A mutant hemolysin with lower biological activity produced by a mutant Vibrio parahaemolyticus

Abstract A mutant toxin (m-TDH) of thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus w was isolated from the culture of a strain of this organism mutagenized with N -methyl- N '-nitro- N -nitrosoguanidine. Although the m-TDH had a molecular structure similar to the native Vp-TDH, the m-TDH retained only about 7% residual hemolytic activity of the native toxin. Furthermore, other biological activities of m-TDH, such as lethality in mice and enterotoxicity in rabbit ileal loops, were also weakened. The m-TDH was immunologically indistinguishable from the native Vp-TDH. These results suggest that the m-TDH is only slightly different in structure from the native Vp-TDH. Also, the mutagenized site in m-TDH, which is not immunogenic, seems to be involved in expressing not only hemolytic activity but also lethal and enterotoxic activity.     

Molecular cloning and nucleotide sequence of the gramicidin S synthetase 1 gene

The entire gene for gramicidin S synthetase 1 (GS 1) was cloned into the plasmid vector pUC18, and the nucleotide sequences of the GS 1 gene and its flanking region were determined. The full-length clone was 4,539 base pairs long and had an open reading frame of 3,294 nucleotides coding for 1,098 amino acids. The calculated molecular weight of 123,474 agreed with the apparent molecular weight of 120,000 found in SDS-PAGE of GS 1 from B. brevis. The nucleotide sequence of GS 1 gene was highly homologous to that of tyrocidine synthetase 1. The overall similarity between the deduced amino acid sequences of the two genes was 57.5%. The gene product of clone GS309 was easily purified to an essentially homogeneous state by ammonium sulfate fractionation followed by DEAE-Sepharose CL-6B, Ultrogel AcA-34, and second DEAE-Sepharose CL-6B column chromatography. The purified protein catalyzed the D-phenylalanine-dependent ATP-32PPi exchange reaction which is specific for GS 1 activity, and the specific activity of the purified product was nearly the same as the purified GS 1 from B. brevis. The product also showed a weak phenylalanine racemase activity.     

Characterization of two glucuronic acid-containing glycosphingolipids in larvae of the green-bottle fly, Lucilia caesar

Two glucuronic acid-containing glycosphingolipids were purified from larvae of the green-bottle fly, Lucilia caesar by DEAE-Sephadex and Iatrobeads column chromatography. Structures of these acidic glycolipids, glycolipids X and Y, were elucidated by means of sugar analysis, permethylation, enzymatic hydrolysis, negative-ion fast atom bombardment mass spectrometry, and NMR studies. Glycolipid X was determined to have the following structure: GlcA beta 1-3Gal beta 1-3GalNAc alpha 1-4 GalNAc beta 1-4 GlcNAc beta 1-3Man beta 1-4Glc beta 1-1 ceramide. The other acidic glycolipid, glycolipid Y contains a phosphoethanolamine residue linked through the 6-hydroxy group of the N-acetyl-glucosamine unit of glycolipid X. The ceramide moieties were composed of saturated fatty acids (16:0-22:0) and tetradeca- and hexadeca-4-sphingenines. Based on the structural similarity of the ceramide moieties it appears likely that glycolipid X is an intermediate from which glycolipid Y is synthesized by addition of a phosphoethanolamine residue.     

Hepatocyte Growth Factor (HGF)-Induced Cell Migration Is Negatively Modulated by Epidermal Growth Factor through Tyrosine Phosphorylation of the HGF Receptor

Hepatocyte growth factor (HGF) stimulated cell migration of human gastric carcinoma cell lines MKN1, MKN7, and MKN28. Epidermal growth factor (EGF) also stimulated the cell migration of these three cell lines. In MKN7 cells, HGF-stimulated cell migration was rather reduced in the presence of EGF, whereas such an observation was not made with MKN1 and MKN28 cells. Therefore, we compared the effect of EGF on HGF-stimulated HGF receptor phosphorylation in these cell lines. HGF induced a rapid tyrosine phosphorylation of the HGF receptor in all these cell lines. In MKN7 cells, the increased phosphorylation was further enhanced by EGF, although EGF alone did not affect tyrosine phosphorylation of the HGF receptor. In MKN1 and MKN28 cells, EGF did not influence tyrosine phosphorylation of the HGF receptor, whether HGF was present or not. The data presented here suggest that EGF negatively modulates the cellular response to HGF by increasing tyrosine phosphorylation of the HGF receptor in certain types of epithelial cells, e.g., MKN7 cells.     

The Om(1e) Mutation in Drosophila Ananassae Causes Compound Eye Overgrowth Due to Tom Retrotransposon-Driven Overexpression of a Novel Gene

Optic morphology (Om) mutations in Drosophila ananassae are a group of retrotransposon (tom)-induced gain-of-function mutations that map to at least 22 independent loci and exclusively affect the compound eye morphology. In marked contrast to other Om mutations, which are characterized by fewer-than-normal and disorganized ommatidia, the Om(1E) mutation exhibits a peculiar phenotype as enlarged eyes with regularly arrayed normal ommatidia. To characterize the Om(1E) mutation, we have carried out molecular analyses. A putative Om(1E) locus cloned by tom tagging and chromosome walking contained two transcribed regions in the vicinity of tom insertion sites of the Om(1E) mutant alleles, and one of these regions was shown to be the Om(1E) gene by P element-mediated transformation experiments with D. melanogaster. The Om(1E) gene encodes a novel protein having potential transmembrane domain(s). In situ hybridization analyses demonstrated that the Om(1E) gene is expressed ubiquitously in embryonic cells, imaginal discs, and the cortex of the central nervous system of third instar larvae, and specifically in lamina precursor cells. Artificially induced ubiquitous overexpression of Om(1E) affected morphogenesis of wing imaginal disc derivatives or large bristle formation. These findings suggest that the Om(1E) gene is involved in a variety of developmental processes.     

