The role of glycerol-3-phosphate dehydrogenase 1 in the progression of fatty liver after acute ethanol administration in mice

Acute ethanol consumption leads to the accumulation of triglycerides (TGs) in hepatocytes. The increase in lipogenesis and reduction of fatty acid oxidation are implicated as the mechanisms underlying ethanol-induced hepatic TG accumulation. Although glycerol-3-phosphate (Gro3P), formed by glycerol kinase (GYK) or glycerol-3-phosphate dehydrogenase 1 (GPD1), is also required for TG synthesis, the roles of GYK and GPD1 have been the subject of some debate. In this study, we examine (1) the expression of genes involved in Gro3P production in the liver of C57BL/6J mice in the context of hepatic TG accumulation after acute ethanol intake, and (2) the role of GPD1 in the progression of ethanol-induced fatty liver using GPD1 null mice. As a result, in C57BL/6J mice, ethanol-induced hepatic TG accumulation began within 2 h and was 1.7-fold greater than that observed in the control group after 6 h. The up-regulation of GPD1 began 2 h after administering ethanol, and significantly increased 6 h later with the concomitant escalation in the glycolytic gene expression. The incorporation of ¹⁴C-labelled glucose into TG glycerol moieties increased during the same period. On the other hand, in GPD1 null mice carrying normal GYK activity, no significant increase in hepatic TG level was observed after acute ethanol intake. In conclusion, GPD1 and glycolytic gene expression is up-regulated by ethanol, and GPD1-mediated incorporation of glucose into TG glycerol moieties together with increased lipogenesis, is suggested to play an important role in ethanol-induced hepatic TG accumulation.     

Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte growth factor

In the present study, we examined the roles of hepatocyte growth factor (HGF) and nitric oxide (NO) in the activation of satellite cells in passively stretched rat skeletal muscle. A hindlimb suspension model was developed in which the vastus, adductor, and gracilis muscles were subjected to stretch for 1 h. Satellite cells were activated by stretch determined on the basis of 5-bromo-2'-deoxyuridine (BrdU) incorporation in vivo. Extracts from stretched muscles stimulated BrdU incorporation in freshly isolated control rat satellite cells in a concentration-dependent manner. Extracts from stretched muscles contained the active form of HGF, and the satellite cell-activating activity could be neutralized by incubation with anti-HGF antibody. The involvement of NO was investigated by administering nitro-L-arginine methyl ester (L-NAME) or the inactive enantiomer N^G-nitro-D-arginine methyl ester HCl (D-NAME) before stretch treatment. In vivo activation of satellite cells in stretched muscle was not inhibited by D-NAME but was inhibited by L-NAME. The activity of stretched muscle extract was abolished by L-NAME treatment but could be restored by the addition of HGF, indicating that the extract was not inhibitory. Finally, NO synthase activity in stretched and unstretched muscles was assayed in muscle extracts immediately after 2-h stretch treatment and was found to be elevated in stretched muscle but not in stretched muscle from L-NAME-treated rats. The results of these experiments demonstrate that stretching muscle liberates HGF in a NO-dependent manner, which can activate satellite cells. muscle regeneration     

Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches

Fourteen strains of fructophilic lactic acid bacteria were isolated from fructose-rich niches, flowers, and fruits. Phylogenetic analysis and BLAST analysis of 16S rDNA sequences identified six strains as Lactobacillus kunkeei, four as Fructobacillus pseudoficulneus, and one as Fructobacillus fructosus. The remaining three strains grouped within the Lactobacillus buchneri phylogenetic subcluster, but shared low sequence similarities to other known Lactobacillus spp. The fructophilic strains fermented only a few carbohydrates and fermented d-fructose faster than d-glucose. Based on the growth characteristics, the 14 isolates were divided into two groups. Strains in the first group containing L. kunkeei, F. fructosus, and F. pseudoficulneus grew well on d-fructose and on d-glucose with pyruvate or oxygen as external electron acceptors, but poorly on d-glucose without the electron acceptors. Strains in this group were classified as “obligately” fructophilic lactic acid bacteria. The second group contained three unidentified strains of Lactobacillus that grew well on d-fructose and on d-glucose with the electron acceptors. These strains grew on d-glucose without the electron acceptors, but at a delayed rate. Strains in this group were classified as facultatively fructophilic lactic acid bacteria. All fructophilic isolates were heterofermentative lactic acid bacteria, but “obligately” fructophilic lactic acid bacteria mainly produced lactic acid and acetic acid and very little ethanol from d-glucose. Facultatively fructophilic strains produced lactic acid, acetic acid and ethanol, but at a ratio different from that recorded for heterofermentative lactic acid bacteria. These unique characteristics may have been obtained through adaptation to the habitat.     

