Habitat Segregation and Biochemical Activities of Marine Members of the Family Vibrionaceae

A comparative study of marine members of the family Vibrionaceae with the technique of numerical taxonomy revealed habitat segregation as well as a cosmopolitan nature of species distribution among the vibrios in different marine environments. The bacterial strains analyzed were isolated from seawater, sediments, phyto- and zooplankton, and fish in the Indian Ocean, the South and East China Sea, and West Pacific Ocean, and coastal areas of Japan. A total of 155 morphological, physiological, and biochemical tests were carried out for each of 405 strains examined. The results showed that most of the large taxonomical clusters which emerged from the computation corresponded to ecological groups which have particular niches. For instance, each group of seawater vibrios inhabited a particular water layer of limited depth range, in spite of the fact that strains of the group were isolated from sampling locations spread over a wide area from the Indian Ocean to Japanese coast. Various vibrio groups showed remarkable differences in their physiological and biochemical activities, and the activities of each group seemed to correspond with its ecological niche. The strains which inhabited surface-water layers grew fast and actively utilized many high-molecular-weight organic compounds and carbohydrates that are derived from fresh, easily degradable organic matter present in the surface waters, whereas the middle- and deep-water vibrios did not decompose most of the high-molecular-weight organic compounds except chitin but, rather, utilized some carbohydrates and organic acids which seemed to be derived from refractory particulate organic matter present in the deeper waters.     

Comparison of the biological properties of purified natural and recombinant human interleukin-2

We compared the biological properties of the purified recombinant human IL-2 derived from E. coli with those of purified natural IL-2. Both had almost the same specific in vitro activities on a weight basis to support long-term proliferation of IL-2 dependent human peripheral blood lymphocytes, a mouse killer T cell line, and a mouse natural killer cell line; induce killer cells in normal mouse spleen cells; and induce antibody forming cells in nude mouse spleen cells. No differences in these biological activities were found between two forms of natural IL-2 that were separable by reverse phase high performance liquid chromatography.     

Extension of the substrate spectrum by an amino acid substitution at residue 219 in the Citrobacter freundii cephalosporinase

The cephalosporinase of Citrobacter freundii GN346 is a class C beta-lactamase, consisting of 361 amino acids and exhibiting the substrate profile of a typical cephalosporinase. On the conversion of a conserved glutamic acid at residue 219 to lysine, the substrate spectrum of the cephalosporinase was extended to oxyimino cephalosporins, aztreonam and carbenicillin, which are essentially undesirable substrates for the enzyme. Escherichia coli cells carrying the mutant gene showed higher resistance levels to cefuroxime, aztreonam, and carbenicillin, but a lower resistance level to cefoxitin, than cells carrying the wild gene. The kcat values of the purified mutant enzyme for ceftazidime, cefuroxime, and cefmenoxime were 77,100, and 300 times those of the wild enzyme, respectively. The relative Vmax values of the mutant enzyme for aztreonam and carbenicillin were determined to be 11 and 23 times those of the wild enzyme, respectively, but the value of the mutant enzyme for cefoxitin was only one-third that of the wild enzyme.     

Two distinct class II molecules encoded by the genes within HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating and restriction molecules

By using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we investigated the difference in the HLA class II molecule between HLA-Dw2 and Dw12, both of which are typed as HLA-DR2 serologically. The anti-HLA-DR framework monoclonal antibody (MoAb) HU-4 precipitated an alpha-chain and two beta-chains of human class II molecules from both Dw2 and Dw12 homozygous B lymphoblastoid cell lines. It was demonstrated clearly that an alpha-chain (alpha 1) and one of the beta-chains (beta 1) showed no difference in mobility in the 2D-PAGE between Dw2 and Dw12, but that another beta chain (beta 2) of Dw2 was distinct from that of Dw12 in the 2D-PAGE profile. Thus, MoAb HU-4 precipitated alpha 1 beta 1 and alpha 1 beta 2 molecules from Dw2 and Dw12, and the alpha 1 beta 1 molecule appears to be an HLA-DR2 molecule. The alpha 1 beta 2 molecule, on the other hand, is a class II molecule distinct from those precipitated with anti-DR2, anti-DQw1 (DC1, MB1, MT1), or anti-FA MoAbs. MoAb HU-4 completely inhibited the mixed lymphocyte culture reaction (MLR) between Dw2 and Dw12, but anti-DR2 MoAb HU-30, which reacts only with the alpha 1 beta 1 molecule, did not show an inhibitory effect on the MLR between Dw2 and Dw12. The alpha 1 beta 2 molecule is therefore the molecule which elicits MLR between Dw2 and Dw12. An IL 2-dependent T cell line established from an HLA-Dw12/D blank heterozygous high responder to the streptococcal cell wall antigen (SCW) clearly distinguished the Dw2 specificity from Dw12 specificity expressed on the antigen-presenting cell (APC). Moreover, MoAb HU-4 markedly inhibited the cooperation between the T cell line and APC to respond to SCW. These observations indicate that the alpha 1 beta 2 molecule is recognized as a restriction molecule by the T cell line at the antigen presentation of SCW through APC MoAb HU-30 on the other hand partially inhibited the MLR between Dw2 or Dw12 homozygous cell as a stimulator cell and non DR2 cell as a responder cell. It markedly inhibited the proliferative response of the Dw12/D- heterozygous T cell line to SCW, presented by Dw2+ but Dw12- allogeneic APC, and the peripheral response of Dw2 or Dw12 homozygous peripheral blood lymphocytes to SCW. Thus, two distinct class II molecules encoded by the genes within the HLA-DR subregion of HLA-Dw2 and Dw12 can act as stimulating molecules in the MLR and as restriction molecules in the antigen presentation by APC.     

