Multiple forms of amylase in leaf extracts: electrophoretic transfer of the enzyme forms into amylose-containing polyacrylamide gels

A method for the analysis of multiple forms of glucan-degrading enzymes is described. The procedure consists of the separation of the proteins by electrophoresis or isoelectric focusing in glucan-free polyacrylamide gels followed by the nondenaturing electrophoretic transfer into a second polyacrylamide layer which contains immobilized glucans. The method combines the resolving power of electrophoretic separations in glucan-free media with the sensitivity of amylase activity detection in amylose-containing polyacrylamide gels. The procedure is especially useful when samples containing low amylase activity, but a large number of multiple enzyme forms, are to be analyzed.     

Action potentials of the rabbit, guinea pig, dog and albino rat working ventricular myocardium under interpolated extrasystole conditions during postnatal ontogenesis

Experiments were carried out on the working myocardium of the right heart ventricle of newborn and adult rabbits, guinea-pigs, dogs and albino rats. In the dog, the guinea-pig and the rabbit, after ten action potentials (AP) elicited with 1 Hz frequency we always interpolated an extrasystole at an interval (TE) of 100-900 ms. In albino rats we used a basic frequency of 2 Hz and a TE of 30-370 ms from the last regular AP. Using glass microelectrodes, we recorded the extrasystolic AP (EAP) and the next subsequent AP (2AP). The results were evaluated by constructing graphs of the correlations of the duration of the plateau phase (D0) to TE and of the duration of repolarization to -60 mV level (D60) to the TE. In the myocardium of newborn rabbits, guinea-pigs and dogs, with short TE both D0 and D60 of the EAP are shorter than in the steady state (SS), while for the 2AP the same parameters are influenced only a little. As the TE lengthens, the EAP gradually acquire a length corresponding more to the SS. With TE longer than half the duration of the cycle in the steady state the EAP return to normal, while the 2AP become shorter. The effect of extrasystole on the rat EAP and 2AP diminished with advancing age. In the myocardium of adult rabbits and adult guinea-pigs, and slightly in the myocardium of adult dogs and newborn rats, we observed that the duration of the EAP, with certain TE, was greater than in the steady state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the S-adenosylhomocysteine hydrolase inhibitors 3-deazaadenosine and 3-deazaaristeromycin on RNA methylation and synthesis

The effects of 3-deazaaristeromycin and 3-deazaadenosine on RNA methylation and synthesis were examined in the mouse macrophage cell line, RAW264. S-Adenosylhomocysteine accumulated in cells incubated with 3-deazaaristeromycin while S-3-deazaadenosylhomocysteine was the major product in cells incubated with 3-deazaadenosine and homocysteine thiolactone. RNA methylation was inhibited to a similar extent by the accumulation of either S-adenosylhomocysteine or S-3-deazaadenosylhomocysteine, with S-adenosylhomocysteine being a slightly better inhibitor. In mRNA, the synthesis of N6-methyladenosine and N6-methyl-2'-O-methyladenosine were inhibited to the greatest extent, while the synthesis of 7-methylguanosine and 2'-O-methyl nucleosides were inhibited to a lesser extent. Incubation of cells with 100 microM 3-deazaaristeromycin or with 10 microM 3-deazaadenosine and 50 microM homocysteine thiolactone produced little inhibition of mRNA synthesis, even though mRNA methylation was inhibited. In contrast, mRNA synthesis was greatly inhibited by treatment of cells with 100 microM 3-deazaadenosine and the inhibition of synthesis was not correlated with an inhibition of methylation.     

Homologies between paraflagellar rod proteins from trypanosomes and euglenoids revealed by a monoclonal antibody

A Nonidet P 40 insoluble fraction was isolated from Trypanosoma brucei and was used to raise a monoclonal antibody (5E9). The antigen was localized by indirect immunofluorescence in the flagellum of T. brucei and of two species of euglenoids, Euglena gracilis and Distigma proteus. In immunoblot analysis, 5E9 appeared to bind to paraflagellar rod proteins PFR1 and PFR2 of T. brucei (72000 and 75000 mol. wt.) and of E. gracilis (67000 and 76000 mol. wt.). The presence of a common epitope in paraflagellar rod proteins from species of trypanosomes and euglenoids shows that despite distinct structures of the rods some identical domain exists in the proteins that could be involved in their supramolecular assembly into a similar organelle. The antigenic determinant defined by 5E9 was also shown to be present in a 87000 molecular weight polypeptide located in the proximal part of the flagellum of Crithidia oncopelti in which a paraflagellar rod is not detectable at the ultrastructural level.     

