Promoter analyses of SCC antigen genes

SCC antigen (SCCA) has been used as a tumor marker for squamous cell carcinoma. Analyses of the SCCA1 and SCCA2 genes, which are almost identical, and their promoters have been reported. Recently it was found that both SCCAs were stimulated by interleukin (IL)-4 and IL-13. Here we analyzed the promoter activity of both SCCAs in the 5'-flanking region, exon 1, and intron 1 to evaluate a putative STAT6 binding site. The addition of intron 1 to the luciferase assay constructs including the 5'-flanking region significantly augmented the promoter activity of both SCCA1 and SCCA2. Furthermore, deletion analyses of intron 1 revealed that a 50-bp fragment of intron 1 that includes putative STAT6 binding site was responsible for the increased promoter activity. Although the sequences of SCCA1 and SCCA2 are very similar in the 5'-flanking region, the analysis of the -337 single nucleotide polymorphism of SCCA2 indicated that this polymorphism may underlie the difference in promoter activity between SCCA1 and SCCA2.     

Attenuated phosphorylation of heat shock protein 27 correlates with tumor progression in patients with hepatocellular carcinoma

Heat shock protein 27 (HSP27) is expressed at high levels in human hepatocellular carcinoma (HCC). We examined correlations of total HSP27 and serine phosphorylated (Ser-15, Ser-78, and Ser-82) HSP27 levels in HCC tissues with clinical and pathologic characteristics in 48 resected HCC specimens. The levels of total and Ser-phosphorylated HSP27 were evaluated by Western blot analysis. Immunohistochemical analysis of HSP27 expression was also performed on some samples. Phosphorylation of HSP27 was detected in all 48 HCC tissues. Levels of phosphorylated HSP27 were correlated inversely with tumor size, microvascular invasion of HCC, and tumor stage by TNM classification. In contrast, only microvascular invasion showed an inverse correlation with total HSP27 levels. The decrease in phosphorylated HSP27 in progressed HCC was also observed by immunohistochemistry. Levels of phosphorylated HSP27 gradually decreased in parallel with HCC progression. Our findings suggest that phosphorylated HSP27 may have a suppressive role in progression of human HCC.     

Genomewide high-density SNP linkage analysis of 236 Japanese families supports the existence of schizophrenia susceptibility loci on chromosomes 1p, 14q, and 20p

The Japanese Schizophrenia Sib-Pair Linkage Group (JSSLG) is a multisite collaborative study group that was organized to create a national resource for affected sib pair (ASP) studies of schizophrenia in Japan. We used a high-density single-nucleotide–polymorphism (SNP) genotyping assay, the Illumina BeadArray linkage mapping panel (version 4) comprising 5,861 SNPs, to perform a genomewide linkage analysis of JSSLG samples comprising 236 Japanese families with 268 nonindependent ASPs with schizophrenia. All subjects were Japanese. Among these families, 122 families comprised the same subjects analyzed with short tandem repeat markers. All the probands and their siblings, with the exception of seven siblings with schizoaffective disorder, had schizophrenia. After excluding SNPs with high linkage disequilibrium, we found significant evidence of linkage of schizophrenia to chromosome 1p21.2-1p13.2 (LOD=3.39) and suggestive evidence of linkage to 14q11.2 (LOD=2.87), 14q11.2-q13.2 (LOD=2.33), and 20p12.1-p11.2 (LOD=2.33). Although linkage to these regions has received little attention, these regions are included in or partially overlap the 10 regions reported by Lewis et al. that passed the two aggregate criteria of a meta-analysis. Results of the present study—which, to our knowledge, is the first genomewide analysis of schizophrenia in ASPs of a single Asian ethnicity that is comparable to the analyses done of ASPs of European descent—indicate the existence of schizophrenia susceptibility loci that are common to different ethnic groups but that likely have different ethnicity-specific effects.     

High-throughput detection of multiple genetic polymorphisms influencing drug metabolism with mismatch primers in allele-specific polymerase chain reaction

Because of genetic polymorphisms of drug-metabolizing enzyme genes, the activities of the enzymes in humans vary widely and alter the metabolism of commonly used clinical agents. Severe adverse effects or resistance to therapy may result. We have developed a rapid and high-throughput genotyping method for detecting polymorphisms of the drug-metabolizing enzyme genes CYP2C9*3, CYP2C19*2, *3, CYP2D6*2, *4, *10, *14, *21, NAT2*5, *6, *7, and TPMT*3 using allele-specific polymerase chain reaction (PCR) with mismatch primers (ASPCR-MP) and CYP2D6*5, *36, and CYP2D6xN using stepdown PCR with detection by SYBR Green I. We analyzed genomic DNA from 139 Japanese volunteers. Identical genotyping results were obtained by using ASPCR-MP, stepdown PCR, and conventional PCR. We found that the methods clearly differentiate three specific profiles with no overlap in the signals. Moreover, both ASPCR-MP and stepdown PCR for genotyping took less than 3-4h. To our knowledge, this is the first report of successful simultaneous detection of multiple genetic polymorphisms with point mutations using ASPCR-MP or multiple genetic polymorphisms with large structural alterations using stepdown PCR. In conclusion, ASPCR-MP and stepdown PCR appear to be suitable for large clinical and epidemiological studies as methods that enable highly sensitive genotyping and yield a high-throughput.     

