Reduced cholecystokinin in the brain of LEC rats with hepatic encephalopathy.

Levels of cholecystokinin (CCK) immunoreactivity and distribution of CCK immunoreactive cells were studied in the cerebral cortex of LEC (Long Evans Cinnamon) rats with hepatic encephalopathy. CCK immunoreactivity in water extract of cerebral cortex of LEC rats with hepatic encephalopathy (n = 7) was 41.5 +/- 2.6 (mean +/- S.E.M. pmol/g wet wt.) and that of LEC rats without encephalopathy (n = 8) was 67.1 +/- 6.9, the difference being significant (P less than 0.01). CCK immunoreactive cells assessed by immunohistochemistry were also markedly decreased in the cortex of LEC rats with hepatic encephalopathy of stage IV. Thus, CCK reduction was observed in the cerebral cortex of LEC rats with hepatic encephalopathy which are provided as a model for analysis of the pathogenesis of acute hepatic encephalopathy.     

An establishment of ELISA for murine IL-6

Summary Murine interleukin-6 (mIL-6) was expressed inEscherichia coli as human growth hormone (hGH) fusion protein. The products were cleaved by thrombin to liberate mIL-6. Monoclonal and polyclonal antibodies specific to mIL-6 were prepared by immunizing rats with mIL-6 thus obtained. ELISA for the quantitation of mIL-6 was also established, which could detect mIL-6 in a quantity as low as 2 ng/ml.     

Effect of dexamethasone on nucleolar casein kinase II activity and phosphorylation of nucleolin in lymphosarcoma P1798 cells.

Ribosomal RNA (rRNA) synthesis in murine P1798 lymphosarcoma cells is reversibly inhibited by glucocorticoids. The effects of dexamethasone upon nucleolin phosphorylation and upon the amount and activity of casein kinase II have been examined. P1798 cells were exposed to 0.1 microM dexamethasone for 36 h. Cells were labeled in vivo with [32P]orthophosphate followed by immunoprecipitation with anti-nucleolin antibody. Nucleolin phosphorylation was reduced by 60% in dexamethasone-treated cells. Nucleoli were isolated and labeled with [gamma-32P]ATP in vitro. Nucleolin protein was reduced to 40% of control in nuclei from dexamethasone-treated cells. Nucleolin phosphorylation was reduced to 20% of control. Nucleolar casein kinase II activity and protein were also reduced (30-55% and 35-50% of control, respectively) by treatment with dexamethasone. Cycloheximide (10 micrograms/ml for 3 h) reduced the amount and activity of casein kinase II, but did not cause a decrease in nucleolin protein. These observations are discussed relative to the hypothesis that glucocorticoids regulate the amount or activity of proteins of short biological half-life that are involved in the regulation of rRNA synthesis.     

A hyperthermostable pullulanase produced by an extreme thermophile,Bacillus flavocaldarius KP 1228, and evidence for the proline theory of increasing protein thermostability

Summary A cell-associated pullalanase (-dextrin 6-glucanohydrolase, EC 3.2.1.41) of an extreme thermophile, Bacillus flavocaldarius KP 1228, was purified to homogeneity. The molecular weight and isoelectric point were estimated to be about 55 000 and 7.0, respectively. The N-terminal sequence was Ala-Try-Tyr-Glu-Gly-Ala-Phe-Phe-Tyr-Gln-Ile-Phe-Pro-Asp-Tyr-Phe-Phe-Tyr-Ala-Gly-. The enzyme was most active at pH 6.3. The activities for 5% pullulan and 5% soluble starch were maximal at 75–80° C and at 80–85° C, respectively. The enzyme was stable up to 90° C for 10 min at pH 6.8. The enzyme had no antigenic determinants shared with pullulanases from the mesophiles Klebsiella pneumoniae and B. acidopullulyticus NCIB 11647. A comparison of amino acid composition demonstrated that the proline content increased greatly in a linear fashion with the rise in thermostability in the order K. pneumoniae B. acidopullulyticus B. flavocaldarius enzymes, as found with Bacillus oligo-1,6-glucosidases.Presented in part at the Annual Meeting of the Agricultural Chemical Society of Japan at Tokyo, April 2, 1987 (Abstracts, p 91)Offprint requests to: Y. Suzuki     

Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth

Summary Tissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm³ per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed. Offprint requests to: S. Mitsuda     

