Biomedical subjects
Publications and source records attributed to S Cohen.
This review summarizes briefly some of the historical aspects of the discovery, isolation, and mode of action of epidermal growth factor (EGF). Also considered are some of the key experiments which relate the biochemical mechanisms of EGF action to the mechanism involved in oncogenic transformation by the retroviruses.
The protein kinase associated with the purified epidermal growth factor (EGF) receptor from membrane (Mr = 150,000) or vesicle (Mr = 170,000) preparations of A-431 cells was shown to catalyze the phosphorylation of the peptide Leu-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly at the tyrosine residue. EGF enhanced peptide phosphorylation by 3-5-fold. The steady state kinetic analysis of the purified kinase from membranes showed that the reaction mechanism was of the sequential type in either the presence of absence of EGF. Thus, the peptide and ATP must bind to the enzyme before any product is released. Both neurotensin 8-13 and kyotorphin were inhibitors but not substrates of the protein kinase. Kyotorphin was a linear noncompetitive inhibitor with ATP as the variable substrate and a linear competitive inhibitor with peptide as the variable substrate. ADP, a product of the kinase reaction, was a linear noncompetitive inhibitor with respect to ATP and a linear competitive inhibitor with respect to peptide. Based on these data, it can be suggested that the tyrosine protein kinase from A-431 cells catalyzes a Ordered Bi Bi reaction where peptide is the first substrate to bind and ADP is the last product to be released.
A rapid and simple method is described to determine the orientation of isolated plasma membrane vesicle populations. The method is based on quantitation of the exposure of actin, an extrinsic membrane component bound exclusively to the cytoplasmic surface. Actin accessibility was determined using DNase I as a nonpermeating probe. The activity of this enzyme is lost upon complexation with actin, and inhibition can be conveniently monitored as the change in the rate of DNase-mediated hyperchromic reaction. Exposed actin is determined in intact vesicles, and total actin in detergent-lysed membranes; the ratio of these values equals the sum of inside-out and leaky vesicles. Sealed right-side-out vesicles are calculated by difference. Using thymocyte plasma membrane vesicles as a model system, we determined that 54% of the membrane is sealed in a right-side-out configuration. This value is consistent with independent determinations of inside-out membranes in the same preparation, obtained by 5'-nucleotidase latency measurements, and with reported values in other lymphocyte membrane preparations.
Cytoplasmic extracts, prepared from continuously proliferating lymphoblastoid cells, as well as mitogen-activated normal lymphocytes, contain an extractable factor capable of inducing DNA synthesis in isolated quiescent nuclei. This factor is not detectable in resting cells. It is nondialyzable, precipitable by 30-50% saturated ammonium sulfate, and inactivated by trypsin. It is heat sensitive, but stable to cold and lyophilization. The molecular weight of the factor is greater than 100,000. This cytoplasmic activator of nuclear DNA replication is not released from the cell, and has no effect on intact cells. This suggests that it serves as an intracellular mitogenic signal in replicating cells.
The aggregations of protoporphyrin IX and haematoporphyrin IX in aqueous solutions were studied by fluorimetric techniques. Porphyrin concentrations were limited to 0.001-0.1 microM and 0.01-1 microM for protoporphyrin and haematoporphyrin respectively, where dimerization is the dominant aggregation process. The dimerization equilibrium constants (at 25 degrees C, neutral pH, 50 mM-Tris/HCl buffer) were determined to be 3 X 10(7) M and 4 X 10(5) M for the proto and the haemato derivatives respectively. The fluorescence intensity of a given protoporphyrin solution (within the range indicated above) was markedly decreased by salts in the system, over the salt concentration range 0.1-7 mM at constant ionic strength, in the sequence CaCl2 greater than MgCl2 greater than KCl greater than NaCl. The direction of this effect, fluorescence quenching, suggests that these salts promote an increase in aggregation. The differences in the magnitudes of the effect, among different salt species sharing a common anion, at constant ionic strength, imply that the effect is cation-specific. In contrast, the fluorescence intensity of a given solution of haematoporphyrin (within the range indicated above) was unaffected by these salts, under similar concentrations, nor was it sensitive to the total buffer concentration, or to the type of buffer in the system.
Sera from patients with progressive systemic sclerosis were compared with sera from normal individuals and from patients with other connective tissue diseases for cytotoxic effects on cultured human cells. More than 40% of the sera from patients with active progressive systemic sclerosis were cytotoxic by several criteria for pulmonary arterial or umbilical venous endothelial cells, foreskin fibroblasts, and neuroblastoma cells. Cytotoxic sera caused morphologic changes, uptake of trypan blue dye, and a decrease in the incorporation of 3H-thymidine into DNA. In contrast, only 4 sera from normal individuals or patients with other rheumatic diseases affected cell morphology, staining, or uptake of 3H-thymidine. Partial characterization of the cytotoxic factor indicated that it is sensitive to proteolysis by trypsin. The molecular weight of the factor was estimated to be similar to that of albumin.
Cell swelling and elevated intracellular Ca2+ increase K+ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low-K+ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K+ (86Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca2+ also increased 86Rb fluxes. Ca2+- and volume-induced 86Rb fluxes were also studied in isolated membranes. A plasma membrane-rich vesicle fraction, enriched over 11-fold in 5'-nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside-out and trapped 86Rb in an osmotically active compartment of approximately 1.3 microliter/mg protein. Equilibrium exchange fluxes of 86Rb in the vesicles were unaffected by Ca2+ with or without A23187. Calmodulin had no effect on 86Rb permeability but stimulated ATP-dependent Ca2+ accumulation. Hypotonic swelling increased both uptake and efflux of 86Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K+ (86Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume- and Ca2+-induced changes in K+ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD.
