The stable maintenance system pem of plasmid R100: degradation of PemI protein may allow PemK protein to inhibit cell growth.

We constructed plasmids carrying heat-inducible pemI and pemK genes, which were fused with the collagen-lacZ sequence in frame. The PemK-collagen-LacZ (PemK*) protein produced from the fusion gene upon heat induction inhibited the growth of cells and killed most of the cells in the absence of the PemI protein but did not do so in the presence of the PemI protein. This supports our previous assumption that the PemK protein inhibits cell division, leading to cell death, whereas the PemI protein suppresses the function of the PemK protein. We also constructed the plasmid carrying the heat-inducible pem operon which consists of the intact pemI gene and the pemK gene fused with collagen-lacZ. The simultaneously induced PemI and PemK* proteins did not inhibit the growth of cells. However, the temperature shift to 30 degrees C after induction of both proteins at 42 degrees C caused inhibition of cell growth and death of most cells. This suggests that the PemI protein is somehow inactivated upon the arrest of de novo synthesis of the PemI and PemK* proteins, allowing the PemK* protein to function. We observed that the PemI-collagen-LacZ (PemI*) protein was degraded faster than the PemK* protein, perhaps by the action of a protease(s). In fact, the lon mutation, which caused no apparent degradation of the PemI* protein, did not allow the PemK* protein to function, supporting the suggestion described above. Instability of the PemI protein would explain why the cells which have lost the pem+ plasmid are preferentially killed.     

Biochemical and immuno- and lectin-histochemical studies of solubility and retention of bone matrix proteins during EDTA demineralization

Summary The present study utilized biochemical and immuno-and lectin-histochemical methods to demonstrate solubility and retention of mineral-binding non-collagenous proteins in rat midshaft subperiosteal bone during EDTA demineralization. A monoclonal antibody (9-A-2) specific for chondroitin 4-sulphate and dermatan sulphate and wheat germ agglutinin (WGA) specific forN-acetyl-d-glucosamine,N-acetylneuraminic acid, andN-acetyl-d-galactosamine were used. Bone proteins were extracted from fresh unfixed or aldehyde-fixed specimens with a three step extraction procedure, 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl, followed by GdnCl. For comparison with the second extraction step, ethanolic trimethylammonium EDTA (ethanolic EDTA) was substituted for aqueous EDTA. Based on protein staining and Western blot analysis of SDS-polyacrylamide gel electrophoresis of each extract using 9-A-2 and WGA, retention of mineral-binding proteins extractable from fresh specimens with aqueous EDTA was greatly increased in tissue when ethanolic EDTA was used. Their retention was even greater with prior aldehyde fixation. Maximum retention with no detectable solubility of 9-A-2 and WGA reactive proteins was obtained after ethanolic EDTA extraction of aldehyde-fixed specimens, which concomitantly provided the strongest immuno- and lectin staining. These results indicate that this combined method dramatically improves retention of PGs and glycoproteins during demineralization of bone tissues and provides the best method for localizing these glycoconjugates.     

Differential establishment and survival of Hymenolepis diminuta in syngeneic and outbred rat strains.

Experimental Hymenolepis diminuta infection was carried out in inbred strains of rats (F344/N, JAR-2, LOU/M, TM, DA and DA-bg/bg) and outbred Wistar rats. All strains became infected with this cestode, but clear strain-dependent variation in the susceptibility to H. diminuta infection was observed. Marked differences in worm persistence and worm weight were found at 6 weeks post-infection in TM and DA rats. These strains would be useful to clarify the interactions between H. diminuta and its rat host.     

An endonuclease activity in human polymorphonuclear neutrophils that removes 8-hydroxyguanine residues from DNA+

An endonuclease that specifically removes 8-hydroxyguanine (oh8Gua) from DNA has been isolated from Escherichia coli. As the amount of oh8Gua produced in DNA of X-ray-irradiated mice is known to decrease with time after irradiation, an attempt was made to find a similar activity in human polymorphonuclear neutrophils (PMNs) using a synthetic dsDNA containing oh8Gua as a substrate. The PMN enzyme was isolated free of other DNases, and found to cleave the substrate DNA simultaneously at 2 sites, the phosphodiester bonds 5' and 3' to oh8Gua, producing free hydroxyl and phosphate groups, respectively. The enzyme showed almost no activity on DNAs containing other kinds of modified base tested or mismatched DNA. Thus human cells also contain an endonuclease that specifically removes oh8Gua residues from DNA.     

