-Dawley rats were studied: group 1, continuous exercise training (n = 13); and group 2, sedentary control (n = 6). Exercise trained animals ran 5 d.wk-1 on a motorized treadmill for 45 min.d-1 at approximately 75-80% VO2max. When compared with controls, exercise training resulted in elevated (P less than 0.05) activities of 3-hydroxy-acyl-Co-A dehydrogenase (HADH) and citrate synthase (CS) in two abdominal expiratory muscles (rectus abdominus and external obliques). In contrast, training did not alter (P greater than 0.05) CS or HADH activity in the internal obliques/transversus abdominus muscles. In general, the training-induced increases in expiratory muscles CS activity were relatively small (approximately 10-13%) when compared with the training-induced increase in CS activity in the plantaris muscle (approximately 44%). These data demonstrate that continuous exercise training results in small but statistically significant improvements in the oxidative and beta oxidation capacities of expiratory muscles.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1991

Characterization of the murine thrombospondin gene.

Thrombospondin is an adhesive glycoprotein that supports cell attachment, spreading, and migration. The murine thrombospondin gene is approximately 18 kb in length and includes 22 exons. Interspecific backcross analysis using progeny derived from matings of (C57BL/6J x Mus spretus) F1 x C57BL/6J mice indicates that the thrombospondin gene is tightly linked to the Fshb, Actcl, Ltk, and B2M loci on murine chromosome 2. The sequence of the murine gene is very similar to that of the human gene in (1) regions of the promoter, (2) the coding region, and (3) the 3'-untranslated region. The predicted amino acid sequence of the mature murine thrombospondin subunit is 95.1% identical to that of the human. The sequences of these two species are most similar at the regions containing the type 1, 2, and 3 repeats as well as the COOH-terminal globular domain. The thrombospondin promoter is similar to the 5' flanking region of some housekeeping and growth control genes in that it contains multiple GC-rich regions and lacks a CAAT box. The presence of various consensus sequences suggests that thrombospondin gene expression is regulated by cAMP, cytokines, and steroid hormones.

Amino Acid Sequence↗
PubMed1991

Evidence for an alveolar-arterial PO2 gradient threshold during incremental exercise.

Examination of the alveolar-to-arterial O2 tension (A-a PO2 difference) provides a method of examining the efficiency of pulmonary gas exchange during exercise. At present, considerable confusion exists as to the exact pattern of the A-a PO2 difference during incremental exercise. We tested the hypothesis that the A-a PO2 difference during incremental exercise is alinear with respect to metabolic rate. Measurements of the A-a PO2 difference were made on six healthy male subjects during incremental exercise under sea level conditions (PIO2 = 149 torr). An alinear model best described the relationship between the A-a PO2 difference and metabolic rate; only small increases in the A-a PO2 difference occurred at low work rates followed by a rapid increase at higher work rates. The existence of a "A-a PO2 difference threshold" was mathematically confirmed by the use of a computer algorithm to define inflection points. These data provide evidence that the relationship between the A-a PO2 difference and metabolic rate is alinear and that a metabolic threshold exists for a rapid increase in the A-a PO2 difference. We conclude that the efficiency of pulmonary gas exchange during exercise is unaltered from rest during low-to-moderate power outputs, however, high intensity exercise compromises pulmonary gas exchange efficiency as evidenced by a significant widening of the A-a PO2 difference.

Adult↗
PubMed1991

Influence of a carbohydrate-electrolyte beverage on performance and blood homeostasis during recovery from football.

This study compared the efficacy of a 7% glucose polymer beverage containing electrolytes (GP) versus a nonnutrient, nonelectrolyte placebo (P) in maintaining blood homeostasis during recovery from football and determined whether consumption of the GP beverage improved anaerobic performance immediately after football competition when compared with the placebo. Forty-four high school football players participated in a 50-play scrimmage designed to simulate game conditions. At each of six periods before and during the scrimmage, players consumed 170 ml of the GP or P beverage. Eight maximal-effort 40-yd sprints (40-sec rest intervals) were performed before and after the scrimmage to assess the decrement in anaerobic performance from the scrimmage. Venous blood samples were drawn before and after the scrimmage and analyzed. The pre- to postscrimmage differences in mean and peak sprint velocities did not differ between treatments, nor did body weight and plasma. In contrast, the percent decrease in plasma volume was significantly greater in the P group. Postscrimmage increases in glucose and insulin were greater in the GP group. These data suggest that CHO-electrolyte drinks do not prevent a decline in anaerobic performance when compared to water, but a CHO-electrolyte drink is more effective in maintaining PV than water during recovery from anaerobic exercise.

Adolescent↗
PubMed1991

Physiological correlates to 800 meter running performance.

Much of the previous research efforts aimed at determining those physiological characteristics that contribute to distance running success have centered around distances greater than 1500 meters with little attention to events such as the 800 meter run. Therefore, this investigation examined the relationship between selected physiological and body composition, characteristics and performance in an 800 meter run. Measurements of body composition, VO2max, running economy, and performance times for 100 and 300 meter dashes were obtained on 11 male track athletes. Stepwise multiple regression analysis was performed using 800 meter race time as the dependent variable. Although the combination of 300 and 100 meter run times, percent body fat, running economy and VO2 max as independent variables accounted for the greatest amount of total variance (r2 = .89), the additional variance explained by the model did not increase significantly (p greater than 0.05), when VO2max, percent body fat, and running economy were added to a model which contained 300 and 100 meter run time (r2 = .85) as the explanatory variables. These data offer additional support for the notion that much of the intramuscular ATP produce and utilized during an 800 meter run comes from anaerobic metabolic pathway.

Adult↗
PubMed1990

cDNA sequence for human erythrocyte ankyrin.

The cDNA for human erythrocyte ankyrin has been isolated from a series of overlapping clones obtained from a reticulocyte cDNA library. The composite cDNA sequence has a large open reading frame of 5636 base pairs (bp) with the complete coding sequence for a polypeptide of 1879 amino acids with a predicted molecular mass of 206 kDa. The derived amino acid sequence contained 194 residues that were identical to those obtained by direct amino acid sequencing of 11 ankyrin proteolytic peptides. The primary sequence contained 23 highly homologous repeat units of 33 amino acids within the 90-kDa band 3 binding domain. Two cDNA clones showed evidence of apparent mRNA processing, resulting in the deletions of 486 bp and 135 bp, respectively. The 486-bp deletion resulted in the removal of a 16-kDa highly acidic peptide, and the smaller deletion had the effect of altering the COOH terminus of the molecule. Radiolabeled ankyrin cDNAs recognized two erythroid message sizes by RNA blot analysis, one of which was predominantly associated with early erythroid cell types. An ankyrin message was also observed in RNA from the human cerebellum by the same method. The ankyrin gene is assigned to chromosome 8 using genomic DNA from a panel of sorted human chromosomes.

Amino Acid Sequence↗