Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP

Hepatitis B virus (HBV) entry has been analyzed using infection-susceptible cells, including primary human hepatocytes, primary tupaia hepatocytes, and HepaRG cells. Recently, the sodium taurocholate cotransporting polypeptide (NTCP) membrane transporter was reported as an HBV entry receptor. In this study, we established a strain of HepG2 cells engineered to overexpress the human NTCP gene (HepG2-hNTCP-C4 cells). HepG2-hNTCP-C4 cells were shown to be susceptible to infection by blood–borne and cell culture-derived HBV. HBV infection was facilitated by pretreating cells with 3% dimethyl sulfoxide permitting nearly 50% of the cells to be infected with HBV. Knockdown analysis suggested that HBV infection of HepG2-hNTCP-C4 cells was mediated by NTCP. HBV infection was blocked by an anti-HBV surface protein neutralizing antibody, by compounds known to inhibit NTCP transporter activity, and by cyclosporin A and its derivatives. The infection assay suggested that cyclosporin B was a more potent inhibitor of HBV entry than was cyclosporin A. Further chemical screening identified oxysterols, oxidized derivatives of cholesterol, as inhibitors of HBV infection. Thus, the HepG2-hNTCP-C4 cell line established in this study is a useful tool for the identification of inhibitors of HBV infection as well as for the analysis of the molecular mechanisms of HBV infection.     

CD98hc regulates the development of experimental colitis by controlling effector and regulatory CD4 T cells

CD4⁺ T cell activation is controlled by signaling through the T cell receptor in addition to various co-receptors, and is also affected by their interactions with effector and regulatory T cells in the microenvironment. Inflammatory bowel diseases (IBD) are caused by the persistent activation and expansion of auto-aggressive CD4⁺ T cells that attack intestinal epithelial cells. However, the molecular basis for the persistent activation of CD4⁺ T cells in IBD remains unclear. In this study, we investigated how the CD98 heavy chain (CD98hc, Slc3a2) affected the development of colitis in an experimental animal model. Transferring CD98hc-deficient CD4⁺CD25⁻ T cells into Rag2^−/− mice did not cause colitis accompanied by increasing Foxp3⁺ inducible regulatory T cells. By comparison, CD98hc-deficient naturally occurring regulatory T cells (nTregs) had a decreased capability to suppress colitis induced by CD4⁺CD25⁻ T cells, although CD98hc-deficient mice did not have a defect in the development of nTregs. Blocking CD98hc with an anti-CD98 blocking antibody prevented the development of colitis. Our results indicate that CD98hc regulates the expansion of autoimmune CD4⁺ T cells in addition to controlling nTregs functions, which suggests the CD98hc as an important target molecule for establishing strategies for treating colitis.     

Luminescence enhancement of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase by three amino acid substitutions

To characterize the luminescence properties of nanoKAZ, a 16 amino acid substituted mutant of the catalytic 19 kDa protein (KAZ) of Oplophorus luciferase, the effects of each mutated amino acid were investigated by site-specific mutagenesis. All 16 single substituted KAZ mutants were expressed in Escherichia coli cells and their secretory expressions in CHO-K1 cells were also examined using the signal peptide sequence of Gaussia luciferase. Luminescence activity of KAZ was significantly enhanced by single amino acid substitutions at V44I, A54I, or Y138I. Further, the triple mutant KAZ-V44I/A54I/Y138I, named eKAZ, was prepared and these substitutions synergistically enhanced luminescence activity, showing 66-fold higher activity than wild-KAZ and also 7-fold higher activity than nanoKAZ using coelenterazine as a substrate. Substrate specificity of eKAZ for C2- and/or C6-modified coelenterazine analogues was different from that of nanoKAZ, indicating that three amino acid substitutions may be responsible for the substrate recognition of coelenterazine to increase luminescence activity. In contrast, these substitutions did not stimulate protein secretion from CHO-K1 cells, suggesting that the folded-protein structure of KAZ might be different from that of nanoKAZ.     