Effect of erythroid differentiation factor on megakaryocytic differentiation of L8057, a murine megakaryoblastic leukemia cell line.

To assess the potent effect of erythroid differentiation factor (EDF) on megakaryocytopoiesis, effect of EDF on megakaryocytic differentiation of L8057, a murine megakaryoblastic cell line, was examined. EDF potentiated AchE induction of L8057 in a dose dependent manner. The potency of EDF on megakaryocytic differentiation is comparable to that on erythroid differentiation reported previously. The present results suggest that EDF may play a regulatory role in megakaryocytopoiesis as well as in erythropoiesis.     

Verbascoside production by plant cell cultures

Verbascoside was found to be produced in all calli derived from eleven species that contained the compound in their leaves. Cell suspension cultures were also established in three species, i.e., Leucosceptrum japonicum f. barbinerve, Syringa josikaea, and Sy. vulgaris, all of which were found to produce verbascoside at more than 1 g/l. Of the three species, suspension cultures of L. japonicum f. barbinerve showed rapid growth and the highest yield of verbascoside (1.89 g/l). In these cultures, the effects of major salt concentration in B5 medium on cell growth and verbascoside production were examined. Maximum cell growth and maximum verbascoside production were both achieved by reducing the major salt concentration to half that of the original medium.     

Synthesis of bombyxin-IV,an insulin superfamily peptide from the silkworm,Bombyx mori,by stepwise and selective formation of three disulfide Bridges

We report the synthesis of bombyxin-IV, a disulfide-linked, heterodimeric, insulin superfamily peptide from the silkworm,Bombyx mori. The two chains (A- and B-chains) were synthesized separately by the solid-phase method using fluoren-9-ylmethoxycarbonyl (Fmoc) group as a protecting group for α-amino group. Three disulfide bonds were bridged step by step (A6–A11, A20–B22, and A7–B10) in a good yield. Synthetic bombyxin-IV was identical with natural one with regard to the retention time on a reversed-phase column and the molecular weight measured by mass spectrometry. Circular dichroism (CD) spectrum of the synthetic bombyxin-IV was very similar to that of the natural one. The specific activity of synthetic bombyxin-IV is equal to that of natural one (0.1 ng/Samia unit). These results suggest that the synthetic bombyxin-IV has the tertiary structure identical with the natural peptide. Our method developed for synthesis of bombyxin-IV would be generally applicable to the synthesis of insulin-like heterodimeric peptides.     

Cloning and nucleotide sequence of cDNA for retinochrome, retinal photoisomerase from the squid retina

The Rhodopsin-retinochrome system is essential for the visual photoreception of molluscs. cDNA coding for retinochrome of the squid (Todarodes pacificus) was cloned and the nucleotide sequence has been determined. The sequence (2.1 kb) covers the whole coding region of 903 bp. The deduced primary sequence suggests that retinochrome contains seven transmembrane spanning domains. The homology with bovine rhodopsin and the possible retinal binding site are also discussed.     

Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli

The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product.     

New method of immobilization of microbial cells by capture on the surface of insoluble pyridinium-type resin

A new method for the immobilization of microbial cells has been developed. Whole cells of Escherichia coli with aspartase activity were immobilized by capture on the surface of cross-linked poly(N-benzyl-4-vinylpyridinium bromide) containing styrene (BVPS resin), an insoluble pyridinium-type resin. When a suspension of the bacterial cells in buffer solution was passed through a glass column containing beads of BVPS resin, the cells were captured on the resin surface and formed an immobilized cell system. A fixed-bed column reactor containing 300 mg of the bacterial cells immobilized by capture on 10 g of BVPS resin beads was used for the preparation of L-aspartic acid from ammonium fumarate. Continuous operation of tne bioreactor produced L-aspartic acid in a quantitative yield when the influent substrate concentration was 0.1M and the flow rate was 0.41-0.83 bed volumes per hour at pH 7.4-7.7 at 30 degrees C.