Characteristics of stable isotope signature of diet in tissues of captive Japanese macaques as revealed by controlled feeding

We determined the magnitude of isotopic fractionation of carbon and nitrogen stable isotope ratios (as enrichment factors, Δδ¹³C and Δδ¹⁵N, respectively) between the tissues and diets of captive Japanese macaques (Macaca fuscata) using a controlled feeding experiment, to provide basic data for reconstructing their feeding habits. The Δδ¹³C and Δδ¹⁵N values, respectively, were 0.9 ± 0.2 ‰ (mean ± standard deviation, SD) and 3.0 ± 0.3 ‰ for whole blood, 1.3 ± 0.2 ‰ and 4.3 ± 0.3 ‰ for plasma, and 0.8 ± 0.2 ‰ and 3.0 ± 0.2 ‰ for red blood cells. However, the Δδ¹³C and Δδ¹⁵N values for hair were 2.8 ± 0.3 ‰ and 3.4 ± 0.2 ‰, respectively. No difference was detected in the δ¹³C and δ¹⁵N values of hair sampled from different parts of the body. We investigated the effects of diet on δ¹³C in growing hair by alternating the diet of the macaques each month between two diets that differed markedly in δ¹³C. Hair regrown after shaving repeatedly recorded the δ¹³C of the diet consumed during the time of hair growth. On the other hand, hair naturally grown during the diet-change experiment did not show a clear pattern. One possible reason is that the hair had grown abnormally under unnatural indoor conditions and showed complicated isotope signatures. To reconstruct the long-term feeding history of Japanese macaques, we need to further clarify the relationships between the stable isotope signature of diet and various body tissues.     

Japanese Encephalitis Virus Core Protein Inhibits Stress Granule Formation through an Interaction with Caprin-1 and Facilitates Viral Propagation

Stress granules (SGs) are cytoplasmic foci composed of stalled translation preinitiation complexes induced by environmental stress stimuli, including viral infection. Since viral propagation completely depends on the host translational machinery, many viruses have evolved to circumvent the induction of SGs or co-opt SG components. In this study, we found that expression of Japanese encephalitis virus (JEV) core protein inhibits SG formation. Caprin-1 was identified as a binding partner of the core protein by an affinity capture mass spectrometry analysis. Alanine scanning mutagenesis revealed that Lys⁹⁷ and Arg⁹⁸ in the α-helix of the JEV core protein play a crucial role in the interaction with Caprin-1. In cells infected with a mutant JEV in which Lys⁹⁷ and Arg⁹⁸ were replaced with alanines in the core protein, the inhibition of SG formation was abrogated, and viral propagation was impaired. Furthermore, the mutant JEV exhibited attenuated virulence in mice. These results suggest that the JEV core protein circumvents translational shutoff by inhibiting SG formation through an interaction with Caprin-1 and facilitates viral propagation in vitro and in vivo.     

Identification of the N-Terminal Domain of the Influenza Virus PA Responsible for the Suppression of Host Protein Synthesis

Cellular protein synthesis is suppressed during influenza virus infection, allowing for preferential production of viral proteins. To explore the impact of polymerase subunits on protein synthesis, we coexpressed enhanced green fluorescent protein (eGFP) or luciferase together with each polymerase component or NS1 of A/California/04/2009 (Cal) and found that PA has a significant impact on the expression of eGFP and luciferase. Comparison of the suppressive activity on coexpressed proteins between various strains revealed that avian virus or avian-origin PAs have much stronger activity than human-origin PAs, such as the one from A/WSN/33 (WSN). Protein synthesis data suggested that reduced expression of coexpressed proteins is not due to PA''s reported proteolytic activity. A recombinant WSN containing Cal PA showed enhanced host protein synthesis shutoff and induction of apoptosis. Further characterization of the PA fragment indicated that the N-terminal domain (PANt), which includes the endonuclease active site, is sufficient to suppress cotransfected gene expression. By characterizing various chimeric PANts, we found that multiple regions of PA, mainly the helix α4 and the flexible loop of amino acids 51 to 74, affect the activity. The suppressive effect of PANt cDNA was mainly due to PA-X, which was expressed by ribosomal frameshifting. In both Cal and WSN viruses, PA-X showed a stronger effect than the corresponding PANt, suggesting that the unique C-terminal sequences of PA-X also play a role in suppressing cotransfected gene expression. Our data indicate strain variations in PA gene products, which play a major role in suppression of host protein synthesis.     

A NADPH-dependent (S)-imine reductase (SIR) from Streptomyces sp. GF3546 for asymmetric synthesis of optically active amines: purification, characterization, gene cloning, and expression

A NADPH-dependent (S)-imine reductase (SIR) was purified to be homogeneous from the cell-free extract of Streptomyces sp. GF3546. SIR appeared to be a homodimer protein with subunits of 30.5 kDa based on SDS-polyacrylamide gel electrophoresis and HPLC gel filtration. It also catalyzed the (S)-enantioselective reduction of not only 2-methyl-1-pyrroline (2-MPN) but also 1-methyl-3,4-dihydroisoquinoline and 6,7-dimethoxy-1-methyl-3,4-dihydroisoquinoline. Specific activities for their imines were 130, 44, and 2.6 nmol?min^?1?mg^?1, and their optical purities were 92.7 % ee, 96.4 % ee, and >99 % ee, respectively. Using a NADPH-regenerating system, 10 mM 2-MPN was converted to amine with 100 % conversion and 92 % ee after 24 h. The amino acid sequence analysis revealed that SIR showed about 60 % identity to 6-phosphogluconate dehydrogenase. However, it showed only 37 % identity with Streptomyces sp. GF3587 (R)-imine reductase. Expression of SIR in Escherichia coli was achieved, and specific activity of the cell-free extract was about two times higher than that of the cell-free extract of Streptomyces sp. GF3546.     