N-linked glycosylation of the superoxide-producing NADPH oxidase Nox1

Nox1 is a membrane-integrated protein that belongs to the Nox family of superoxide-producing NADPH oxidases. Here we show that human Nox1 undergoes glycosylation at Asn-162 and Asn-236 in the second and third extracellular loops, respectively. Simultaneous threonine substitution for these residues completely abrogates the glycosylation, but does not prevent Nox1 from forming a heterodimer with p22^phox, trafficking to the cell surface, or producing superoxide. In the absence of p22^phox, Nox1 is transported to the plasma membrane mainly as a form with high mannose N-glycans, although their conversion into complex N-glycans is induced by expression of p22^phox. These findings indicate that glycosylation and subsequent N-glycan maturation of Nox1 are both dispensable for its cell surface recruitment. Superoxide production by unglycosylated Nox1 is largely dependent on p22^phox, which is abrogated by glutamine substitution for Pro-156 in p22^phox, a mutation leading to a defective interaction with the Nox1-activating protein Noxo1. Thus p22^phox directly contributes to Nox1 activation in a glycosylation-independent manner, besides its significant role in Nox1 glycan maturation.     

Differences in the heat stress associated with white sportswear and being semi-nude in exercising humans under conditions of radiant heat and wind at a wet bulb globe temperature of greater than 28 °C

This study investigated whether wearing common white sportswear can reduce heat stress more than being semi-nude during exercise of different intensities performed under radiant heat and wind conditions, such as a hot summer day. After a 20-min rest period, eight male subjects performed three 20 min sessions of cycling exercise at a load intensity of 20 % or 50 % of their peak oxygen uptake (VO_2peak) in a room maintained at a wet bulb globe temperature (WBGT) of 28.7?±?0.1 °C using two spot lights and a fan (0.8 m/s airflow). Subjects wore common white sportswear (WS) consisting of a long-sleeved shirt (45 % cotton and 55 % polyester) and short pants (100 % polyester), or only swimming pants (SP) under the semi-nude condition. The mean skin temperature \( \left(\overline{T} sk\right) \) was greater when subjects wore SP than WS under both the 20 % and 50 % exercise conditions. During the 50 % exercise, the rating of perceived exertion (RPE) and thermal sensation (TS), and the increases in esophageal temperature (ΔTes) and heart rate were significantly higher (P?<?0.001–0.05), or tended to be higher (P?<?0.07), in the WS than SP trials at the end of the third 20-min exercise session. The total sweat loss (m _sw,tot) was also significantly higher in the WS than in the SP trials (P?<?0.05). However, during the 20 % exercise, the m _sw,tot during exercise, and the ΔTes, RPE and TS at the end of the second and third sessions of exercise did not differ significant between conditions. The heat storage (S), calculated from the changes in the mean body temperature (0.9Tes + 0.1 \( \overline{T} sk \) ), was significantly lower in the WS trials than in the SP trials during the 20 min resting period before exercise session. However, S was similar between conditions during the 20 % exercise, but was greater in the WS than in the SP trials during 50 % exercise. These results suggest that, under conditions of radiant heat and wind at a WBGT greater than 28 °C, the heat stress associated with wearing common WS is similar to that of being semi-nude during light exercise, but was greater during moderate exercise, and the storage of body heat can be reduced by wearing WS during rest periods.     

