Specific detection of α-amylase activity in crude plant extracts after isoelectric focusing

A new method which utilizes Procion Red MX 2B amylopectin for the detection of α-amylase in crude plant extracts is described. The substrate is specific only against α-amylase hydrolysis and β-amylase does not attack it. Paper containing Procion Red MX 2B amylopectin applied to gels after isoelectric focusing reveals α-amylase isoenzymes as white bands. When this technique is used, heat-inactivation of β-amylase is not required.     

Stoichiometric and catalytic hydrogenation of the [Co2(CO)6(μ2-η2-alkyne)] complexes

The [Co₂(CO)₆(RC₂R′)] complexes (R, R′ = H, Me, Et, Prⁿ) react with molecular hydrogen under mild conditions of temperature and pressure, at low but appreciable rates. The effect of the steric hindrance of the substituents and the strength of the metalcarbon bonds are discussed. The kinetic data measured for [Co₂(CO)₆(HC₂H)], suggest that both H₂-coordination and CO-dissociation are involved in the rate-determining step of the overall hydrogenation process.The catalytic activity of [Co₂(CO)₆(HC₂H)] in the homogeneous hydrogenation of acetylene is described. At low substrate/catalyst ratio the initial hydrogenation rate is equal, within experimental error, to that found for the stoichiometric reaction; on increasing the acetylene concentration, cyclotrimerization to benzene becomes the dominant process. Interestingly C₄ hydrocarbons (mainly butadiene and 1-butene) are produced in measurable yield (?8%). The formation of these products is interpreted as the result of the hydrogenation of the elusive [Co₂(CO)₅(HC₂H)₂] complex, an unstable intermediate in the cyclotrimerization chain.     

Effect of various drugs producing convulsive seizures on rat brain glycerolipid metabolism

Convulsive seizures were elicited in the rat by the injection of several different drugs (pyridoxal phosphate, bicuculline, penicillin and ouabain). Glycerolipid metabolism was studied after the intraventricular injection of [2-³H]glycerol, which was incorporated into rat brain glycerides. The percentage of total lipid label found in each lipid class (phosphatidylethanolamine, PE; phosphatidylcholine, PC; phosphatidylserine, PS; phosphatidic acid, PA; phosphatidylinositol, PI; diacylglycerol (+ monoacylglycerol), DG and triacylglycerol, TG) depended on the time elapsed from the injection of the labeled precursor. The percent of total lipid radioactivity as PE and PC increased with time (3–60 min), whereas the opposite was true for the radioactivity of DG and PA. The radioactivity of other lipid classes did not appreciabily vary between 3 and 60 min from the injection of the labeled glycerol. The intraventricular administration of pyridoxal phosphate together with labeled glycerol decreased the percent of lipid radioactivity as PE and increased that as DG. This lipid effect was detected also after the administration of other convulsants, such as ouabain and penicillin. The intraperitoneal administration of bicuculline affected lipid metabolism in cerebellum.     

Amino acid sequence of rabbit ventricular myosin light chain-2: Identity with the slow skeletal muscle isoform

Many studies have established a correlation of differences in the activities of various muscle types with differences in the expression of myosin isoforms. In this paper we report the sequence determination of myosin light chain-2 from rabbit slow skeletal (LC2s) and ventricular (LC2v) nmscles. We sequenced tryptic peptides from LC2v which account for all except a few terminal amino acid residues. The major part (87 residues) of the rabbit LC2s sequence, obtained from tryptic and cyanogen bromide (CNBr) peptides, was found to be identical to rabbit LC2v. Our results provide the first sequence information on LC2s from any species, and lend strong support to the hypothesis that LC2s and LC2v are identical. Comparisons of rabbit LC2v and LC2s with rabbit LC2f (from fast skeletal muscle), and also with chicken LC2f and LC2v, show clearly that LC2s and LC2v from mammalian and avian species are more closely related to each other than they are to LC2f isoforms from the same species.     

Die Diffusion des 59Fe-markierten Hämoglobins,ein Artefakt der Glutaraldehyd-Fixierung

Zusammenfassung Ratten wurden 50–80 c ⁵⁹Fe-Citrat in die caudale Vene injiziert. Nach 3–5tägigem Einbau des markierten Eisens in das Hämoglobin wurden Milz-Blöckchen (2 × 2 × 5 mm) 2 Std in GA vorfixiert oder in Hanks-Lösung (= Kontrolle) überführt. Ein Teil der Blöckchen wurde anschließend in OsO₄-Lösung nachfixiert.Die autoradiographischen Ergebnisse zeigen eine Diffusion des Hämoglobins vom unfixierten Zentrum zur Peripherie des Blöckchens.Aktivitätsbestimmungen, die am ganzen Blöckchen, dessen abgetrennten zentralen und peripheren Anteilen, sowie im Überstand vorgenommen wurden, bestätigen diese Diffusion. Während der OsO₄-Nachfixierung erfolgte ein weiterer Verlust des markierten Hämoglobins aus dem zentralen Teil des Blöckchens, nicht aber aus der vorfixierten Peripherie.

