Detection of Mycoplasma fermentans in saliva sampled from infants, preschool and school children, adolescents and adults by a polymerase chain reaction-based assay.

Attempts were made to detect Mycoplasma fermentans in saliva sampled from 201 subjects (108 males and 93 females) aged from 4 months to 59 years by a polymerase chain reaction-based assay. M. fermentans was detected in saliva from 110 (54.7%) of 201 subjects, and 10 (28.6%) of 35 subjects aged from 4 months to 3 years. Of ten positive subjects, three were aged from 16 to 23 months and five were from 26 to 31 months. The incidence tended to increase with age up to the teens. The incidence was significantly greater in teenagers than in subjects aged from 7 to 12 years, but there was no significant difference in the incidence between the group of teenagers and each of the groups of subjects older than the teenagers. Thus, it was suggested that M. fermentans colonized the mouth at the age of about 16 months up to the age of 19 years.     

Expression and function of inducible co-stimulator in patients with systemic lupus erythematosus: possible involvement in excessive interferon-gamma and anti-double-stranded DNA antibody production

Inducible co-stimulator (ICOS) is the third member of the CD28/cytotoxic T-lymphocyte associated antigen-4 family and is involved in the proliferation and activation of T cells. A detailed functional analysis of ICOS on peripheral blood T cells from patients with systemic lupus erythematosus (SLE) has not yet been reported. In the present study we developed a fully human anti-human ICOS mAb (JTA009) with high avidity and investigated the immunopathological roles of ICOS in SLE. JTA009 exhibited higher avidity for ICOS than a previously reported mAb, namely SA12. Using JTA009, ICOS was detected in a substantial proportion of unstimulated peripheral blood T cells from both normal control individuals and patients with SLE. In CD4+CD45RO+ T cells from peripheral blood, the percentage of ICOS+ cells and mean fluorescence intensity with JTA009 were significantly higher in active SLE than in inactive SLE or in normal control individuals. JTA009 co-stimulated peripheral blood T cells in the presence of suboptimal concentrations of anti-CD3 mAb. Median values of [3H]thymidine incorporation were higher in SLE T cells with ICOS co-stimulation than in normal T cells, and the difference between inactive SLE patients and normal control individuals achieved statistical significance. ICOS co-stimulation significantly increased the production of IFN-gamma, IL-4 and IL-10 in both SLE and normal T cells. IFN-gamma in the culture supernatants of both active and inactive SLE T cells with ICOS co-stimulation was significantly higher than in normal control T cells. Finally, SLE T cells with ICOS co-stimulation selectively and significantly enhanced the production of IgG anti-double-stranded DNA antibodies by autologous B cells. These findings suggest that ICOS is involved in abnormal T cell activation in SLE, and that blockade of the interaction between ICOS and its receptor may have therapeutic value in the treatment of this intractable disease.     

Polystyrene microgel amphiphiles with maltohexaose. Synthesis, characterization, and potential applications

4-Vinylbenzyl maltohexaoside peracetate (1) was copolymerized with divinylbenzene (DVB) using 1-phenyl-1-(2',2',6',6'-tetramethyl-1'-piperidinyloxy)ethane (2) in m-xylene. The copolymerizations were performed at 138 degrees C for 20 h using the mole fraction of 1 in the total feed of 1 and DVB (F1: [1]/[1]+[DVB]) varying from 0.11 to 0.38, affording polymeric products in yields ranging from 32 to 40%. The characterizations by linear PS-calibrated size exclusion chromatography (SEC), dynamic laser light scattering (DLS) measurements, and 1H NMR spectroscopy indicated that the product was assignable to the cross-linked poly(4-vinylbenzyl maltohexaoside peracetate) particle which is able to produce stable solutions, i.e., the PSt microgel with acetyl maltohexaose, 3. The specific rotations ([alpha]D23, c = 1.0 CHCl3) of 3 ranged from +43.3 degrees to +85.6 degrees. The average molar masses determined by the static laser light scattering (SLS) measurement of 3, M(w,SLS)'s, were from 64,700 to 118,000, which were calculated using the respective refractive index increments, dn/dc's, ranging from 0.03387 to 0.08340. The apparent numbers of the 1, 2, and DVB units in 3, N1, N2, and N(DVB), which were estimated from the respective [alpha]D23 values, M(w,SLS)'s, and real yields, ranged from 22 to 35, from 7 to 26, and from 146 to 506, respectively. The deacetylation of 3 was achieved by treatment with sodium methoxide in dry 1,4-dioxane to produce the PSt microgel with maltohexaose as the hydrophilic segment, 4, as a white solid. The solubility of 4 in various solvents was examined, indicating that a hydrophilic property was effectively introduced. Notably, 4 gave clear solutions in the mixed solvent of 1,4-dioxane and H2O. The ability to solubilize fullerite (mixture of fullerenes, C60/C70 = ca. 9/1) in aqueous solutions was examined according to the literature method. Approximately, 100 mg of 4 (1.7 micromol) solubilizes 1.3 mg of fullerite (1.7 micromol).     

