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Biomedical subjects

R M Smith

Publications and source records attributed to R M Smith.

At least 181 records · Page 10Linked to original sources
PubMed1995

Demonstration of specific insulin binding to cytosolic proteins in H35 hepatoma cells, rat liver and skeletal muscle.

We previously demonstrated that internalized insulin enters the cytoplasm before accumulating in nuclei of H35 rat hepatoma cells. This finding raises the possibility that insulin may interact with cytosolic proteins in addition to insulin-degrading enzyme (IDE). In the present study, cytosol from H35 hepatoma cells, rat liver or muscle was incubated with A14- or B26-125I-insulin at 4 degrees C for 5-120 min in the absence or presence of 25 micrograms/ml unlabelled insulin. 125I-insulin was cross-linked to cytosolic proteins by disuccinimidyl suberate and analysed by reducing or non-reducing SDS/PAGE and autoradiography. Our results demonstrate the presence of both tissue-specific and common cytosolic proteins which specifically bind insulin. In muscle cytosol, only two proteins of 27 and 110 kDa were specifically labelled with B26-125I-insulin. Seven major bands, of 27, 45, 55, 60, 76, 82 and 110 kDa, were labelled in rat liver cytosol. Detection of cytosolic insulin-binding proteins in H35-cell cytosol was dependent on cell-culture conditions. Labelling in cytosol from serum-deprived cells was decreased or absent compared with cytosol prepared from serum-fed or serum-deprived cells treated with 100 ng/ml insulin for 1 h before preparation of the cytosol, in which six bands, of 32, 41, 45, 55, 82 and 110 kDa, were specifically labelled with B26-125I-insulin. This result suggests that the concentration or binding activity of some cytosolic insulin-binding proteins is rapidly regulated. Labelling of both rat liver and H35 cytosolic insulin-binding proteins was time-dependent, and decreased or disappeared at 120 min in parallel with the degradation of labelled insulin. Fewer bands were specifically labelled with A14-125I-insulin than with B26-125I-insulin. The number of labelled bands observed under reducing and non-reducing conditions was not different in any of the cytosols. The 110 kDa band in all cytosols was identified as IDE by Western-blot analysis; the other proteins did not react with anti-IDE antibody and remain unidentified. 1,10-Phenanthroline (2 mM) increased IDE labelling, but decreased the labelling of 82 and 27 kDa bands. The marked difference in the number of cytosolic insulin-binding proteins in muscle and either H35 cells or liver suggests both that the labelling is specific and that these proteins serve a function and may be involved in some heretofore unknown mechanism of the signalling pathway by which insulin regulates cell growth or differentiation.

Animals↗
PubMed1995

Low avidity recognition of self-antigen by T cells permits escape from central tolerance.

The immunodominant epitope of myelin basic protein, Ac1-9, is encephalitogenic in H-2u mice. We have previously demonstrated that this epitope displays low affinity for I-Au and have suggested that the avidity of T cell recognition in the thymus may be compromised, enabling autoreactive T cells to escape self-tolerance. We have addressed this hypothesis directly by constructing transgenic mice expressing an encephalitogenic T cell receptor (TCR). Parenteral administration of Ac1-9 had no discernable impact on developing thymocytes. In contrast, peptide analogs displaying far higher affinity for I-Au, provoked deletion of CD4+ CD8+ cells and transient down-regulation of the TCR by mature CD4+ CD8- thymocytes. The use of analogs of intermediate affinity permitted a margin of error to be defined for the induction of tolerance and confirmed that the affinity of Ac1-9 lies well below the critical threshold.

Animals↗
PubMed1995

Discriminant analysis of biomechanical differences between novice, good and elite rowers.

Oar force and oar angle data resulting from a 6-min maximal rowing ergometer test undertaken by novice (n = 9), good (n = 23) and national (n = 9) level male rowers, were used to identify biomechanical performance variables which accurately discriminated between rowers of differing ability levels. The variables included two work capacity measures, mean propulsive power output per kilogram of body mass (W kg-1) and propulsive work consistency (%), and two skill variables, stroke-to-stroke consistency (%) and stroke smoothness (%). Discriminant function analysis indicated the presence of two functions, both of which clearly indicated the importance of mean propulsive power output per kilogram of body mass as a discriminating variable. Function 2 gave greater weight to stroke-to-stroke consistency and stroke smoothness than function 1; however, function 1 was the most powerful discriminator. Classification procedures were used to predict the ability level to which a rower most likely belonged and involved defining the 'distance' between each rower and each ability level centroid, with the rower being classified into the 'nearest' ability level. These procedures indicated that 100% of the elite, 73.9% of the good, 88.9% of the novice and 82.9% of all rowers were correctly classified into their respective skill levels. Stepwise discriminant analysis included the variables in the following order: mean propulsive power output per kilogram of body mass, stroke-to-stroke consistency, stroke smoothness and propulsive work consistency (P < 0.001). The results of this study indicate that biomechanical performance variables related to rowing capacity and skill may be identified and used to discriminate accurately between rowers of differing skill levels, and that, of these variables, propulsive work consistency is the least effective discriminator.

