X-ray Crystallographic Study of an HIV-1 Fusion Inhibitor with the gp41 S138A Substitution

The S138A substitution of fusion inhibitory peptides derived from the C-terminal heptad repeat (C-HR) of the human immunodeficiency virus type 1 (HIV-1) gp41 leads to enhanced binding affinity to the N-terminal heptad repeat (N-HR). As such, these peptides exhibit highly potent anti-HIV-1 activity. X-ray crystallographic analysis was performed to understand the effect of the substitution on binding affinity. The comparison of the native and S138A crystal structures indicated that the increase in the hydrophobicity of the S138A substitution may aid the stabilization of the N-HR/C-HR complex through additional hydrophobic contacts. Free-energy calculations suggest that the difference between the desolvation free energies of the C-HR-derived peptides with and without the S138A mutation dominates the observed difference in anti-HIV-1 activity.     

Functional polymorphisms of HSPA5: possible association with bipolar disorder

Altered endoplasmic reticulum stress (ER) response signaling is suggested in bipolar disorder. Previously, we preliminarily reported the genetic association of HSPA5 (GRP78/BiP) with bipolar disorder. Here, we extended our analysis by increasing the number of Japanese case-control samples and NIMH Genetics Initiative bipolar trio samples (NIMH trios), and also analyzed schizophrenia samples. In Japanese, nominally significant association of one haplotype was observed in extended samples of bipolar disorder but not in schizophrenia. In NIMH trios, no association was found in total samples. However, an exploratory analysis suggested that the other haplotype was significantly over-transmitted to probands only from the paternal side. The associated haplotype in Japanese or NIMH pedigrees shared three common polymorphisms in the promotor, which was found to alter promotor activity. These findings suggested promotor polymorphisms of HSPA5 may affect the interindividual variability of ER stress response and may confer a genetic risk factor for bipolar disorder.     

Characterization of recombinant amyloidogenic chicken cystatin mutant I66Q expressed in yeast

Amyloidogenic chicken cystatin mutant I66Q (cC I66Q) was successfully secreted by yeasts Pichia pastoris and Saccharomyces cerevisiae. The soluble monomer and dimer forms of amyloidogenic cC I66Q were found in the culture medium, while large amounts of insoluble aggregate and polymeric form cC I66Q besides the monomer and dimer forms were secreted into the culture medium. The amyloidogenic cC I66Q showed a comparable circular dichroism spectrum to that of the wild cystatin, and the monomer form exhibited a similar level of inhibitory activity toward papain, but the dimmer form did not. During storage of amyloidogenic cC I66Q under physiological and acidic conditions, typical binding with Congo red and thioflavin T, and the formation of amyloid fibrils were observed, whereas the characteristic of similar amyloidosis was hardly detected for the wild recombinant cystatin.     

Radioimmunoassay for non-enzymatically glycated serum proteins

We have developed a radioimmunoassay (RIA) for nonenzymatically glycated serum proteins. The polyclonal antibodies prepared against reduced glycated human albumin were specific for the glucitollysine residues of serum proteins. Serum proteins from diabetic patients (n = 25) contained 5.3 +/- 2.8 nmoles of glucitollysine/mg protein, compared to 2.0 +/- 0.2 in controls (n = 20). The intra- and inter-assay variables were 3.2-6.2% and 4.4-8.6%, respectively. Results from this assay procedure correlated well with those from the boronate affinity chromatography procedure (r = 0.94; P less than 0.001). The data suggested that diabetic serum proteins contained at least 2.5 times as much immunochemically detectable glucitollysine residures as normal serum proteins after reduction of the proteins with sodium borohydride.     

Microsatellite mapping of the genes for brittle rachis on homoeologous group 3 chromosomes in tetraploid and hexaploid wheats

The brittle rachis character, which causes spontaneous shattering of spikelets, has an adaptive value in wild grass species. The loci Br1 and Br2 in durum wheat (Triticum durum Desf.) and Br3 in hexaploid wheat (T. aestivum L.) determine disarticulation of rachides above the junction of the rachilla with the rachis such that a fragment of rachis is attached below each spikelet. Using microsatellite markers, the loci Br1, Br2 and Br3 were mapped on the homoeologous group 3 chromosomes. The Br2 locus was located on the short arm of chromosome 3A and linked with the centromeric marker, Xgwm32, at a distance of 13.3 cM. The Br3 locus was located on the short arm of chromosome 3B and linked with the centromeric marker, Xgwm72 (at a distance of 14.2 cM). The Br1 locus was located on the short arm of chromosome 3D. The distance of Br1 from the centromeric marker Xgdm72 was 25.3 cM. Mapping the Br1, Br2 and Br3 loci of the brittle rachis suggests the homoeologous origin of these 3 loci for brittle rachides. Since the genes for brittle rachis have been retained in the gene pool of durum wheat, the more closely linked markers with the brittle rachis locus are required to select against brittle rachis genotypes and then to avoid yield loss in improved cultivars.     