A procedure is described for the simultaneous determination of molal volumes (v02) and solubility parameters (delta) of compounds of medicinal interest. These include alkanoic acids of various chain length and branching (some solid at room temperature), cholesterol, and cholesteryl esters. The procedure is based on the determination of partial molal volumes (v2) from high-precision density measurements of dilute solutions of these compounds in reference solvents, which range in polarity from carbon tetrachloride (delta = 8.6) to nitrobenzene (delta = 10.0). In some cases, the present results do not agree with values of delta published in the literature. Values calculated from group contributions proposed by other authors are prone to error particularly in the case of branched acids and cholesteryl esters.
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Nine patients with severe symptoms of diffuse esophageal spasm and lower esophageal sphincter dysfunction who were unresponsive to medical therapy and bougienage dilatation were treated by forceful pneumatic dilatation. Treatment with pneumatic dilatation in eight of the nine patients produced a marked improvement in dysphagia and regurgitation (average follow-up of 37.4 months). Esophageal motility performed up to three years (average 12.4 months) after clinically successful pneumatic dilatation revealed a decrease in lower esophageal sphincter pressure from 34.0 +/- 4.0 mm Hg (mean +/- standard error) to 19.2 +/- 2.7 mm Hg (P less than 0.01). There were no significant changes in either the percentage of lower esophageal sphincter relaxation or the type of esophageal motor pattern. We conclude from this study that pneumatic dilatation is an effective form of therapy for a select group of patients with severe symptomatic diffuse esophageal spasm with lower esophageal sphincter dysfunction who are unresponsive to conventional medical therapy.
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Alternate medications for the treatment of Tourette syndrome are required because haloperidol in some patients either may be ineffective or may cause disturbing side effects. Propranolol, a beta-adrenergic blocking agent, has been reported as effective, in uncontrolled trials, in ameliorating symptoms of tic disorder, tardive dyskinesia, and drug-induced extrapyramidal syndrome. Propranolol, in doses up to 120 mg per day, was administered to five patients with Tourette syndrome in a placebo-controlled study and was found ineffective in ameliorating symptoms of Tourette syndrome. Results underscored the importance of placebo-controlled investigation when evaluating the effects of drugs in Tourette syndrome.
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P. knowlesi parasites with a maximum age distribution of 3 h were metabolically labelled with [35S]methionine during 9 sequential non-overlapping intervals, from young rings to mature segmented schizonts. The proteins synthesised at the different stages were compared using sodium dodecyl sulphate-polyacrylamide gel electrophoresis; more than 40 polypeptides (Mr 20 000 to over 200 000) were identified in the different parasite preparations. The major polypeptides synthesised by rings and trophozoites of different ages were similar, but differences in minor polypeptides could always be recognised. At the onset of schizogony ring and trophozoite specific proteins ceased to be synthesised and proteins specific to schizogony emerged. In general, schizont-specific proteins were of higher molecular weight than ring stage proteins. Details of the morphological changes which occurred during the metabolic labelling episode permits correlation between parasite structure and synthesis of particular polypeptides. Comparison of parasite components metabolically labelled with [3H]glucosamine during defined periods of development also revealed stage-specific synthesis of glycoproteins. The extent to which proteins are altered after synthesis has been investigated by pulse-chase experiments.
Highly synchronised cultures of cloned Plasmodium falciparum (clone T9-94) were metabolically radiolabelled with [35S]methionine during eight consecutive non-overlapping intervals, while parasites developed from young rings to mature schizonts. Analysis of equal amounts of trichloroacetic acid precipitable radioactivity from each interval by sodium dodecyl sulphate-polyacrylamide gel electrophoresis fluorography allowed the stage specificity of protein synthesis to be investigated. More than forty polypeptides with molecular weights of 20 000 to 200 000 can be distinguished. While some proteins are synthesised throughout erythrocytic schizogony many are shown to be stage-specific. Among these are a range of high molecular weight proteins synthesised only during nuclear division. Detailed morphological information permits correlations to be made between synthesis of particular polypeptides and parasite structure.
We recently established a multidisciplinary center for the evaluation and treatment of male sexual dysfunction. The patients are seen in a single day by the endocrinologist, psychiatrist, psychologist, neurophysiologist and urologist. Laboratory studies include chemical and hormonal profiles, pudendal nerve latency testing and determination of the penile blood pressure. Nocturnal penile tumescence studies are done in selected cases. A diagnosis was made in 95 of 106 patients (90 per cent). The problem was psychogenic in 42 per cent of the patients, vascular and neurologic in 34 per cent, hormonal in 15 per cent, urologic in 7 per cent and medication-related in 2 per cent. The interdisciplinary approach to this problem allows for a more comprehensive and accurate evaluation, while providing a convenient service to the patient.
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Is stressor exposure necessary to produce "stress" effects, or can these effects result from stressor anticipation alone? The present research explores whether it is possible to obtain "stress responses" during and after the period in which stressor exposure is being anticipated. In the first study, the expectation of submerging one's hand in ice water resulted in decreased frustration tolerance and increased blood pressure when compared with control groups not expecting this stressor. A second study replicated and extended these results to show that the expectation of control over the stressor ameliorates the negative impact of stressor expectation. The second study also examined the aftereffects of expectations. Particularly, it found that despite being relieved of the expectation that they would immerse their hand in ice water, subjects who had expected stressor exposure had decreased frustration tolerance when compared with either subjects who had expected a nonstressful procedure or those who had expected to have control over stressor termination. A third study, using noise as the expected stressor, replicated both the aftereffect of the anticipation period and the moderation of that effect by perceived control. The discussion (a) focuses on the implications of this work for understanding why aftereffects occur and (b) proposes that previously observed stressor exposure effects may in fact be postexpectation effects.