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The diffusion of ⁵⁹Fe-labelled hemoglobin, an artefact of the fixation with glutaraldehyde

Summary Injections of 50–80 c ⁵⁹Fe-citrate into the caudale vein of rats were performed. After 3–5 days of ⁵⁹Fe incorporation into the hemoglobin the spleen was taken off, cut in small blocks (2 × 2 × 5 mm) and prefixed for 2 hours in GA or transfered into Hankssolution (= control). Later on some blocks of the spleen were postfixed in OsO₄ solution.A diffusion of the hemoglobin from the unfixed center to the peripheral tissue of the spleen-block is demonstrated by autoradiographic results.After measuring the radioactivity of the total spleen-block of the separated central and peripherical parts as well as their supernatants a diffusion was confirmed. A further loss of the labelled hemoglobin has been observed during the OsO₄ postfixation from the central part of the spleen-block, but not from the prefixed periphery.

     

A re-evaluation of the mesangial cells of the renal glomerulus

Summary The topographical localization of the mesangial cells in renal glomeruli of rats, and their relationships with the structures at the hilus of the glomerulus were studied in this investigation. It was observed that the mesangial cells occupy a parietal position in the wall of the glomerular capillaries, and that they are anatomically continuous with the smooth muscle cells of the tunica media of the glomerular arterioles.This study was supported by a United States Public Health Service Grant AM 08628 (Institute of Arthritis and Metabolic Diseases).     

Hydrolysis of bis(5''-nucleosidyl) polyphosphates by Escherichia coli 5''-nucleotidase.

Two enzymatic activities that split diadenosine triphosphate have been reported in Escherichia coli: a specific Mg-dependent bis(5'-adenosyl) triphosphatase (EC 3.6.1.29) and the bis(5'-adenosyl) tetraphosphatase (EC 3.6.1.41). In addition to the activities of these two enzymes, a different enzyme activity that hydrolyzes dinucleoside polyphosphates is described. After purification and study of its molecular and kinetic properties, we concluded that it corresponded to the 5'-nucleotidase (EC 3.1.3.5) that has been described in E. coli. The enzyme was purified from sonic extracts and osmotic shock fluid. From sonic extracts, two isoforms were isolated by chromatography on ion-exchange Mono Q columns; they had a molecular mass of about 100 kilodaltons (kDa). From the osmotic shock fluid, a unique form of 52 kDa was recovered. Mild heating transformed the 100-kDa isoform to a 52-kDa form, with an increase in activity of about threefold. The existence of a 5'-nucleotidase inhibitor described previously, which associates with the enzyme and is not liberated in the osmotic shock fluid, may have been responsible for these results. The kinetic properties and substrate specificities of both forms (52 and 100 kDa) were almost identical. The enzyme, which is known to hydrolyze AMP and uridine-(5')-diphospho-(1)-alpha-D-glucose, but not adenosine-(5')-diphospho-(1)-alpha-D-glucose, was also able to split adenosine-(5')-diphospho-(5)-beta-D-ribose, ribose-5-phosphate, and dinucleoside polyphosphates [diadenosine 5',5'-P1,P2-diphosphate,diadenosine 5',5'-P1,P3-triphosphate, diadenosine 5',5'-P1,P4-tetraphosphate, and bis(5'-guanosyl) triphosphate]. The effects of divalent cations and pH on the rate of the reaction with different substrates were studied.     

Substance P affects the GABAergic system in the hypothalamo-pituitary axis

In the present work we examined the effect of the neutralization of endogenous substance P by the administration of an anti-substance P serum (ASPS) on GABA concentration in the anterior pituitary in hyperprolactinemic conditions induced by 5-hydroxytryptophan or by grafting anterior pituitaries. ASPS reduced the increase in the anterior pituitary GABA concentration induced by hyperprolactinemia. In vitro experiments showed that substance P inhibited K⁺-evoked GABA efflux from hypothalamic fragments and decreased GABA concentration in the anterior pituitary but ASPS increased it. Our results demonstrate that substance P modifies hypothalamic GABA release and anterior pituitary GABA concentration and suggest that an interaction exists between substance P and GABA.     

Candida albicans stress mannoproteins expression in superficial and systemic candidiasis

The presence of heat shock mannoproteins (HSMPs) reactive with sIgA was demonstrated in several C. albicans strains. The subculture of the C. albicans isolated from mucosal surfaces on Sabouraud's dextrose agar at 25 °C switched off the HSMP expression. A re-expression of the HSMPs was obtained in the same medium by shifting the temperature of incubation to 37 °C. However, expression of HSMPs in two strains isolated from deep infections was maintained during several subcultures on Sabouraud's dextrose agar at 25 °C. A glycoprotein of 200 kDa seemed to be the main HSMP reacting with vaginal sIgA. The data presented in this study suggest that factors other than temperature can influence the expression of C. albicans HSMPs and therefore these antigens should be referred as stress mannoproteins.Abbreviations HSMPs heat shock mannoproteins - MAb monoclonal antibody - sIgA secretory IgA