The carbohydrate of human thrombin: Structural analysis of glycoprotein oligosaccharides by mass spectrometry

The carbohydrate structure of human thrombin has been determined by direct probe mass spectrometry of the oligosaccharides released by trifluoroacetolysis from the asialo glycoprotein. The free oligosaccharides were studied as permethylated and N-trifluoroacetylated oligosaccharide alditols. The structure was confirmed by sequential exoglycosidase digestion of intact thrombin and sugar and methylation analysis of the oligosaccharides by gas-liquid chromatography-mass spectrometry. The results indicate the following structure:

with Fuc present on only about 50% of the oligosaccharides.     

ADP- and epinephrine-elicited release of [3H]guanylylimododiphosphate from platelet membranes: Implications for receptor-Ni stoichiometry

Epinephrine-promoted release of [³H]guanylylimidodiphosphate ([³H]Gpp(NH)p) from human platelet membranes has been used to probe the interactions between alpha₂-adrenergic recpetors and N_i, the guanine nucleotide binding protein that couples those receptors to an inhibition of adenylate cyclase activity. We show here that ADP, which also acts through specific platelet receptors to inhibit adenylate cyclase activity, also promotes the release of [³H]Gpp(NH). The amount of [³H]Gpp(NH)-release elicited by epinephrine and by ADP together is equal to the sum of the amounts released by the two agents acting individually. Furthermore the maximal amounts of [³H]Gpp(NH)-release elicited by each of the two agents approximates the numbers of receptors for ADP and epinephrine present in the platelet membranes. These results suggest that the two receptor types interact with distinct portions of the pool of N_i molecules and that each receptor initiates guanine-nucleotide exchange on a single molecule of N_i.     

Fluorescence and photoelectron studies of the intercalative binding of benz(a)anthracene metabolite models to DNA

An analog of the peptidyl transferase inhibitor sparsomycin was a competitive inhibitor (Ki = 1.8 microM) of peptidyl-puromycin synthesis on E. coli polysomes. Preincubation of polysomes with the compound enhanced the degree of inhibition of peptide bond formation. A model for the involvement of a histidine residue in peptidyl transferase activity is presented as a result of our observations which include direct association of [3H] labelled analog with 70S ribosomes. The correct oxidation state of sulfur in the compound was necessary for the "preincubation effect" and entry of the compound into bacterial cells.     

Clinical trial of chromium and yeast supplements on carbohydrate and lipid metabolism in diabetic men

Chromium (Cr) deficiency in experimental animals and in humans sustained by prolonged total parenteral nutrition has been shown to cause diabetes mellitus. Prior trials in humans indicated that Cr supplements, in either inorganic or organic form, may improve carbohydrate utilization. We report here a clinical double-blind, random cross-over trial of inorganic chromium trichloride, a brewer’s yeast that contained Cr as glucose-tolerance-factor (GTF), a brewer’s yeast extract without GTF, and a placebo. Forty-three outpatient diabetic men received three of these supplements for 4 months each. Subgroups included 21 ketosis-prone, 7 ketosis-resistant non-obese, and 15 ketosis-resistant obese men. Cr levels were followed pre- and post-treatment in hair, red blood cells, plasma, and urine. Response of carbohydrate metabolism to treatment was assessed in terms of change in insulin requirements, fasting plasma glucose, plasma cholesterol, and triglycerides, as well as the change in plasma glucose, glucagon, and insulin or C-peptide levels in response to a standard meal. In some men, these parameters were also measured after iv tolbutamide. Both the inorganic and organic oral Cr supplements increased measurable body pools of Cr in hair and red blood cells by about 25%. However, fasting plasma glucose and lipids and the glucose response to either the standard meal or to tolbutamide were not significantly altered by any of the treatments.     

The N-Shaped Current-Potential Characteristic in Frog Skin: III. Ionic Dependence

A ramp voltage clamp measurement described previously is used to detect alterations in the frog skin current-potential (I-V) characteristic following removal or replacement of various ions in the solutions bathing the skin. The ionic requirements for the maintenance of a negative-slope I-V property are the following: Ca⁺⁺, Na⁺, and Cl^- must be in the outside solution; K⁺ and Cl^- must be in the inside solution. Removal of any one of these ions from its respective solution results in the decay and eventual disappearance of the negative slope.

The similarity between the I-V characteristic following Ca⁺⁺ removal with EDTA from the outside solution and the I-V relation in a refractory skin suggests that the loss (refractory state) and recovery of the negative slope is a consequence of unbinding and subsequent rebinding of Ca⁺⁺ to membrane sites. The role of the univalent ions is not clear—presumably some or all of these ions constitute the current through the skin; however, some of these ions may also be involved in maintaining a membrane condition necessary for the existence of a negative slope I-V relation. Further, excitation does not appear to be a direct consequence of the Na⁺ pump.

     

Statistical-Mechanical Studies of the α⇄β Transformation in Keratin: IV. The Hill Model

The relationship between the two-dimensional Hill model and the David-Schor extension of the one-dimensional Zimm-Bragg model for the alpha right arrow over left arrow beta transformation in keratins is developed. On the basis of the assumptions of the David Schor model, it appears unlikely that the Hill model in its present form can give detailed agreement with the experimental tension-length isotherms.     

The N-Shaped Current-Potential Characteristic in Frog Skin: II. Kinetic Behavior during Ramp Voltage Clamp

Previous step voltage-clamp measurements on frog skin showed the presence of an N-shaped current-potential (I-V) relation in excitable skin. However, the collection and reconstruction of I-V data using discrete step changes of skin potential was tedious because of the long refractory period (up to 1 min) in frog skin. A direct and rapid (5 msec) method for recording the N-shaped I-V characteristic in real time is presented. Ramp functions are used as the command to the clamp system instead of a step function. Consequently the skin potential is forced to change in a linear manner (as commanded) and the skin current can be recorded as a continuous function of the controlled change of skin potential. With the ramp clamp, a low-resistance membrane state ( 10 Omega . cm(2)) resembling a breakdown phenomenon was observed at high skin potential ( 300 mv). Entry into the low resistance state resulted in a collapse of the N-shaped I-V relation to a nearly linear function. The utility of the ramp measurement is demonstrated by predicting (1) that the maximum rate of rise of the spike occurs at a voltage corresponding to the valley (local minimum) in the N-shaped I-V curve, (2) that the rate of rise of the spike increases with increasing clamp currents, (3) the voltage peak of the spike, and (4) the time course of the rising phase of the spike.     

BONE MARROW RESTORATION AFTER LOCALIZED DEPLETION

Studies have been made of marrow restoration after localized depletion of the rabbit femur by dextran perfusion. Restoration was shown to involve an initial period of reorganization which blends with a more prolonged period of hemic cell repopulation. Cellularity returned to normal levels by 35 days, the recovery of myeloid cells being somewhat more rapid than that of the erythroid elements. In either case, the evolution of immature hemic cells was soon followed by the appearance of more mature forms even at the earliest stages of marrow repopulation. ³H-TdR uptake per cell increased rapidly to a level approximately twice normal after the first week. The augmented incorporation of thymidine, revealed by scintillation spectrometry and confirmed upon autoradiography, was shown to be due to an increase in DNA synthesis rate as well as in the fraction of participating cells. It is suggested that the enhanced cell production is brought about by a decrease in the proliferative cell cycle and an increase in the growth fraction. The origin of the repopulating cells remains a moot point. Cell migration from the epiphyseal marrow is apparently not involved. Irrespective of the source of stem-type cells, the stimulus for regeneration appears to be locally determined.