Coupling ion specificity of chimeras between H(+)- and Na(+)-driven motor proteins, MotB and PomB, in Vibrio polar flagella

We have shown that a hybrid motor consisting of proton-type Rhodobacter sphaeroides MotA and sodium-type VIBRIO: alginolyticus PomB, MotX and MotY, can work as a sodium-driven motor in VIBRIO: cells. In this study, we tried to substitute the B subunits, which contain a putative ion-binding site in the transmembrane region. Rhodobacter sphaeroides MotB did not work with either MotA or PomA in Vibrio cells. Therefore, we constructed chimeric proteins (MomB), which had N-terminal MotB and C-terminal PomB. MomB proteins, with the entire transmembrane region derived from the H(+)-type MotB, gave rise to an Na(+) motor with MotA. The other two MomB proteins, in which the junction sites were within the transmembrane region, also formed Na(+) motors with PomA, but were changed for Na(+) or Li(+) specificity. These results show that the channel part consisting of the transmembrane regions from the A and B subunits can interchange Na(+)- and H(+)-type subunits and this can affect the ion specificity. This is the first report to have changed the specificity of the coupling ions in a bacterial flagellar motor.     

Identification of a response element for vitamin D3 and retinoic acid in the promoter region of the human fructose-1,6-bisphosphatase gene

Fructose-1,6-bisphosphatase (FBPase) is a key gluconeogenic enzyme. The data herein show that both the enzyme activity and mRNA level of the human FBPase gene are enhanced by 9-cis retinoic acid (9cRA) and all-trans retinoic acid (atRA) as well as by 1,25-dihydroxyvitamin D3 (VD3) in human promyelocytic HL60 cells and normal monocytes in peripheral blood, which were used as an alternative source to liver for the DNA diagnosis of FBPase deficiency. To understand the molecular mechanism of this enhancing action, the 2.4 kb 5'-regulatory region of the human FBPase gene was isolated and sequenced. Using luciferase reporter gene assays, a 0.5 kb FBPase basal promoter fragment was found to confer induction by VD3, 9cRA, and atRA that was mediated by the vitamin D3 receptor (VDR), retinoid X receptor (RXR), and retinoic acid receptor (RAR). Within this region, a direct repeat sequence, 5'-TAACCTttcTGAACT-3' (-340 to -326), which functions as a common response element for VD3, 9cRA, and atRA, was identified. The results of electrophoretic mobility shift assays indicated that VDR-RXR and RAR-RXR heterodimers bind this response element. Collectively, these observations indicate that VD3 and RA are important modulators of the expression of the human FBPase gene in monocytic cells.     

Characterization of a flagellar sheath component, PF60, and its structural gene in marine Vibrio

The Polar flagella (Pof) of Vibrio alginolyticus are surrounded by a membrane structure called a sheath. Five major proteins, whose molecular masses are 60, 47, 45, 44, and 18 kDa (named PF60, PF47, PF45, PF44, and PF18, respectively), have been detected in polar flagella. PF47 and PF45 have been identified as flagellins while the other proteins are thought to be sheath-associated ones. In this study, we isolated and partially characterized a major sheath protein, PF60. We found that PF60 can be solubilized by Triton X-100 treatment, but not by heat or acid treatment. After digestion with a peptidase, the N-terminal sequences of several fragments were determined and the N-terminus of intact PF60 seemed to be blocked. Through PCR in conjunction with oligonucleotide primers deduced from the peptide sequences, a DNA fragment of PF60 was amplified. A 4.5 kb HindIII restriction fragment was cloned by colony hybridization using the PCR fragment. Subsequent sequence analysis revealed three complete and one partial open reading frame (ORFs). The three ORFs, which exhibit sequence homology, correspond to PF60 (named pfsA), an amino acid transport ATP-binding protein, and an amino acid binding periplasmic protein. The single pfsA gene constitutes an operon and encodes a protein of 491 amino acids containing a putative signal peptide sequence at the N-terminal. A sequence database search revealed no homologous protein. However, PfsA seems to resemble lipoproteins in the N-terminal signal sequence and the biochemical data obtained in this study are consistent with that PfsA is a lipoprotein. The expression of the pfsA gene may be coordinately regulated with flagellar formation and similarly regulated to PF47 flagellin.     