The Aromatic Ring of Phenylalanine 334 Is Essential for Oligomerization of Vibrio vulnificus Hemolysin

Vibrio vulnificus hemolysin (VVH) is thought to be a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins. To date, the structure-function relationships of CDCs produced by Gram-negative bacteria remain largely unknown. We show here that the aromatic ring of phenylalanine residue conserved in Vibrionaceae hemolysins is essential for oligomerization of VVH. We generated the VVH mutants; substituted Phe 334 for Ile (F334I), Ala (F334A), Tyr (F334Y), or Trp (F334W); and tested their binding and oligomerizing activity on Chinese hamster ovary cells. Binding in all mutants fell by approximately 50% compared with that in the wild type. Oligomerizing activities were completely eliminated in F334I and F334A mutants, whereas this ability was partially retained in F334Y and F334W mutants. These findings indicate that both hydrophobicity and an aromatic ring residue at the 334th position were needed for full binding activity and that the oligomerizing activity of this toxin was dependent on the existence of an aromatic ring residue at the 334th position. Our findings might help further understanding of the structure-and-function relationships in Vibrionaceae hemolysins.Vibrio vulnificus hemolysin (VVH) is a pore-forming toxin produced by the Gram-negative bacterium Vibrio vulnificus (6, 11). VVH binds directly to cholesterol and is oligomerized in vitro. Once VVH forms the VVH-cholesterol complex, it can no longer bind to susceptible cells (10). Therefore, VVH could be considered a member of the cholesterol-dependent cytolysin (CDC) toxin family (35).A wide variety of Gram-positive and some Gram-negative bacteria produce CDCs, which require cellular cholesterol to exert their cytotoxicity (22, 38). Structure-function relationships between CDCs produced by Gram-positive bacteria (gpCDCs) have been studied intensively for over a decade, whereas CDCs produced by Gram-negative bacteria remain largely unknown. On the other hand, it is well known that some Vibrionaceae bacteria, such as Vibrio vulnificus, Vibrio cholerae, Aeromonas hydrophila, and Aeromonas sobria, produce pore-forming toxins/hemolysins. Among them, it was reported that VVH and Vibrio cholerae cytolysin (VCC) required cholesterol to exert their activity (12, 35). Thus, Vibrionaceae hemolysins are thought to be members of the CDC family. The generalized toxic steps are thought to be similar for both gpCDCs and Vibrionaceae hemolysins (22); i.e., monomers interact with a susceptible cell membrane, these monomers are assembled to form oligomers by membrane fluidity, and transmembrane pore formation follows (5, 22, 27, 30, 37). Although gpCDCs and Vibrionaceae hemolysins have common toxic steps, the following differences exist between them. (i) There is no similarity in amino acid sequences. (ii) gpCDCs have a highly conserved tryptophan-rich motif, which is involved in membrane recognition (3, 9, 27), whereas this motif does not exist in Vibrionaceae hemolysins. (iii) gpCDCs, such as perfringolysin and intermedilicine, are composed of four domains, whereas Vibrionaceae hemolysins are composed of two or three domains (21, 24, 25). (iv) Vibrionaceae hemolysins form pores that are smaller (2 to 3 nm in diameter) (33, 36) than those formed by gpCDCs (approximately 30 nm) (1, 2, 19).Recently, the crystal structure of VCC was determined (21). VCC is composed of three domains, namely the cytolysin domain, the β-trefoil lectin domain, and the β-prism lectin domain (21). The proposed mechanisms of action of VCC are as follows: (i) monomer binding to cell surfaces via interactions with the cytolysin domain, (ii) binding to carbohydrate receptors by the β-prism lectin domain, (iii) oligomerization via the cytolysin domain, and (iv) pore formation by insertion of a stem-loop from the cytolysin domain into the cellular membrane (21). On the other hand, from the analysis of the VVH amino acid sequence, it has been predicted that VVH is composed of two domains (21) and is missing the β-prism lectin domain, which binds to carbohydrate receptors on the cellular membrane (21). Therefore, the structure and functions of VVH are thought to be slightly different from those of VCC. Thus, analysis of the structure-function relationship of VVH will aid in the understanding of the evolutionary process of CDCs as well as of the toxic mechanism of VVH.In this study, we show that phenylalanine in the 334th position (F334) is required for the binding and oligomerizing ability of VVH. In particular, the benzene ring of this phenylalanine is a prerequisite for its oligomerizing ability. Because of the high conservation of this phenylalanine in other Vibrionaceae hemolysins, our results will contribute to a better understanding of the structure-function relationships of Vibrionaceae hemolysins.     