Adult↗
PubMed1995

Relining an ocular prosthesis: a case report.

There are times when a patient who has an ocular prosthesis may need surgery to correct an anophthalmic socket defect or to replace an implant. When such a situation arises, it may not be possible to construct a new prosthesis soon after surgery. In this article, a technique for relining an ocular prosthesis is described.

Acrylic Resins↗
PubMed1995

Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides.

An enhanced appreciation of uptake mechanisms and intracellular trafficking of phosphorothioate modified oligodeoxynucleotides (P-ODN) might facilitate the use of these compounds for experimental and therapeutic purposes. We addressed these issues by identifying cell surface proteins with which P-ODN specifically interact, studying P-ODN internalization mechanisms, and by tracking internalized P-ODN through the cell using immunochemical and ultrastructural techniques. Chemical cross-linking studies with a biotin-labeled P-ODN (bP-ODN), revealed the existence of five major cell surface P-ODN binding protein groups ranging in size from approximately 20-143 kD. Binding to these proteins was competitively inhibited with unlabeled P-ODN, but not free biotin, suggesting specificity of the interactions. Additional experiments suggested that binding proteins likely exist as single chain structures, and that carbohydrate moieties may play a role in P-ODN binding. Uptake studies with 35S-labeled P-ODN revealed that endocytosis, mediated by a receptor-like mechanism, predominated at P-ODN concentrations < 1 microM, whereas fluid-phase endocytosis prevailed at higher concentrations. Cell fractionation and ultrastructural analysis demonstrated the presence of ODN in clathrin coated pits, and in vesicular structures consistent with endosomes and lysosomes. Labeled ODN were also found in significant amounts in the nucleus, while none was associated with ribosomes, or ribosomes associated with rough endoplasmic reticulum (ER). Since nuclear uptake was not blocked by wheat germ agglutinin or concanavalin A, a nucleoporin independent, perhaps diffusion driven, import process is suggested. These data imply that antisense DNA may exert their effect in the nucleus. They also suggest rational ways to design ODN which might increase their efficiency.

Biological Transport↗
PubMed1995

Inhibitory effect of porcine surfactant on the respiratory burst oxidase in human neutrophils. Attenuation of p47phox and p67phox membrane translocation as the mechanism.

Surfactant has been shown to inhibit the production of reactive oxygen intermediates by various cells including alveolar macrophages and peripheral blood neutrophils. Superoxide O2-. production by the respiratory burst oxidase in isolated plasma membranes prepared from PMA-treated human neutrophils was significantly attenuated by prior treatment with native porcine surfactant. The effect was concentration dependent with half-maximal inhibition seen at approximately 0.050 mg surfactant phospholipid/ml. Kinetic analyses of the membrane-bound enzyme prepared from neutrophils stimulated by PMA in the presence or absence of surfactant demonstrated that surfactant treatment led to a decrease in the maximal velocity of O2-. production when NADPH was used as substrate, but there was no effect on enzyme substrate affinity. Immunoblotting studies demonstrated that surfactant treatment induced a decrease in the association of two oxidase components, p47phox and p67phox, with the isolated plasma membrane. In contrast, surfactant treatment of the cells did not alter the phosphorylation of p47phox. A mixture of phospholipids (phosphatidylcholine and phosphatidylglycerol in a 7:3 ratio) showed similar inhibition of the PMA-induced O2-. generation. Taken together, these data suggest the mechanism of surfactant-induced inhibition of O2-. production by human neutrophils involves attenuation of translocation of cytosolic components of the respiratory burst oxidase to the plasma membrane. The phospholipid components of surfactant appear to play a significant role in this mechanism.

Animals↗
PubMed1995

Electron microscopic visualization of insulin translocation into the cytoplasm and nuclei of intact H35 hepatoma cells using covalently linked Nanogold-insulin.

