Continuous acetic acid production by the bioreactor system loading a new ceramic carrier for microbial attachment

Summary We have developed a bioreactor system for aerobic fermentation, using a new ceramic carrier APHROCELL which has a suitable shape for liquid and gas passage. In acetic acid fermentation byAcetobacter cells from ethanol, as a typical example of aerobic fermentation, a productivity of 17.25 g/l h was attained at continuous production of 23 g-acetic acid/l; at an acetic acid concentration around 53 g/l, the productivity was 6.4 g/l h. Thus a marketable vinegar can be obtained continuously by this bioreactor system. Because of the simplicity of the APHROCELL reactor, scale up should be relatively easy.     

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.     

Vasoactive intestinal peptide stimulates ciliary motility in rabbit tracheal epithelium: modulation by neutral endopeptidase.

We studied the effect of vasoactive intestinal peptide (VIP) on ciliary activity in rabbit cultured tracheal epithelium by a photoelectric method in vitro. Administration of VIP (10(-7) M) elicited an increase in ciliary beat frequency (CBF) from the baseline values of 970 +/- 52 to 1139 +/- 75 beats/min (mean +/- S.E., P less than 0.01). This ciliostimulatory effect was dose-dependent, with the maximal increase and EC50 value being 17.4 +/- 1.0% (P less than 0.05) and 6.10(-11) M, respectively. The VIP-induced increase in CBF was abolished by pretreatment of cells with [4-Cl-D-Phe6, Leu17]-VIP, a VIP receptor antagonist. The neutral endopeptidase inhibitor phosphoramidon (10(-5) M) potentiated the effect of VIP, so that the CBF dose-response curve for VIP was shifted to lower concentrations by 0.5 log U. The administration of VIP increased cyclic AMP levels in epithelial cells, an effect that was also potentiated by phosphoramidon. These results suggest that VIP may interact with its specific receptors and stimulate airway ciliary activity probably through the activation of adenylate cyclase, and that neutral endopeptidase may play a role in modulating this effect of VIP.     

Gene structure and expression of a tobacco endochitinase gene in suspension-cultured tobacco cells

We have isolated and characterized the genomic clone CHN50 corresponding to tobacco basic endochitinase (E.C.3.2.1.14). DNA sequence and blotting analysis reveal that the coding sequence of the gene present on CHN50 is identical to that of the cDNA clone pCHN50 and, moreover, the CHN50 gene has its origin in the progenitor of tobacco, Nicotiana sylvestris. Tobacco basic chitinases are encoded by a small gene family that consists of at least two members, the CHN50 gene and a closely related CHN17 gene which was characterized previously. By northern blot analysis, it is shown that the CHN50 gene is highly expressed in suspension-cultured tobacco cells and the mRNA accumulates at late logarithmic growth phase. To identify cis-DNA elements involved in the expression of the CHN50 gene in suspensioncultured cells, the chimeric gene consisting of 1.1 kb CHN50 5 upstream region fused to the coding sequence of -glucuronidase (GUS) was introduced by electroporation into protoplasts isolated from suspension-cultured tobacco cells. Transient GUS activity was found to be dependent on the growth phase of the cultured cells, from which protoplasts had been prepared. Functional analysis of 5 deletions suggests that the distal region between -788 and -345 contains sequences that potentiate the high-level expression in tobacco protoplasts and the region (-68 to -47) proximal to the TATA box functions as a putative silencer.     

Requirement of c-kit for development of intestinal pacemaker system.

A discovery that the protooncogene encoding the receptor tyrosine kinase, c-kit, is allelic with the Dominant white spotting (W) locus establishes that c-kit plays a functional role in the development of three cell lineages, melanocyte, germ cell, and hematopoietic cell which are defective in W mutant mice. Recent analyses of c-kit expression in various tissues of mouse, however, have demonstrated that c-kit is expressed in more diverse tissues which are phenotypically normal in W mutant mice. Thus, whether or not c-kit expressed outside the three known cell lineages plays a functional role is one of the important questions needing answering in order to fully elucidate the role of c-kit in the development of the mouse. Here, we report that some of the cells in smooth muscle layers of developing intestine express c-kit. Blockade of its function for a few days postnatally by an antagonistic anti-c-kit monoclonal antibody (mAb) results in a severe anomaly of gut movement, which in BALB/c mice produces a lethal paralytic ileus. Physiological analysis indicates that the mechanisms required for the autonomic pacing of contraction in an isolated gut segment are defective in the anti-c-kit mAb-treated mice, W/Wv mice and even W/+ mice. These findings suggest that c-kit plays a crucial role in the development of a component of the pacemaker system that is required for the generation of autonomic gut motility.     

Histochemical study on the maturation of human megakaryocytes using microfluorometry

Summary A microcytofluorometrical DNA measurement was basically studied and was applied to single megakaryocytes previously identified on a Wright-Giemsa stained smear. The smear was first photographed and the location of each megakaryocyte was recorded on a cell map. The smear was then bleached with 50% acid ethanol and absolute methanol, and re-stained with 4,6-diamidino-2-phenylindole (DAPI) reagent (pH 7.4) at 4° C. Nuclear blue fluorescence was observed and the intensity of this fluorescence was proportional to the amount of DNA with the coefficient of variation (CV) of 3.6% when stained for 30 min. After 30 min DAPI staining, the DNA measurement was microcytofluorometrically performed in single megakaryocytes which had been morphologically classified into 4 groups on the basis of cytoplasmic maturation, Bessis' classification, assessed on Wright-Giemsa-stained bone-marrow smears from normal human beings. The histograms of the cells did not show any difference in DNA ploidy distribution among the classes: that is, the DNA histograms disclosed ploidy distribution from 4 N to 64 N with the largest population of 16 N. These findings suggest that nuclear DNA synthesis is completed before platelet production starts. This method is useful for comparing the morphological features and DNA content of single megakaryocytes.     

Application of the tryptophanase promoter to high expression of the tryptophan synthase gene inEscherichia coli

Summary The application of an inducible regulation system using the trytophanase operon promoter (TPase promoter; P_tna) was examined for its high expression of the tryptophan synthase (TS) gene in Escherichia coli. The main problem in the application of P_tna for industrial purposes is catabolite repression by glucose, since glucose is the most abundant carbon source. However, this problem could be avoided by changing glucose to an organic acid, such as succinate, fumarate, malate and acetate, in the course of cultivation after glucose initially added was completely consumed. Under these conditions, l-tryptophan was also used to induce tryptophan synthase. Thus, the specific activity of TS in E. coli strain no. 168 harbouring pBR322F-P_tnaTS was increased 500-fold compared to that of the cultured host strain. About 1 mol l-tryptophan/l reaction mixture was formed from indole and l-serine at 37° C for 3.5 h. Offprint requests to: H. Yukawa     

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     

Identification of (Na,K)ATPase inhibitor in brine shrimp,Artemia salina,as long-chain fatty acids

Summary An inhibitory activity to (Na,K)ATPase was found in cell extracts of the brine shrimp, Artemia salina, irrespective of its developmental stages. Organic solvent extraction together with gas chromatographic analysis reveals that the inhibitory activity is due to long-chain, non-esterified fatty acids and their derivatives. Unsaturated fatty acids, especially with cis-configuration, are more effective in inhibition than saturated ones.Abbreviations ATPase adenosine triphosphatase - EDTA ethylenediamine-tetraacetate - TLC thin-layer chromatography     

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.