Nucleotide sequence of the maltohexaose-producing amylase gene from an alkalophilic Bacillus sp. #707 and structural similarity to liquefying type alpha-amylases

The nucleotide sequence of the gene for maltohexaose-producing amylase from an alkalophilic Bacillus sp. #707 was determined. Starting at an ATG initiation codon, an open reading frame was composed of 1554 bp (518 amino acids). The NH2-terminal portion encoded a 33 amino acid-long signal peptide. The deduced amino acid sequence of the extracellular mature enzyme was more than 60% homologous to those of the liquefying type alpha-amylases but not to those of the saccharifying type alpha-amylases. The sequence of its signal peptide was completely different from those of other alpha-amylases.     

Allelic differentiation at the <Emphasis Type="Italic">Sg-1</Emphasis> locus for the terminal sugar of the C-22 position of group A saponin in Chinese wild soybean (<Emphasis Type="Italic">Glycine soja</Emphasis> Sieb. &amp; Zucc.)

Group A saponins are thought to be the cause of bitter and astringent tastes in processed foods of soybean (Glycine max), and the elimination of group A saponins is an important breeding objective. The group A saponins include two main Aa and Ab types, controlled by codominant alleles at the Sg-1 locus that is one of several key loci responsible for saponin biosynthesis in the subgenus Glycine soja. However, A0 mutant lacking group A saponin is a useful gene resource for soybean quality breeding. Here, eight Chinese wild soybean A0 accessions were sequenced to reveal the mutational mechanisms, and the results showed that these mutants were caused by at least three kinds of mechanisms involving four allelic variants (sg-1^0-b2, sg-1^0-b3, Sg-1^b-0, and Sg-1^b-01). The sg-1^0-b2 had two nucleotide deletions at positions +?72 and +?73 involving in the 24th and 25th amino acids. The sg-1^0-b3 contained a stop codon (TGA) at the 254th residue. The Sg-1^b-0 and Sg-1^b-01 were two novel A0-type mutants, which likely carried normal structural alleles, and nevertheless did not encode group A saponin due to unknown mutations beyond the normal coding regions. In addition, to reveal the structural features, allelic polymorphism, and mechanisms of the abiogenetic absence of group A (i.e., A0 phenotype), nucleotide sequence analysis was performed for the Sg-1 locus in wild soybean (Glycine soja). The results showed that Sg-1 alleles had a lower conservatism in the coding region; as high as 18 sequences were found in Chinese wild soybeans in addition to the Sg-1^a (Aa) and Sg-1^b (Ab) alleles. Sg-1^a and Sg-1^b alleles were characterized by eight synonymous codons and nine amino acid substitutions. Two evolutionarily transitional allelic sequences (Sg-1^a7 and Sg-1^b2) from Sg-1^a toward Sg-1^b were detected.     

Age and growth of migrating tropical eels,Anguilla celebesensis and Anguilla marmorata

The age and growth of migrating tropical eels, Anguilla celebesensis and Anguilla marmorata from central Sulawesi, Indonesia, were examined. Migrating eels (63 A. celebesensis and 38 A. marmorata ) were obtained from weirs near the Poso Lake outlet and non‐migrating eels (35 A. celebesensis and 119 A. marmorata ) were captured by baited hooks, eel pots, scoop net and electro‐fishing in the Poso River system, Laa River system, Baluga River, Tongku River and Padapu River from February 2009 to October 2010. In both species, the proportion of eels with opaque otolith edges showed a single peak in July, suggesting that one annulus (a pair of translucent and opaque zones) was formed each year in their otoliths. Mean ± s.d . and range of total length (L _T) and age was 785·2 ± 114·9 (585–1083) mm and 7·5 ± 1·6 (5–11) years in migrating female A. celebesensis and 1132·2 ± 173·7 (800–1630) mm and 11·6 ± 3·3 (7–23) years in A. marmorata . The age of migrating female eels was negatively correlated with annual growth rate, 100·7 ± 17·2 (68·1–145·0) mm year^?1 in A. celebesensis and 97·9 ± 19·3 (66·6–131·6) mm year^?1 in A. marmorata , but there was no significant correlation between the L _T and annual growth rate in either species. The annual growth rates of these female tropical eels were typically higher than those of temperate anguillid species, suggesting a latitudinal cline in growth rate in the genus Anguilla reflecting the environmental conditions of their growth habitat.     

