Enhancingin vitro antibody production rate per cell by applying mouse peritoneal factors

Summary Monoclonal antibody production rateper hybridoma cell was measured as much larger in mouse peritoneal cavity thanin vitro culture. To identify factors enhancing antibody productivity per cell, a hybridoma was culturedin vitro with ascites and peritoneal exudate cells(PEC). Both the ascites and the PEC improved the productivity per cellin vitro. Each chromatograph-fraction of ascites was assayed for the enhancing activity.     

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.     

Outgrowth dependency of forelimb regeneration on nerves in adult African clawed frog,Xenopus laevis

Summary The relationship between the size and shape of regenerative outgrowth and the quantity of innervation was studied in adult Xenopus laevis. The forelimbs, of which the nerve supply was artificially altered, were amputated midway through the stylopodium and were kept for 1 year. The regenerative outgrowths that formed in normal limbs with an intact nerve supply were mainly spike-shaped and occasionally rod-shaped. However, when the nerve supply to the distal part of the forelimb was augmented by surgically diverting ipsilateral sciatic nerve bundles, the quantity of innervation was increased to about two and a half times that of the normal limb. These hyperinnervated outgrowths were somewhat larger than those of the normally innervated outgrowths and the majority of them were oar-shaped, a type hardly ever encountered in normal regeneration. In contrast, when partial denervation was performed concomitantly with limb amputation, by ablation of the N. radialis at the shoulder joint, the quantity of innervation decreased to about one half that of the normal limb. The outgrowths obtained were spike-shaped in all cases, with their size being about half that of the normally innervated outgrowths. Furthermore, when both the N. radialis and N. ulnaris were ablated in the same way, the amputated limbs were mostly non-regenerative, but some of them regenerated small conical outgrowths. Based on these results, a discussion is presented concerning the relationship between a regenerative outgrowth and the innervation of the forelimb in Xenopus.     

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     

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.     

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.