A comparison of raw starch-digesting glucoamylase production in liquid and solid cultures of Rhizopus strains

Maximum growth for Rhizopus sp. A-11 was obtained at a zinc ion concentration of 0.7 ppm in a liquid medium. Glucoamylase (GA, EC 3.2.1.3) production in Rhizopus sp. A-11 was maximized at 710 U/ml, at the presence of 75 ppm for calcium and 0.7 ppm of zinc ions in liquid medium. Zinc ion is known as an essential biometal for Rhizopus growth; however, growth was inhibited by the zinc ion concentration, not maximized. Although calcium ion was not necessary to Rhizopus growth, GA production using Rhizopus sp. A-11 was markedly stimulated by calcium ion concentration over 75 ppm in the liquid medium. The GA productivity of the present liquid culture was about 4.4 times higher than that of the solid state culture, based on the unit starch amount in the liquid and solid media carbon source. The characteristics of the GA produced by the Rhizopus sp. A-11 liquid culture were interesting; that is, almost all the GA produced was classified as raw starch-digesting GA (GA-I). Secreted protein in the culture liquid after 30 h was nearly GA, and had a limited amount of impure protein. As a result, it was found that using a Rhizopus culture in a specified metal-ion regulated medium was an effective method for producing GA. Thus the present culture method was renamed the "metal-ion-regulated liquid culture method".     

Hepatitis E Virus in Cambodia: Prevalence among the General Population and Complete Genome Sequence of Genotype 4

Hepatitis E virus (HEV) is a growing public health problem in many countries. In this study, we investigated HEV seroprevalence among the general population in the Siem Reap province, Cambodia, and performed HEV genetic analysis with the aim to develop an HEV prevention strategy. This seroepidemiological cross-sectional study conducted from 2010 to 2014 included 868 participants from four different locations in Siem Reap province, Cambodia. They answered questionnaires and provided blood samples for the analysis of hepatitis virus infections. Among the participants (360 men and 508 women; age range, 7–90 years), the prevalence of anti-HEV IgG was 18.4% (95% confidence interval: 15.9–21.0); HEV RNA was detected in two participants (0.23%) and was classified as genotype 3 and 4. Full-length genome of the genotype 4 isolate, CVS-Sie10, was sequenced; it contained 7,222 nucleotides and three ORFs and demonstrated high sequence identity with the swine China isolates swGX40 (95.57%), SS19 (94.37%), and swDQ (91.94%). Multivariate logistic regression analysis revealed that men, elderly people, and house workers were risk groups significantly associated with the positivity for anti-HEV IgG. This is the first report on the detection of HEV genotype 4 in humans in Cambodia and on the complete genome sequence of HEV genotype 4 from this country. Our study demonstrates that new HEV infection cases occur frequently among the general population in Cambodia, and effective preventive measures are required.     

Age-related disruption of autophagy in dermal fibroblasts modulates extracellular matrix components

Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5—genes essential for autophagosome formation—was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.     

Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway

Previously, we achieved approximately 30-fold enhanced secretion of the protease-sensitive model protein human growth hormone (hGH) by multiple gene deletion of seven obstructive proteases in the fission yeast Schizosaccharomyces pombe. However, intracellular retention of secretory hGH was found in the resultant multiprotease-deficient strains. As a solution, genetic modification of the intracellular trafficking pathway that is related to intracellular retention of hGH was attempted on a protease octuple deletant strain. Vacuolar accumulation of the intracellularly retained hGH was identified by secretory expression of hGH fused with EGFP, and three vacuolar protein sorting (vps)-deficient strains, vps10Δ, vps22Δ, and vps34Δ, were determined on account of their hGH secretion efficiency. The mutant vps10Δ was found to be effective for hGH secretion, which suggested a role for vps10 in the vacuolar accumulation of the intracellularly retained hGH. Finally, vps10 deletion was performed on the protease octuple deletant strain, which led to an approximately 2-fold increase in hGH secretion. This indicated the possible application of secretory-pathway modification and multiple protease deletion for improving heterologous protein secretion from the fission yeast S. pombe.