The relationship of hormone-sensitive and hormone-insensitive phosphatidylinositol to phosphatidylinositol 4,5-bisphosphate in the WRK-1 cell

We have previously characterized two distinct pools of phosphatidylinositol (PI) in the WRK-1 rat mammary tumor cell, one whose metabolism is enhanced in response to vasopressin and another which is insensitive to hormonal manipulation. The purpose of the present study was to examine the relationship between cellular phosphatidylinositol 4,5-bisphosphate (PIP2) and each of the two PI pools. We have found that in WRK-1 cells, vasopressin induces the rapid loss of PIP2 and the accumulation of inositol phosphates. By making use of kinetic differences in 32Pi uptake into the two pools of PI and assessing radioactivity levels in the 1-phosphate of PIP2, we have determined that hormone-sensitive PI is the precursor of approximately 60% of the cellular PIP2; the remainder is synthesized from the hormone-insensitive pool. Additional data indicate that PIP2 derived from hormone-sensitive PI is likewise hormone-sensitive, while that synthesized from hormone-insensitive PI remains stable over a long period of time and is not affected by the presence of vasopressin.     

The HsdS polypeptide of the Type IC restriction enzyme Eco R124 is a sequence-specific DNA-binding protein

The HsdS and HsdM polypeptides of the type IC restriction enzyme EcoR124 have been purified independently and used in a set of gel retardation experiments to determine the minimum requirements for sequence-specific recognition of DNA by this enzyme. The HsdS polypeptide alone is able to bind to DNA in a sequence-specific manner. In addition, whilst the presence of the HsdM polypeptide gives rise to a stimulation of DNA binding by the HsdS subunit it is not clear whether, under the conditions of the experiments reported here, the HsdS subunit maintains the same interactions with the HsdM subunits observed in the absence of DNA.     

Intracellular and Extracellular Uptake of Cadmium by the Moss Rhytidiadelphus squarrosus

Intra- and extracellular Cd uptake were investigated in themoss Rhytidiadelphus squarrosus. Intracellular Cd uptake displayedMichaelis–Menten kinetics and showed that the moss hada high uptake rate and high affinity for Cd. Extracellular Cdbinding capacity was also high. The anion used had little effecton Cd uptake to either location but both intra- and extracellularCd uptake were reduced by equimolar concentrations of Ca, Mgor Zn, although inhibition was not wholly competitive. IntracellularCd uptake was not significantly affected by the supply of energygenerating reserves but, like extracellular Cd uptake, showedaltered characteristics following growth under controlled laboratoryconditions. The results are compared with data on Cd uptakeby the lichen genus Peltigera. Rhytidiadelphus squarrosus, moss, cadmium, intra- and extracellular uptake characteristics, kinetics     

Different secretory pathways of renin from mouse cells transfected with the human renin gene

Mammalian cells in culture, transfected with human renin gene, can provide a useful tool for studying renin biosynthesis and secretion. We transfected fibroblast cells (mouse L929 and Chinese hamster ovary cells) and pituitary tumor cells (mouse AtT-20) with the human renin gene and a selectable plasmid (pSV2Neo). Transfected fibroblasts synthesize prorenin only. Prorenin is secreted by fibroblasts constitutively and the secretion is not influenced by 8-bromo-cAMP. On the other hand, transfected AtT-20 cells synthesized both prorenin and mature active renin. Transfected AtT-20 cells release prorenin by constitutive secretion but mature renin is secreted by a regulated mechanism since the secretion of the former is not influenced by 8-bromo-cAMP but the release of the latter is significantly stimulated. Our studies demonstrate that human renin may be secreted by at least two cellular pathways: prorenin by a constitutive pathway and mature renin by a regulated pathway. These transfected cells may provide useful models for studies of human renin synthesis, processing, and secretion.