Advanced glycation end products suppress osteoblastic differentiation of stromal cells by activating endoplasmic reticulum stress

Advanced glycation end products (AGEs) are involved in bone quality deterioration in diabetes mellitus. We previously showed that AGE2 or AGE3 inhibited osteoblastic differentiation and mineralization of mouse stromal ST2 cells, and also induced apoptosis and decreased cell growth. Although quality management for synthesized proteins in endoplasmic reticulum (ER) is crucial for the maturation of osteoblasts, the effects of AGEs on ER stress in osteoblast lineage are unknown. We thus examined roles of ER stress in AGE2- or AGE3-induced suppression of osteoblastogenesis of ST2 cells. An ER stress inducer, thapsigargin (TG), induced osteoblastic differentiation of ST2 cells by increasing the levels of Osterix, type 1 collagen (Col1), alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA. AGE2 or AGE3 suppressed the levels of ER stress sensors such as IRE1α, ATF6 and OASIS, while they increased the levels of PERK and its downstream molecules, ATF4. A reduction in PERK level by siRNA did not affect the AGEs-induced suppression of the levels of Osterix, Col1 and OCN mRNA. In conclusion, AGEs inhibited the osteoblastic differentiation of stromal cells by suppressing ER stress sensors and accumulating abnormal proteins in the cells. This process might accelerate AGEs-induced suppression of bone formation found in diabetes mellitus.     

Mutations in SERPINB7, Encoding a Member of the Serine Protease Inhibitor Superfamily,Cause Nagashima-type Palmoplantar Keratosis

“Nagashima-type” palmoplantar keratosis (NPPK) is an autosomal recessive nonsyndromic diffuse palmoplantar keratosis characterized by well-demarcated diffuse hyperkeratosis with redness, expanding on to the dorsal surfaces of the palms and feet and the Achilles tendon area. Hyperkeratosis in NPPK is mild and nonprogressive, differentiating NPPK clinically from Mal de Meleda. We performed whole-exome and/or Sanger sequencing analyses of 13 unrelated NPPK individuals and identified biallelic putative loss-of-function mutations in SERPINB7, which encodes a cytoplasmic member of the serine protease inhibitor superfamily. We identified a major causative mutation of c.796C>T (p.Arg266^∗) as a founder mutation in Japanese and Chinese populations. SERPINB7 was specifically present in the cytoplasm of the stratum granulosum and the stratum corneum (SC) of the epidermis. All of the identified mutants are predicted to cause premature termination upstream of the reactive site, which inhibits the proteases, suggesting a complete loss of the protease inhibitory activity of SERPINB7 in NPPK skin. On exposure of NPPK lesional skin to water, we observed a whitish spongy change in the SC, suggesting enhanced water permeation into the SC due to overactivation of proteases and a resultant loss of integrity of the SC structure. These findings provide an important framework for developing pathogenesis-based therapies for NPPK.     

Conformational restriction approach to BACE1 inhibitors II: SAR study of the isocytosine derivatives fixed with a cis-cyclopropane ring

To improve the efficacy of the conformationally restricted BACE1 inhibitors, structural modifications were investigated using two strategies: (a) modification of the terminal aromatic ring and (b) insertion of a spacer between the aromatic rings. In the latter approach, another type of inhibitor 17 bearing an ethylene spacer between two aromatic rings was found to exhibit good BACE1 inhibitory activity, while the corresponding conformationally unrestricted compound 25 showed no activity. This result revealed an interesting effect of a conformational restriction with a cyclopropane ring.     

CXCR4-derived synthetic peptides inducing anti-HIV-1 antibodies

Despite almost 30 years since the identification of the human immunodeficiency virus type I (HIV-1), development of effective AIDS vaccines has been hindered by the high mutability of HIV-1. The HIV-1 co-receptors CCR5 and CXCR4 are genetically stable, but viral proteins may mutate rapidly during the course of infection. CXCR4 is a seven transmembrane G protein-coupled receptor, possessing an N-terminal region (NT) and three extracellular loops (ECL1-3). Previous studies have shown that the CXCR4-ED-derived peptides inhibit the entry of HIV-1 by interacting with gp120, an HIV-1 envelope glycoprotein. In the present study, antigenicity of CXCR4-derived peptides has been investigated and the anti-HIV-1 effects of induced antisera have been assessed. It was found that CXCR4-ED-derived antigen molecules immunize mice, showing that the linear peptides have higher antigenicity than the cyclic peptides. The L1- and L2-induced antisera inhibited the HIV-1 entry significantly, while anti-N1 antibodies have no inhibitory activity. This study produced promising examples for the design of AIDS vaccines which target the human protein and can overcome mutability of HIV-1.