Molecular typing of Campylobacter jejuni and C. coli from chickens and patients with gastritis or Guillain‐Barré syndrome based on multilocus sequence types and pulsed‐field gel electrophoresis patterns

Campylobacter jejuni has recently been noted as the most common cause of bacterial foodborne diseases in Japan. In the present study, we determined ST types of C. jejuni and Campylobacter coli isolated from chickens and patients with enteritis or GBS in Japan and Thailand. C. jejuni from chickens, enteritis, and GBS exhibited divergent ST types and included several novel types in addition to worldwide common types. C. coli from enteritis was also divergent. Novel ST types may represent unidentified native clones in each country. Pulsed‐field gel electrophoresis confirmed the above typing and demonstrated long‐term persistence and transmission.     

Life history of the endangered Japanese striped loach,Cobitis kaibarai (Cypriniformes: Cobitidae), with special reference to its reproductive ecology and the influence of creek reshapings on its population density

In recent years, the biodiversity of freshwater fishes has been markedly decreasing worldwide because of anthropogenic activities. The Japanese striped loach, Cobitis kaibarai (Cypriniformes: Cobitidae), is a primary freshwater fish endemic to northern Kyushu, Japan. This species is designated as endangered IB class in the Red List by the Japan Ministry of the Environment. Its population is decreasing, possibly because of habitat loss and degradation. To conserve C. kaibarai populations, information on its basic ecology is necessary; nonetheless, its detailed life history and reproductive ecology have yet to be clarified. In this study, the authors conducted monthly capture–mark–recapture surveys and periodical observations to investigate the life history, spawning sites and season of C. kaibarai. They also evaluated the influence of creek reshaping (concrete revetment) on the C. kaibarai population in Saga Plain, northern Kyushu. Between 2015 and 2018, more individuals were captured during winter than summer. The average body width of females peaked in early June and small immatures were confirmed from July. Some individuals were captured across 15 or more months after their initial marking. In the survey of reproductive sites, eggs and larvae of C. kaibarai were found in shallow areas in mid-June; these were temporarily submerged following the increase in water level from early June. Therefore, C. kaibarai spawns in shallow areas during this season. Based on the capture–mark–recapture surveys, the estimated population density of C. kaibarai significantly decreased in a census site that had undergone creek reshaping, which contrasted with the results in a control site, where no significant difference was detected. The standard length of C. kaibarai increased following creek reshaping, suggesting that the proportion of C. kaibarai postponing spawning had increased, possibly because of degradation of spawning environments. The results of this study provide important ecological knowledge for the conservation of C. kaibarai and emphasize the importance of shallow waters for floodplain spawners.     

Effect of albumin on the solubility of cholesterol in bile

Despite the fact that a considerable amount of albumin is present in bile, little is known about the effect of albumin on micellar solubility of cholesterol. The effect of albumin on solubility of cholesterol in various micellar bile salt solutions was studied using Millipore filtration after equilibration. In addition, partitioning of cholesterol from micellar solution was studied using a polyethylene disc method. Decrease of the solubility of cholesterol by the presence of albumin was observed only in unconjugated bile salt solution. The lowering effect of albumin on the cholesterol solubility was found to be proportional to the hydrophobicity of bile salt. In contrast, albumin had almost no effect on cholesterol solubility, either in conjugated bile salt solution or in micellar bile salt solution containing phosphatidylcholine. Addition of albumin enhanced the partitioning of cholesterol out of the micelles in sodium chenodeoxycholate solution as a result of decreased micellar solubility and increased the aqueous solubility of cholesterol in the presence of albumin. Therefore, conjugated bile salt and phosphatidylcholine exert a buffering action on the albumin-induced adverse effect on cholesterol solubility, thus stabilising bile against inadvertent precipitation of cholesterol.     

Electron-microscopic investigations of vasoactive intestinal peptide (VIP)-like immunoreactive terminal formations in the lateral septum of the pigeon

Vasoactive intestinal peptide (VIP)-like immunoreactive terminal fields were examined in the lateral septum of the pigeon by means of immunocytochemistry. According to light-microscopic observations, these projections originated from VIP-like immunoreactive cerebrospinal fluid (CSF)-contacting neurons, which are located in the ependymal layer of the lateral septum and form a part of the lateral septal organ. The processes of these cells gave rise to dense terminal-like structures in the lateral septum. Pre-embedding immuno-electron microscopy revealed that VIP-like immunoreactive axon terminals had synaptoid contacts with perikarya of small VIP-immunonegative neurons of the lateral septum, which were characterized by an invaginated nucleus, numerous mitochondria, a well-developed Golgi apparatus, endoplasmic reticulum and a small number of dense-core vesicles (about 100 nm in diameter). VIP-like immunoreactive axons were also seen in contact with immunonegative dendrites in the lateral septum. In both axosomatic and axodendritic connections, VIP-like immunoreactive presynaptic terminals contained large dense-core vesicles, clusters of small vesicles and mitochondria. These findings suggest that VIP-immunoreactive neurons of the lateral septal organ project to small, presumably peptidergic nerve cells of the lateral septum and that the VIP-like neuropeptide serves as a neuromodulator (-transmitter) in this area.