Changes of carp myosin subfragment-1 induced by temperature acclimation

Summary Heavy meromyosin subfragment-1 (S1) was prepared by -chymotrypsin from myosin of carp acclimated to either 10°C or 30°C for a minimum of 5 weeks. The objective of these studies was to document thermally-induced changes in the myosin molecule and to extend previous observations. Ca²⁺- and K⁺ (EDTA)-ATPase activities of cold-acclimated carp S1 were 1.1 and 0.8 mol P_i·min^-1·mg^-1, respectively, and these values did not differ significantly from those of warm-acclimated carp. The inactivation rate constant (K_D) of S1 from cold-acclimated carp was 32.1x10^-4· s^-1, compared to 13.2x10^-4·s^-1 for warm-acclimated carp. The maximum initial velocity of acto-S1 Mg²⁺-ATPase activity at pH 7.0 in 0.05 M KCl was 9.3 s^-1 with cold-acclimated carp, about 3.7 times higher than that for warm-acclimated carp. However, no significant difference was observed in the apparent affinity of S1 to actin. Peptides maps of the heavy chain of S1 were different and suggested distinct isoforms for the myosins from warm- and cold-acclimated muscle.Abbreviations ATPase adenosine 5-triphosphatase - DTNB 5,5-dithiobis (2-nitrobenzoic acid) - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol bis (-aminoethylether)-N,N,N,N-tetraacetic acid - K _D inactivation rate constant - K _m apparent dissociation constant - P _i inorganic -phosphate - PMSF phenylmethane-sulfonyl fluoride - S ₁ heavy meromyosin subfragment-1 - SDS sodium dodecyl sulfate - SDS-PAGE SDS-polyacrylamide gel electrophoresis - TPCK N-tosyl-l-phenylalanyl chloromethyl ketone - V _max maximum initial velocity     

Xenopus M phase MAP kinase: isolation of its cDNA and activation by MPF.

MAP kinase is activated and phosphorylated during M phase of the Xenopus oocyte cell cycle, and induces the interphase-M phase transition of microtubule dynamics in vitro. We have carried out molecular cloning of Xenopus M phase MAP kinase and report its entire amino acid sequence. There is no marked change in the MAP kinase mRNA level during the cell cycle. Moreover, studies with an anti-MAP kinase antiserum indicate that MAP kinase activity may be regulated posttranslationally, most likely by phosphorylation. We show that MAP kinase can be activated by microinjection of MPF into immature oocytes or by adding MPF to cell-free extracts of interphase eggs. These results suggest that MAP kinase functions as an intermediate between MPF and the interphase-M phase transition of microtubule organization.     

Morphology of the granular hemocytes of the Japanese horseshoe crabTachypleus tridentatus and immunocytochemical localization of clotting factors and antimicrobial substances

Summary The structure of hemocytes in the normal state and during blood coagulation, and the intracellular localization of three clotting factors and two antimicrobial factors were examined in the Japanese horseshoe crabTachypleus tridentatus. Two types of hemocytes were found in the circulating blood: non-granular and granular hemocytes. The latter contained numerous dense granules classed into two major types: L- and D-granules. The L-granules were larger (up to 1.5 m in diameter) and less electron-dense than the D-granules (less than 0.6 m in diameter). The L-granules contained three clotting factors and one antimicrobial factor, whereas the D-granules exclusively contained the other antimicrobial factor. After treatment with endotoxin, the L-granules were released more rapidly than the D-granules, although almost all granules were finally exocytosed. The granular hemocyte possessed a single Golgi complex; possible precursor granules of L-granules and D-granules contained tubular and condensed dense material, respectively. These data are discussed in relation to the self-defense mechanisms of the horseshoe crab.     

A convenient approach to the synthesis of medium size oligodeoxyribonucleotides by improved new phosphite method.

Improvement of the new phosphite method for the synthesis of oligodeoxyribonucleotides using the deoxyribonucleoside 3'-bis(1,1,1,3,3,3- hexafluoro-2-propyl) phosphite unit has been carried out via the hydrolysis and capping steps, without any side reaction products. The new phosphite unit and capping agent, bis(1,1,1,3,3,3-hexafluoro-2-propyl)-2-propyl phosphite, is readily activated by N-methylimdazole under very mild condition on a solid support. This operation involves a one pot reaction, which is an advantage over both the phosphite and H- phosphonate approaches. The mechanism of internucleotidic bond formation of the new phosphite method is also discussed.     

Interstrain differences in red cell enzyme activities in mice and rats.

1. Interstrain differences in red blood cell enzyme activities were studied in mice (BALB/c, C57BL/6, C3H/He, DBA/2 and ddY) and rats (Donryu, F344/N, SD, Wistar and Wistar/ST), and were also compared with hamster, guinea-pig and rabbit. 2. The enzyme activities measured were: glutathione S-transferase (GST), glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase (6-PGD), NADPH-diaphorase (ND), hexokinase (Hx), glutamate oxaloacetate transaminase (GOT), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE). 3. There were marked variations in the activities of some red cell enzymes (e.g. GST, Hx, ND), while others (e.g. G-6-PD, 6-PGD) were much less variable both within different strains and species.