Sequence of insulin effects on cytoskeletal and cytosolic phosphofructokinase, mitochondrial hexokinase, glucose 1,6-bisphosphate and fructose 2,6-bisphosphate levels, and the antagonistic action of calmodulin inhibitors, in diaphragm muscle.

1. Time-curves of insulin effects on energy-producing systems in different cellular compartments of rat diaphragm muscle have revealed: (a) a rapid (within minutes) and transient stimulatory effect of insulin on cytoskeletal phosphofructokinase and aldolase and mitochondrial hexokinase. (b) A slower and consistent stimulatory effect on glucose 1,6-bisphosphate level, with concomitant gradual activation of cytosolic phosphofructokinase. Fructose 2,6-bisphosphate levels were not changed by insulin. (c) Lactate concentration correlated with the stimulation of cytoskeletal and cytosolic glycolysis. 2. Calmodulin antagonists, trifluoperazine or CGS 9343B, prevented all these effects of insulin. 3. These results suggest that cytoskeletal glycolysis and mitochondrial oxidation are the source of ATP for the rapid actions of insulin, whereas cytosolic glycolysis is the source of ATP for the slow actions of insulin. Calmodulin is involved in all these effects of insulin.     

Isolation of cDNA for bovine stomach 155 kDa protein exhibiting myosin light chain kinase activity.

Two proteins with myosin light chain kinase activity and electrophoretic molecular weights of 155,000 and 130,000 were each isolated from bovine stomach smooth muscle [Kuwayama, H., Suzuki, M., Koga, R., & Ebashi, S. (1988) J. Biochem. 104, 862-866]. The 155 kDa component showed a much higher superprecipitation-inducing activity than the 130 kDa component, when compared on the basis of equivalent myosin light chain kinase activity. In this study, we isolated a cDNA for the entire coding region of the 155 kDa protein. The deduced amino acid sequence revealed a high degree of similarity to those of chicken and rabbit smooth muscle myosin light chain kinases. Multiple motifs, such as three repeats of an immunoglobulin C2-like domain, a fibronectin type III domain, and unusual 20 repeats of 12 amino acids were detected in the sequence. Part of the amino-terminal sequence was similar to that of the actin- and calmodulin-binding domain of smooth muscle caldesmon. These observations suggest that the 155 kDa protein has additional functions other than its enzymatic activity. Two mRNAs of 6.0 and 2.6 kb in length in the bovine stomach smooth muscle RNAs were hybridized with cDNA probes. The 2.6-kb RNA probably encodes telokin, which is the carboxyl terminus of smooth muscle myosin light chain kinase. mRNAs with identical lengths were also detected in bovine aorta.     

Site-directed mutagenesis in the uracil-repair system. Preparation of mutated forms of human alpha 2 interferon

The mutant forms of human IFN-alpha 2 gene are obtained by oligonucleotide-directed mutagenesis with the use of uracil-repair system. To intensify the process the procedure of the uracil-containing DNA template preparation is modified. It was determined that when mutagenesis is performed in the uracil-repair system the yield of the process depends on the mutant DNA-strand in vitro synthesis efficiency. It is shown that the stability of the 5'-end primer-template complex and the level of the endogenic primers elongation are the basis factors, that determine induction mutations.     

Vertical distribution and cellular heat resistance of bottom animals from the Possyet Bay (Japan Sea)

Summary 1. Investigations on 15 mass species of bottom animals (Bivalvia, Gastropoda, Asteroidea, Ascidia), performed by means of aqualungs in semi-enclosed bays of the Possyet Bay (Peter the Great Bay of the Japan Sea), have revealed a clear zonality in their vertical distribution. This finding is in contrast to the situation found near islands and capes of Peter the Great Bay, where species distributions are weakly connected with depth, but to a considerable degree determined by the nature of substrate, degree of tides and presence of predators.2. These differences may be explained by differences in summer temperature conditions, considerable decrease of the temperature gradient with increase of depth in semi-enclosed bays, insignificant surface temperature differences and, in depths of about 20–30 m, by intensive water mixing near islands and capes.3. Comparison of the vertical distribution of species from the Possyet Bay with their cellular heat resistance (species-specific feature, used for characterizing genetic heat adaptation) reveals conformity between these indices.4. These results and literature data suggest that the differences in vertical distribution of the bottom invertebrates living in coastal waters are the result of the species-specific adaptation of their cells to environmental temperature; differences in protein structure of different animals are considered to provide the basis of this phenomenon.

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Vertikalverbreitung und zelluläre Hitzeresistenz von Bodentieren aus der Possjet-Bucht (Japanisches Meer)

Kurzfassung Die vertikale Zonierung verschiedener benthonischer Tiere, die in halbgeschlossenen Buchten des Japanischen Meeres untersucht wurden, weicht von den im gleichen Gebiet an Inseln und Steilabhängen festgestellten Tiefenverbreitungen ab. Diese Unterschiede können von der Art des Substrats, der Gezeitenwirkung, den Strömungsverhältnissen und insbesondere von den Temperaturbedingungen abhängen. Ein experimenteller Vergleich der zellulären Hitzeresistenz von Cilienepithelien verschiedener Muschel- und Ascidien-Arten und deren Vertikalverbreitung konnte vor allem den Einfluß der Temperatur deutlich machen. Es wird die Schlußfolgerung gezogen, daß artspezifische Anpassungen an die Umgebungstemperatur, die offensichtlich auf Unterschieden in der Proteinstruktur beruhen, die Vertikalverbreitung von Bodentieren entscheidend mitbestimmen.