Polyphenol‐solubility alters amyloid fibril formation of α‐synuclein

Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer''s and Parkinson''s diseases. Amyloid fibrils form above the solubility of amyloidogenic proteins or peptides upon breaking supersaturation, followed by a nucleation and elongation mechanism, which is similar to the crystallization of solutes. Many additives, including salts, detergents, and natural compounds, promote or inhibit amyloid formation. However, the underlying mechanisms of the opposing effects are unclear. We examined the effects of two polyphenols, that is, epigallocatechin gallate (EGCG) and kaempferol‐7─O─glycoside (KG), with high and low solubilities, respectively, on the amyloid formation of α‐synuclein (αSN). EGCG and KG inhibited and promoted amyloid formation of αSN, respectively, when monitored by thioflavin T (ThT) fluorescence or transmission electron microscopy (TEM). Nuclear magnetic resonance (NMR) analysis revealed that, although interactions of αSN with soluble EGCG increased the solubility of αSN, thus inhibiting amyloid formation, interactions of αSN with insoluble KG reduced the solubility of αSN, thereby promoting amyloid formation. Our study suggests that opposing effects of polyphenols on amyloid formation of proteins and peptides can be interpreted based on the solubility of polyphenols.     

The inhibitory effects of mushroom extracts on sucrose-dependent oral biofilm formation

Mushrooms contain large quantities of α-glucans. Shiitake (Lentinula edodes), Japan’s most popular edible mushroom, has been reported to contain about 6% (weight/dried weight) of α-(1,3)-glucan. This glucan is one of the major components of oral biofilm formed by the cariogenic bacteria Streptococcus mutans and Streptococcus sobrinus. We found that extracts from shiitake and other edible mushrooms could reduce preformed biofilms of S. mutans and S. sobrinus in the presence of dextranase. We also investigated the α-glucanase activities of shiitake mushroom extracts and their effects on biofilm formation. The extracts possessed α-glucanase activity and degraded water-insoluble glucans from mutans streptococci. The extracts strongly inhibited the sucrose-dependent formation of biofilms by S. mutans and S. sobrinus in the presence of dextranase. Our results suggest that some components of mushrooms, including α-glucanases, might inhibit the sucrose-induced formation of oral biofilms.     

Surfactant-modified yeast whole-cell biocatalyst displaying lipase on cell surface for enzymatic production of structured lipids in organic media

The cell surface engineering system, in which functional proteins are genetically displayed on microbial cell surfaces, has recently become a powerful tool for applied biotechnology. Here, we report on the surfactant modification of surface-displayed lipase to improve its performance for enzymatic synthesis reactions. The lipase activities of the surfactant-modified yeast displaying Rhizopus oryzae lipase (ROL) were evaluated in both aqueous and nonaqueous systems. Despite the similar lipase activities of control and surfactant-modified cells in aqueous media, the treatment with nonionic surfactants increased the specific lipase activity of the ROL-displaying yeast in n-hexane. In particular, the Tween 20-modified cells increased the cell surface hydrophobicity significantly among a series of Tween surfactants tested, resulting in 8–30 times higher specific activity in organic solvents with relatively high log P values. The developed cells were successfully used for the enzymatic synthesis of phospholipids and fatty acid methyl esters in n-hexane, whereas the nontreated cells produced a significantly low yield. Our results thus indicate that surfactant modification of the cell surface can enhance the potential of the surface-displayed lipase for bioconversion.     

Overexpression of protein disulfide isomerases enhances secretion of recombinant human transferrin in Schizosaccharomyces pombe

Although the fission yeast Schizosaccharomyces pombe has been used for high-level heterologous protein production, the productivity of secreted human serum transferrin (hTF) has been low, presumably, because the protein harbors twenty disulfide bonds and two N-glycosylation sites. In the present study, we found that overexpression of endogenous putative protein disulfide isomerase (PDI) improved productivity. Whole genome sequence analysis of S. pombe revealed five putative PDI genes and overexpression of two of them, SPAC17H9.14c and SPBC3D6.13c (SpPdi2p or SpPdi3p, respectively), significantly improved the productivity of secreted hTF. GFP-fused SpPdi2p and SpPdi3p were found to localize to the endoplasmic reticulum. Co-overexpression of SpPdi2p or SpPdi3p with hTF coupled with modifications to the growth medium reported in our previous study were able to increase the level of secreted hTF approximately 30-fold relative to conventional conditions.