Ca2+‐dependent down‐regulation of human histamine H1 receptors in Chinese hamster ovary cells

G_q/11 protein‐coupled human histamine H₁ receptors in Chinese hamster ovary cells stimulated with histamine undergo clathrin‐dependent endocytosis followed by proteasome/lysosome‐mediated down‐regulation. In this study, we evaluated the effects of a sustained increase in intracellular Ca²⁺ concentrations induced by a receptor‐bypassed stimulation with ionomycin, a Ca²⁺ ionophore, on the endocytosis and down‐regulation of H₁ receptors in Chinese hamster ovary cells. All cellular and cell‐surface H₁ receptors were detected by the binding of [³H]mepyramine to intact cells sensitive to the hydrophobic and hydrophilic H₁ receptor ligands, mepyramine and pirdonium, respectively. The pretreatment of cells with ionomycin markedly reduced the mepyramine‐ and pirdonium‐sensitive binding sites of [³H]mepyramine, which were completely abrogated by the deprivation of extracellular Ca²⁺ and partially by a ubiquitin‐activating enzyme inhibitor (UBEI‐41), but were not affected by inhibitors of calmodulin (W‐7 or calmidazolium) and protein kinase C (chelerythrine or GF109203X). These ionomycin‐induced changes were also not affected by inhibitors of receptor endocytosis via clathrin (hypertonic sucrose) and caveolae/lipid rafts (filipin or nystatin) or by inhibitors of lysosomes (E‐64, leupeptin, chloroquine, or NH₄Cl), proteasomes (lactacystin or MG‐132), and a Ca²⁺‐dependent non‐lysosomal cysteine protease (calpain) (MDL28170). Since H₁ receptors were normally detected by confocal immunofluorescence microscopy with an antibody against H₁ receptors, even after the ionomycin treatment, H₁ receptors appeared to exist in a form to which [³H]mepyramine was unable to bind. These results suggest that H₁ receptors are apparently down‐regulated by a sustained increase in intracellular Ca²⁺ concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.

     

Microglia Endocytose Amyloid β Through the Binding of Transglutaminase 2 and Milk Fat Globule EGF Factor 8 Protein

Activation of glial cells has been observed in neurodegenerative diseases including Alzheimer’s disease (AD). Aggregation of amyloid β (Aβ) is profusely observed as characteristic pathology in AD brain. In our previous study using microglial cell line BV-2, tissue-type transglutaminase (TG2) was found to be involved in phagocytosis (Kawabe et al., in Neuroimmunomodulation 22(4):243–249, 2015; Kawabe et al., Neurochem Res 2017). In the present study, we examined whether TG2 and milk fat globule EGF factor 8 protein (MFG-E8), an adaptor protein promotes macrophage to engulf apoptotic cells, were involved in Aβ endocytosis. When the neuronal/glial mixed culture was stimulated freshly prepared Aβ_1?42 for 3 days, the incorporation of Aβ was observed by immunofluorescence staining technique in Iba-1-positive microglia. Cystamine, a broad competitive inhibitor of TGs, suppressed it. When aggregated Aβ was added to the mixed culture, the immunoreactivity of MFG-E8 surrounding Aβ was observed, and then followed by microglial endocytosis. Using western blotting technique, MFG-E8 was detected in cell lysate of astrocyte culture, and was also detected in the medium. When microglia culture was incubated with astrocyte conditioned medium, MFG-E8 levels in microglia tended to increase. It is likely that microglia might utilize MFG-E8 released from astrocytes as well as that expressed in themselves in order to endocytose Aβ aggregation. Furthermore, we confirmed that MFG-E8 could bind with TG2 in microglia culture by immunoprecipitate technique. These results suggest that microglia might uptake Aβ as a complex of aggregated Aβ/MFG-E8/TG2.     

Inhibition of Gap Junction Elevates Glutamate Uptake in Cultured Astrocytes

Glutamate uptake is a main function of astrocytes to keep extracellular glutamate levels low and protect neurons against glutamate-induced excitotoxicity. On the other hand, astrocyte networks formed by gap junctions, which are consisted with connexins and connecting neighboring cells, are reported to play a critical role in maintaining the homeostasis in the brain. In the present study, we examined the effects of gap junction inhibitors on the glutamate uptake activity in cultured rat cortical astrocytes. At first, we confirmed the effects of gap junction inhibitors, 1-octanol and carbenoxolone, on cell–cell communication by the scrape-loading assay using a fluorescent dye Lucifer yellow. Both of 1-octanol and carbenoxolone treatments for 20 min in cultured astrocytes significantly suppressed the cell–cell communication assessed as the distance of dye-spreading. 1-octanol and carbenoxolone increased the glutamate uptake by astrocytes and glutamate aspartate transporter (GLAST) expression on the cell membrane. These results suggest that gap junction inhibitors increase the glutamate uptake activity through the increase of GLAST proteins located on the cell membrane. The regulation of gap junction in astrocytes might protect neurons against glutamate-induced excitotoxicity.