Effects of estradiol and testosterone on the synthesis,expression and degradation of androgen receptor in human uterine endometrial fibroblasts

The mechanism of known receptor-mediated androgen effects on the endometrial stroma was studied in endometrial fibroblasts derived from human uterus. 17-Estradiol (E) induced the expressions of androgen receptor (AR) mRNA, and predominantly increased the level of testosterone-binding sites (TBS) in uterine endometrial fibroblasts. The effect on the level of dihydrotestosterone-binding sites (DHTBS) was similar but smaller. This result suggests that the AR mRNA expressed might encode TBS, but probably not DHTBS. The TBS level increased by estrogen was down-regulated by testosterone (T) + E, but the AR mRNA expression increased by E was not down-regulated by E + T in the fibroblasts. Although the synthesis rate of AR was slightly increased (p<0.05) by E alone or E + T, the degradation rate of AR was significantly accelerated (p<0.05) by E + T in the fibroblasts. This result suggests that T might stimulate the metabolic rate of TBS, but does not inhibit the synthesis rate of AR mRNA to TBS in endometrial fibroblasts.     

Further confirmation of the presence of DNA in the pyrenoid core of the siphonous green algal genus Caulerpa (Caulerpales, Ulvophyceae, Chlorophyta)

We re-examined the distribution of chloroplast DNA (ct-DNA) in the pyrenoid core of Caulerpa okamurae Weber van Bosse and C. lentillifera J. Agardh by fluorescence microscopy after staining the squashes and Technovit sections with DNA fluorochromes such as 4′6-diamidino-2-phenylmdole (DAPI), ethidium bromide, Hoechst 33258 and chromomycin A3. All fluorochromes stained specifically the pyrenoid core on the squashes and Technovit sections. In addition, we present new data on the localization of ct-DNA in the pyrenoid core of two other species of the genus Caulerpa: C. cactoides (Turner) Agardh and C. geminata Harvey.     

Immunocytochemical detection of Ca2+-dependent subspecies of protein kinase C in mouse embryos before and during compaction

Summary To confirm the possibility that protein kinase C is involved in compaction of mouse embryos, the presence and distribution pattern of Ca²⁺-dependent subspecies of this enzyme in mouse embryos, before and during compaction, were examined immunocytochemically with three different monoclonal antibodies. These were MC-1a, MC-2a and MC-3a, which selectively interact with the subspecies of the enzyme known as types I, II and III, respectively. Only when embryos were incubated with MC-3a, was immunofluorescence clearly detected in all cells of embryos before and during compaction. This result demonstrates the presence of type III protein kinase C in embryos before and during compaction and suggests the possibility that the type III enzyme may be involved in compaction. No marked differences were found in the distribution pattern of the type III enzyme between embryos examined before and during compaction.     

Regulation by Androgen of Levels of the β Subunit of Nerve Growth Factor and Its mRNA in Selected Regions of the Mouse Brain

Abstract: Our previous studies showed that the concentration of the β subunit of nerve growth factor (β-NGF) in nervous tissues is higher in male than in female mice. To identify the brain regions that are affected by androgens, the amounts of β-NGF protein and its mRNAs were measured in male, female, and castrated male CD-1 mice and testicular feminization mice at 3–4 months of age. Among tissues examined, the hypophysis of males contained the highest average concentration of β-NGF protein. In most regions of the brain, individual levels were more variable in males than in females. However, after the castration, such variations in β-NGF levels disappeared. Average levels of β-NGF protein in males were higher in the cerebellum (eightfold higher), olfactory bulb (12-fold higher), hypothalamus (sixfold higher), and hypophysis (72-fold higher) than thope in corresponding regions of females. No significant differences were observed in levels of β-NGF protein in the hippocampus, cerebral cortex, striatum, septum, and brainstem. The castration of male mice caused a reduction in levels of β-NGF protein in the hypothalamus and hypophysis, but not in the cerebellum and olfactory bulb, to the femgle levels. The concentrations of β-NGF protein in testicular feminization mice were similar to those in female CD-1 mice in all regions. The concentrations of mRNA for β-NGF in the olfactory bulb and hypophysis from males were higher than those from females. By contrast, northern blots showed no remarkable differences in the amounts of brain-derived neurotrophic factor and neurotrophin-3 between the two sexes. Thus, in some regions of the brain, the production of β-NGF appears to be regulated by testosterone, but the regulatory mechanisms do not appear to be simple. Our present results indicate that the binding of testosterone to its receptor is an important step in the regulation of the level of β-NGF in these region.     

Production of poly(3-hydroxyalkanoates-co-3-hydroxy-ω-fluoroalkanoates) byPseudomonas oleovorans from 1-fluorononane and gluconate

Summary Pseudomonas oleovorans grew well and synthesized copolyesters of 3-hydroxyalkanoates and 3-hydroxy--fluoroalkanoates in the mineral medium containing 1-fluorononane and sodium gluconate. The content of fluorinated units in copolyesters could be controlled from 0 to 40 mol%. The copolyesters were shown to have a random sequence distribution of different monomeric units by analysis of the¹³C NMR spectra. The melting temperatures of copolyesters were 52–58°C, and the enthalpy of fusion decreased with the content of fluorinated units.