Insulin affects numerous metabolic processes as well as nuclear events such as gene transcription. Our previous ultrastructural and biochemical studies demonstrated insulin accumulation in nuclei of cultured and rapidly proliferating cells, and biochemical evidence suggested that insulin entered the cell cytoplasm before accumulating in the nucleus. The present study was undertaken to develop a covalently linked electron-dense insulin complex that could be used to visualize the intracellular translocation of insulin and confirm that insulin enters the cytoplasm of cells. Insulin was cross-linked to 1.4-nm diameter Nanogold particles. The complex binds to the plasma membrane insulin receptor, is biologically active, and is degraded by cellular insulin-degradative enzymes. Ultrastructural analysis after silver intensification of the gold particles confirmed that insulin internalization culminates in the translocation of some internalized insulin to the cytoplasm and nuclei. When cytoplasmic insulin-degrading enzyme (IDE) activity was inhibited with 1,10-phenanthroline, an increase in the number of cytoplasmic and nuclear Nanogold-insulin particles was observed. The results of this and previous studies suggest that 1) the translocation of insulin to the cytoplasm, 2) the regulation of insulin degradation in the cytoplasm by IDE, 3) the possible interaction of insulin with cytoplasmic proteins other than IDE, and 4) the subsequent accumulation of intact insulin or insulin complexed with cytoplasmic proteins in nuclei may play a role in insulin's regulation of gene transcription and cell proliferation.

Adipose Tissue↗
PubMed1995

Fish oils modulate blood pressure and vascular contractility in the rat and vascular contractility in the primate.

The effect of dietary fish oils on development of hypertension and vascular response in vitro were studied in rats and a primate. Dietary fish oils (MaxEPA and an n-3 ethyl ester concentrate of higher EPA and DHA content) were administered to spontaneously hypertensive (SHR), stroke-prone spontaneously hypertensive (SHR-SP) and a backcross of SHR and Wistar Kyoto (SHR/WKY) rats from 4-16 weeks of age. Blood pressure was monitored during the feeding period and vascular responses measured in the aorta and mesenteric vascular bed in vitro. Depending on the strain of rat used and the composition of the fish oil the attenuation in blood pressure was 10-26 mmHg. Fish oils attenuated the response mediated by sympathetic nerve stimulation or intralumenal norepinephrine in the perfused mesenteric vascular bed preparation from the SHR. This attenuation was more pronounced for fish oils enriched with eicosapentaenoic acid and docosahexaenoic acid and was more prominent in the SHR and SHR/WKY backcross than it was in the SHR-SP. Prostanoid synthesis or nitric oxide modulation of alpha-adrenoceptor responses were shown not to be involved in the attenuation of vascular responses produced by fish oil. The maximum contraction of aortic ring preparations in response to norepinephrine (NE) was significantly smaller in SHR than WKY rats fed olive oil and for SHR rats maintained on fish oils the contraction was close to WKY olive oil values. Evidence was obtained also for a modulation of vasoconstrictor responses by dietary fish oils in the perfused mesenteric bed of the marmoset monkey.

Animals↗
PubMed1995

Nebulized bacteriostatic saline as a cause of bronchitis.

BACKGROUND: The purpose of this study was to determine whether nebulized bacteriostatic saline, which contains the preservative benzyl alcohol, is an irritant to the tracheobronchial mucosa in healthy adults. METHODS: A randomized, double-blind, placebo-controlled study was conducted with 10 healthy adults who inhaled 3 mL of either bacteriostatic saline or saline placebo by nebulizer four times a day for 2 weeks. Pulmonary function tests and bronchoscopy with biopsy were performed immediately before and after the 2-week nebulization period. RESULTS: Nine of 10 volunteers were compliant with the study protocol. Four of five volunteers who used nebulized bacteriostatic saline developed bronchitic symptoms and had bronchoscopic evidence of bronchitis. The fifth remained healthy. Four volunteers used nebulized saline (placebo). Two of these four became ill, one with a much more severe bronchitis than any of the bacteriostatic saline volunteers, and one with pharyngitis. Bronchoscopic biopsies showed a lymphocytic mucosal infiltrate in those who became ill while using nebulized bacteriostatic saline and a polymorphonuclear mucosal infiltrate in those who became ill while using nebulized saline placebo. CONCLUSIONS: Nebulization of bacteriostatic saline, containing benzyl alcohol as its preservative, causes bronchitis in healthy adults. Even nebulization of sterile saline may be associated with bronchitis and pharyngitis. Physicians who prescribe nebulized medications must pay close attention to the bronchodilator diluent.

Adult↗
PubMed1994

Elevation of serum creatine kinase in divers with arterial gas embolization.