The N- and C-terminal mutations in tryptophan permease Tat2 confer cell growth in<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis> under high-pressure and low-temperature conditions

Tryptophan uptake appears to be the limiting factor in growth of tryptophan auxotrophic Saccharomyces cerevisiae strains under the conditions of high hydrostatic pressure and low temperature. When the cells are subjected to a pressure of 25 MPa, tryptophan permease Tat2 is degraded in a manner dependent on ubiquitination by Rsp5. One of the high-pressure growth-conferring genes, HPG2, was shown to be allelic to TAT2. The HPG2-1 (Tat2^E27F) mutation site is located within the ExKS motif in the N-terminus, and the HPG2-2 (Tat2^D563N) and HPG2-3 (Tat2^E570K) mutation sites are located at the KQEIAE sequence in the C-terminus. The HPG2 mutations enhance the stability of Tat2 during high-pressure or low-temperature incubation, leading to cell growth under these stressful conditions. These results suggest that the cytoplasmic tails are involved in Rsp5-mediated ubiquitination of Tat2 under high-pressure or low-temperature conditions.Communicated by K. Horikoshi     

Estrogen regulation of nitric oxide synthesis in the porcine oocyte

The endothelial type (NOS-3) of three isoforms of nitric oxide (NO) synthase occurs in porcine oocytes and granulosa cells, but the regulation of NO synthesis in oocytes remains unknown. The present study was designed to evaluate steroid control in the process of oocyte NO synthesis. Cumulus-oocyte complexes (COCs), obtained from small-sized antral follicles of immature porcine ovaries, were cultured in estrogen-deprived medium, and the effect of steroids or steroid-free porcine follicular fluids on the NO release from oocytes was investigated. Oocytes that were isolated from cultured COCs were incubated with 1 microM ionomycin. The NO metabolites were identified using a NO detector-high-pressure liquid chromatography system. Oocytes from COCs cultured with 10 nM 17beta-estradiol (E2) released NO in response to ionomycin, whereas progesterone and testosterone had little effect on the synthesis of NO. An inhibitor of NOS suppressed the synthesis of NO. The maximal synthesis was observed after a 15 h-culture with E2. However, oocytes freshly obtained from antral follicles did not response to ionomycin, and the E2 action was suppressed by the addition of steroid-free follicular fluids. Analyses of RT-PCR and Western blotting showed that E2 did not increase NOS-3 expression. In addition, estrogen receptor beta was detected in oocytes and cumulus cells, and estrogen receptor alpha was detected only in cumulus cells. These findings suggest that oocyte NOS-3 is promoted for the synthesis of NO by E2 without increases in NOS-3 expression, but the synthesis of NO is suppressed, at least in the oocytes of early antral follicles.     

Identification of beta-tubulin isoform V as an autoantigen in allergic rhinitis by a proteomic approach

Autoantibodies to IgE and beta2-adrenergic receptor have been reported in patients with allergic rhinitis. To investigate whether autoimmunity in allergic rhinitis is directed to such limited molecules or directed to a wide range of self proteins, we here attempted to survey autoantigens/autoantibodies comprehensively, using proteomics. Specifically, we separated proteins extracted from peripheral blood mononuclear cells by 2-dimensional electrophoresis and then detected autoantigens by subsequent western blotting with sera from patients with allergic rhinitis. As a result, we detected multiple autoantigens, some of which were further identified by mass fingerprinting. Next, we confirmed antigenicity of one of the identified autoantigens, beta-tubulin isoform V (beta-tubV), using a recombinant protein and then measured prevalence of the anti-beta-tubV autoantibodies. As a result, 52% of the tested patients with allergic rhinitis were found to possess anti-beta-tubV autoantibodies. Our study indicates that autoimmunity is a common phenomena and beta-tubV is one of the major autoantigens in allergic rhinitis.     

Thermococcus siculi sp. nov., a novel hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent at the Mid-Okinawa Trough

A novel coccoid-shaped, hyperthermophilic, anaerobic archaeon, strain RG-20, was isolated from a deep-sea hydrothermal vent fluid sample taken at 1394-m depth at the Mid-Okinawa Trough (27°32.7′N, 126°58.5′E). Cells of this isolate occur singly or in pairs and are about 0.8 to 2 μm in diameter. Growth was observed at temperatures between 50° and 93°C, with an optimum at 85°C. The pH range for growth is 5.0–9.0, with an optimum around 7.0. Strain RG-20 requires 1%–4% of NaCl for growth, and cell lysis occurs at concentrations below 1%. The newly isolated strain grows preferentially in the presence of elemental sulfur on proteinaceous substrates such as yeast extract, peptone, or tryptone, and no growth was observed on carbohydrates, carboxylic acids, alcohols, or lipids. This microorganism is resistant to streptomycin, chloramphenicol, ampicillin, and kanamycin at concentrations up to 150 μg/ml, but is susceptible to rifampicin. Analysis of the hydrolyzed core lipids by thin-layer chromatography (TLC) revealed the presence of archaeol and caldarchaeol. The mol% G+C content of the DNA is 55.8. Partial sequencing of the 16S rDNA indicates that strain RG-20 belongs to the genus Thermococcus. Considering these data and on the basis of the results from DNA-DNA hybridization studies, we propose that this strain should be classified as a new species named Thermococcus siculi (si′cu.li. L. gen. n. siculi, of the deep-sea [siculum, deep-sea in literature of Ovid], referring to the location of the sample site, a deep-sea hydrothermal vent). The type strain is isolate RG-20 (DSM No. 12349). Received: May 11, 1998 / Accepted: July 24, 1998