Development of a black gram [Vigna mungo (L.) Hepper] linkage map and its comparison with an azuki bean [Vigna angularis (Willd.) Ohwi and Ohashi] linkage map

The Asian Vigna group of grain legumes consists of six domesticated species, among them black gram is widely grown in South Asia and to a lesser extent in Southeast Asia. We report the first genetic linkage map of black gram [Vigna mungo (L.) Hepper], constructed using a BC₁F₁ population consisting of 180 individuals. The BC₁F₁ population was analyzed in 61 SSR primer pairs, 56 RFLP probes, 27 AFLP loci and 1 morphological marker. About 148 marker loci could be assigned to the 11 linkage groups, which correspond to the haploid chromosome number of black gram. The linkage groups cover a total of 783 cM of the black gram genome. The number of markers per linkage group ranges from 6 to 23. The average distance between adjacent markers varied from 3.5 to 9.3 cM. The results of comparative genome mapping between black gram and azuki bean show that the linkage order of markers is highly conserved. However, inversions, insertions, deletions/duplications and a translocation were detected between the black gram and azuki bean linkage maps. The marker order on parts of linkage groups 1, 2 and 5 is reversed between the two species. One region on black gram linkage group 10 appears to correspond to part of azuki bean linkage group 1. The present study suggests that the azuki bean SSR markers can be widely used for Asian Vigna species and the black gram genetic linkage map will assist in improvement of this crop.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.The first three authors contributed equally to this research     

Involvement of heparan sulfate proteoglycan in sensing hypotonic stress in bovine aortic endothelial cells

Hypotonic stress (HTS) induces various responses in vascular endothelium, but the molecules involved in sensing HTS are not known. To investigate a possible role of heparan sulfate proteoglycan (HSPG) in sensing HTS, we compared the responses of control bovine aortic endothelial cells (BAECs) with those of cells treated with heparinase III, which exclusively degrades HSPG. Tyrosine phosphorylation of 125 kDa FAK induced by HTS (-30%) in control cells was abolished in heparinase III-treated BAECs. The amplitude of the volume-regulated anion channel (VRAC) current, whose activation is regulated by tyrosine kinase, was significantly reduced by the treatment with heparinase III. Also, HTS-induced ATP release through the VRAC pore and the concomitant Ca(2+) transients were significantly reduced in the heparinase III-treated BAECs. In contrast, exogenously applied ATP evoked similar Ca(2+) transients in both control and heparinase III-treated BAECs. The transient formation of actin stress fibers induced by HTS in control cells was absent in heparinase III-treated BAECs. Lysophosphatidic acid (LPA) also induced FAK phosphorylation, actin reorganization and ATP release in control BAECs, but heparinase III did not affect these LPA-induced responses. We conclude from these observations that HSPG is one of the sensory molecules of hypotonic cell swelling in BAECs.     

Mutagenicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and benzo[a]pyrene (BaP) in the gill and hepatopancreas of rpsL transgenic zebrafish

We examined the in vivo mutagenicity of 2-[2-(acetylamino)-4-[bis(2-hydroxyethyl)amino]-5-methoxyphenyl]-5-amino-7-bromo-4-chloro-2H-benzotriazole (PBTA-6) and benzo[a]pyrene (BaP) by using transgenic (Tg) zebrafish carrying the mutational target gene rpsL. PBTA-6 is one of the PBTA-type compounds that were recently identified in highly mutagenic river water in Japan. BaP is a well-known contaminant that is frequently found in polluted water. Both compounds are potent mutagens, as determined by using the Ames test employing S9 mix and Salmonella. Adult rpsL Tg zebrafish were exposed to 0, 7, or 10 mg/L PBTA-6 or 0, 1.5, or 3 mg/L BaP for 96 h in a water bath and the mutations in their gills and hepatopancreata were measured 2-4 weeks later. At 3 weeks after exposure, 3 mg/L BaP significantly increased the rpsL mutant frequency (MF) in the gill and hepatopancreas by 5- and 2.3-fold, respectively, as compared to control fish. Sequence analysis showed that BaP mainly induced G:C to T:A and G:C to C:G transversions, which is consistent with the known mutagenic effects of BaP. In contrast, despite its extremely high mutagenic potency in Salmonella strains, PBTA-6 did not significantly increase the MF in the zebrafish gill or hepatopancreas. Although PBTA-6 is 300 times more mutagenic than BaP in the Ames test [T. Watanabe, H. Nukaya, Y. Terao, Y. Takahashi, A. Tada, T. Takamura, H. Sawanishi, T. Ohe, T. Hirayama, T. Sugimura, K. Wakabayashi, Synthesis of 2-phenylbenzotriazole-type mutagens, PBTA-5 and PBTA-6, and their detection in river water from Japan, Mutat. Res. 498 (2001) 107-115], calculation of the mutagenicity per mole of compound indicated that PBTA-6 was 33- and <3.7-fold less mutagenic in the zebrafish gill and hepatopancreas, respectively, than BaP.     

The genetics of domestication of the azuki bean (Vigna angularis)

Genetic differences between azuki bean (Vigna angularis var. angularis) and its presumed wild ancestor (V. angularis var. nipponensis) were resolved into QTL for traits associated with adaptation to their respective distinct habits. A genetic linkage map constructed using progenies from a cross between Japanese cultivated and wild azuki beans covers 92.8% of the standard azuki bean linkage map. A reciprocal translocation between cultivated and wild azuki bean parents was identified on the basis of the linkage map having a pseudolinkage group and clustering of seed productivity-related QTL with large effect near the presumed breakpoints. In total, 162 QTL were identified for 46 domestication-related traits. Domestication of azuki bean has involved a trade-off between seed number and seed size: fewer but longer pods and fewer but larger seeds on plants with shorter stature in cultivated azuki bean being at the expense of overall seed yield. Genes found related to germination and flowering time in cultivated azuki bean may confer a selective advantage to the hybrid derivatives under some ecological conditions and may explain why azuki bean has evolved as a crop complex in Japan.     

GJB2-associated hearing loss undetected by hearing screening of newborns

The hearing loss caused by GJB2 mutations is usually congenital in onset, moderate to profound in degree, and non-progressive. The objective of this study was to study genotype/phenotype correlations and to document 14 children with biallelic GJB2 mutations who passed newborn hearing screening (NHS). Genetic testing for GJB2 mutations by direct sequencing was performed on 924 individuals (810 families) with hearing loss, and 204 patients (175 families) were found to carry biallelic GJB2 mutations. NHS results were obtained through medical records. A total of 18 pathological mutations were identified, which were subclassified as eight inactivating and 10 non-inactivating mutations. p.I128M and p.H73Y were identified as novel missense GJB2 mutations. Of the 14 children with biallelic GJB2 mutations who passed NHS, eight were compound heterozygotes and 3 were homozygous for the c.235delC mutation in GJB2, and the other three combinations of non-c.235delC mutations identified were p.Y136X-p.G45E/p.V37I heterozygous, c.512ins4/p.R143W heterozygous, and p.V37I/p.R143W heterozygous. These 14 cases demonstrate that the current NHS does not identify all infants with biallelic GJB2 mutations. They suggest that the frequency of non-penetrance at birth is approximately 6.9% or higher in DFNB1 patients and provide further evidence that GJB2 hearing loss may not always be congenital in onset.