BACKGROUND: Arterial gas embolism due to pulmonary barotrauma and the resultant cerebral gas embolism are catastrophic complications of diving. Previous studies have only rarely noted evidence of gas embolism to noncranial sites. METHODS: Among 142 persons with diving-related injuries evaluated between January 1982 and July 1991, we identified 29 who had arterial gas embolism and who underwent biochemical studies indicative of muscle injury. Of the 29 patients, 4 were excluded because cardiopulmonary resuscitation had been performed and 3 were excluded because the duration of their dives met or exceeded standard limits set for dives not requiring staged decompression. The outcome at the time of hospital discharge in the remaining 22 patients was correlated with clinical factors and the results of biochemical studies. We also studied 22 subjects after uncomplicated dives and 11 patients who had sustained blunt trauma. RESULTS: All the patients with diving-associated gas embolism had elevated serum creatine kinase activity (normal, < or = 175 U per liter); the values were markedly elevated (> 900 U per liter) in 14. The MB isoenzyme of creatine kinase was detected in the serum of 13 of 20 patients in whom it was measured and was > or = 4 percent of total creatine kinase activity in 6 patients. In three patients electrocardiography showed myocardial injury. Changes in serum creatine kinase activity of similar magnitude were not present in the subjects who had uncomplicated dives or in the patients with blunt trauma. Thirteen patients recovered fully, four had minor residual neurologic deficits, three were severely impaired, and two died. Logistic-regression analysis revealed a significant correlation between peak serum creatine kinase values and clinical outcome. CONCLUSIONS: Biochemical evidence of muscle injury is frequently found after diving-associated arterial gas embolism. The correlation between serum creatine kinase activity and outcome suggests that serum creatine kinase is a marker of the size and severity of arterial gas embolism.

Adolescent↗
PubMed1994

1,10-Phenanthroline increases nuclear accumulation of insulin in response to inhibiting insulin degradation but has a biphasic effect on insulin's ability to increase mRNA levels.

Previous reports demonstrated that insulin is translocated through the cytoplasm to the nucleus of H35 hepatoma cells and suggested that nuclear insulin may be involved in stimulating transcription of immediate-early genes. In a recent study, inhibition of insulin-degrading enzyme with 1,10-phenanthroline, a Zn2+ chelator, caused a significant increase in the nuclear accumulation of insulin. The present study characterized the effects of 1,10-phenanthroline and its nonchelating isomer, 1,7-phenanthroline, on insulin degradation, nuclear accumulation, and stimulation of immediate-early gene expression. 1,10- but not 1,7-phenanthroline inhibited insulin degradation and increased nuclear accumulation of insulin in a dose-dependent manner. 1,7-phenanthroline caused a dose-dependent decrease in the expression of insulin-stimulated immediate-early genes, but had no significant effect on alpha-tubulin mRNA levels. In the presence of insulin, Northern analysis revealed that 1,10-phenanthroline at all concentrations tested increased alpha-tubulin mRNA levels, but had a biphasic effect on insulin-stimulated immediate-early gene expression. At low concentrations (5-200 microM), 1,10-phenanthroline increased the expression of insulin-stimulated g33, c-fos, and Egr-1 mRNA. At concentrations greater than 1 mM, insulin-stimulated immediate-early gene expression was decreased similar to the effect seen with 1,7-phenanthroline. Nuclear run-on analysis demonstrated that high concentrations of 1,10-phenanthroline decreased insulin-stimulated immediate-early gene transcription but had no effect on transcription of alpha-tubulin. However, low concentrations of 1,10-phenanthroline did not increase transcription of any genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Thiamin deficiency impairs endotoxin-induced increases in hepatic glucose output.

We addressed the role of thiamin, a cofactor for several enzymes involved in glucose metabolism, in the glucose metabolic response to endotoxin. Characterized by hyperglycemia, increased hepatic glucose production exceeding elevated rates of whole-body glucose utilization, this response is mediated by hormones and cytokines and is dependent on the immune and nutritional status of the host. We hypothesized that a thiamin-deficient state would impair the metabolic response to endotoxin. Rats were fed a thiamin-deficient or control diet for 6 wk before in vivo assessment of glucose kinetics. In control rats, Escherichia coli endotoxin increased the rate of glucose appearance (+76%), disappearance (+70%), and metabolic clearance (+50%). Thiamin deficiency resulted in increased plasma glucose (18%) and lactate (3- to 4-fold) as well as in a 30% decrease in insulin and an increase in glucagon (2.6-fold) and corticosterone (3.6-fold). Thiamin deficiency inhibited the endotoxin-induced hyperglycemia and the rise in hepatic glucose production, glucose utilization, and metabolic clearance rate.

Animals↗