Recreational drug use and related social factors among HIV-positive men in Japan.

This study aims to determine the relationship between recreational drug use in HIV-positive males in the past year and socio-economic factors and/or social support networks in Japan. A national online survey in a cross-sectional study was conducted by HIV Futures Japan project from July 2013 to February 2014. Of the 1095 HIV-positive individuals who responded, 913 responses were determined to be valid; responses from the 875 males were analysed. A total of 282 participants used addictive drugs (32.2%) in past year. New psychoactive substances were used by 121 participants (13.8%), methamphetamine or amphetamine by 47 (5.4%), air dusters/sprays/gas by 31 (3.5%), 5-methoxy-N,N-diisopropyltryptamine (5MeO-DIPT) by 16 (1.8%) and cannabis (1.0%) by 9. Multiple logistic regression analysis was performed with the use of alkyl nitrites, addictive drugs, air dusters and thinners, which are low illegality, as dependent variables. We found that the odds ratio (95% confidence interval) for use among participants with full-time and temp/contracted/part-time employees compared to management/administration professions were 2.59 (0.99-6.77) and 2.61 (0.91-7.51). Also, a correlation was observed between alkyl nitrites and new psychoactive substances and usage rates in people engaged in few HIV-positive networks. It is necessary to develop targeted policies for drug use prevention and user support among HIV-positive men and to support and provide care for drug users who are isolated or have a narrow HIV/AIDS support network.

Protein stabilization utilizing a redefined codon.

Recent advances have fundamentally changed the ways in which synthetic amino acids are incorporated into proteins, enabling their efficient and multiple-site incorporation, in addition to the 20 canonical amino acids. This development provides opportunities for fresh approaches toward addressing fundamental problems in bioengineering. In the present study, we showed that the structural stability of proteins can be enhanced by integrating bulky halogenated amino acids at multiple selected sites. Glutathione S-transferase was thus stabilized significantly (by 5.2 and 5.6 kcal/mol) with 3-chloro- and 3-bromo-l-tyrosines, respectively, incorporated at seven selected sites. X-ray crystallographic analyses revealed that the bulky halogen moieties filled internal spaces within the molecules, and formed non-canonical stabilizing interactions with the neighboring residues. This new mechanism for protein stabilization is quite simple and applicable to a wide range of proteins, as demonstrated by the rapid stabilization of the industrially relevant azoreductase.

Transcriptional repressor TIEG1 regulates Bmal1 gene through GC box and controls circadian clockwork.

The circadian clock controls daily rhythms in many physiologic processes, and the clock oscillation is regulated by external time cues such as light, temperature, and feeding. In mammals, the transcriptional regulation of clock genes underlies the clock oscillatory mechanism, which is operative even in cultured fibroblasts. We previously demonstrated that glucose treatment of rat-1 fibroblasts evokes circadian expression of clock genes with a rapid induction of Tieg1 transcript encoding a transcriptional repressor. Here, we found diurnal variation of both Tieg1 mRNA and nuclear TIEG1 protein levels in the mouse liver with their peaks at day/night transition and midnight, respectively. In vitro experiments showed that TIEG1 bound to Bmal1 gene promoter and repressed its transcriptional activity through two juxtaposed GC boxes near the transcription initiation site. The GC box/TIEG1-mediated repression of Bmal1 promoter was additive to RORE-dependent repression by REV-ERBalpha, a well-known repressor of Bmal1 gene. In cell-based real-time assay, siRNA-mediated knock-down of TIEG1 caused period shortening of cellular bioluminescence rhythms driven by Bmal1-luciferase and Per2-luciferase reporters. These findings highlight an active role of TIEG1 in the normal clock oscillation and GC box-mediated regulation of Bmal1 transcription.

Purification, crystallization and preliminary X-ray analysis of a deletion mutant of a major buckwheat allergen.

A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis. A deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active. A three-wavelength MAD data set was collected from a crystal of selenomethionine-labelled rBWp16DeltaN. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 A, alpha = 111.92, beta = 108.91, gamma = 98.74 degrees . One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 A(3) Da(-1).

Identification of a second, non-conserved amino acid that contributes to the unique sugar binding properties of the nematode galectin LEC-1.

The basic disaccharide structure recognized by galectin family members is the lactosamine-like structure Galbeta1-4(3)Glc(NAc). The 32-kDa galectin LEC-1 of the nematode Caenorhabditis elegans is composed of two domains, each of which is homologous to vertebrate 14-kDa-type galectins. The N-terminal lectin domain of LEC-1 recognizes blood group A saccharide (GalNAcalpha1-3(Fucalpha1-2)Galbeta1-3GlcNAc), whereas this saccharide is poorly recognized by the C-terminal domain. Using a combination of site-directed mutagenesis and analysis of the sugar-binding profile by frontal affinity chromatography, we previously found that Thr41 in the N-terminal lectin domain of LEC-1 is important for its affinity for A-hexasaccharide. Thr41 is located on beta-strand S3, next to the three beta-strands S4-S6, where the conserved amino acids form the binding site for the basic Galbeta1-4(3)Glc(NAc) structure. Here, we report that a second amino acid, Asp133, in the N-terminal lectin domain of LEC-1, located on the beta-strand S2 adjacent to that containing Thr41, is important for LEC-1-specific recognition of A-hexasaccharide. These results suggest that amino acid residues other than those located on the three beta-strands S4-S6, contribute to the unique sugar binding specificity of individual galectins.

Crystallization and preliminary x-ray crystallographic analysis of galectin LEC-1 from Caenorhabditis elegans.

Galectin LEC-1 isolated from the nematode Caenorhabditis elegans was the first galectin found in invertebrates and also the first tandem-repeat-type galectin identified, containing two homologous carbohydrate-binding sites. This galectin is localized most abundantly in the adult cuticle and possibly plays a role in the formation of epidermal layers. We succeeded in crystallizing LEC-1 composed of 279 amino acids with a calculated molecular weight of 31,809 Da under two independent sets of conditions as a result of extensive screening. The crystals grown under one set of conditions belong to the triclinic space group P1, with unit-cell parameters a = 48.44, b = 52.13, c = 64.24 A, alpha = 108.73, beta= 91.39, and gamma = 98.45 degrees and two protein molecules per unit cell. The crystals grown under the other set of conditions which included lactose belong to the monoclinic space group P2(1), with unit-cell parameters a = 52.90, b = 47.01, c = 66.16 A, and beta= 113.30 degrees and one protein molecule per asymmetric unit.