Recolonization of laser-ablated bacterial biofilm

The recolonization of laser-ablated bacterial monoculture biofilm was studied in the laboratory by using a flow-cytometer system. The marine biofilm-forming bacterium Pseudoalteromonas carrageenovora was used to develop biofilms on titanium coupons. Upon exposure to a low-power pulsed irradiation from an Nd:YAG laser, the coupons with biofilm were significantly reduced both in terms of total viable count (TVC) and area cover. The energy density used for a pulse of 5 ns was 0.1 J/cm(2) and the durations of irradiation exposure were 5 and 10 min. When placed in a flow of dilute ZoBell marine broth medium (10%) the laser-destructed bacterial film in a flow-cytometer showed significant recovery over a period of time. The flow of medium was regulated at 3.2 ml/min. The increase in area cover and TVC, however, was significantly less than that observed for nonirradiated control (t-test, P< 0.05). The coupons were observed for biofilm area cover and TVC at different intervals (3, 6, and 9 h) after irradiation. While the biofilm in the control coupon at the end of 9 h of exposure showed 95.6 +/- 4.1% cover, the 5- and 10-min irradiated samples after 9 h showed 60.3 +/- 6.5 and 37.4 +/- 12.1% area cover, respectively. The reduced rate of recolonization compared to control was thought be due to the lethal and sublethal impacts of laser irradiation on bacteria. This observation thus provided data on the online recolonization speed of biofilm, which is important when considering pulsed laser irradiation as an ablating technique of biofilm formation and removal in natural systems.     

In vitro laser ablation of laboratory developed biofilms using an Nd:YAG laser of 532 nm wavelength

We studied the laser ablation of laboratory-developed biofilm on titanium and glass surfaces. Specifically, Pseudoalteromonas carrageenovora, a marine biofilm forming bacterium was used to generate laboratory biofilm. Two fluences, 0.05 and 0.1 J/cm(2) and three durations of irradiation, 30 s, 5 min, and 10 min were tested using an Nd;YAG laser of 532 nm wavelength (in the green light area). Nonirradiated coupons with biofilm served as control. The biofilm removal efficiency increased with the increase in laser fluence and duration of irradiation. The maximum biofilm area cover on control coupons of glass and titanium was 62.5 and 76.0%, respectively. Upon irradiation with fluence 0.1 J/cm(2) for the very short duration of 30 s, this reduced to 5.6 and 12.4% and at 10 min to 2.17 and 0.7% on glass and titanium coupons, respectively, while the controls did not show any reductions (62.5 and 76.0% respectively, for glass and titanium coupons). The biofilm TRC (Total Resuscitated Cells) reduction during this period was even more prominent than the area cover, indicating that the remaining biofilm portions on coupons after irradiation were largely composed of dead bacterial cells. The TRC in the irradiation chamber medium for short durations of irradiation showed a significant increase, indicating that the laser irradiation removed live bacteria from the biofilm. The re-growth of the resuscitated cells showed they could grow like the control cells but with a significant lag. The laser's efficiency in the removal of biofilm was better seen on titanium coupons than on glass. Our results showed that a low-power pulsed laser irradiation could be used to remove biofilm formed on hard surfaces.     

Essential role of GATA3 for the maintenance of type 2 helper T (Th2) cytokine production and chromatin remodeling at the Th2 cytokine gene loci

GATA3 expression is essential for type-2 helper T (Th2) cell differentiation. GATA3-mediated chromatin remodeling at the Th2 cytokine gene loci, including Th2-specific long range histone hyperacetylation of the interleukin (IL)-13/IL-4 gene loci, occurs in developing Th2 cells. However, little is known about the role of GATA3, if any, in the maintenance of established remodeled chromatin at the Th2 cytokine gene loci. Here, we established a Cre/LoxP-based site-specific recombination system in cultured CD4 T cells using a unique adenovirus-mediated gene transfer technique. This system allowed us to investigate the effect of loss of GATA3 expression in in vitro differentiated Th2 cells. After ablation of GATA3, we detected reduced production of all Th2 cytokines, increased DNA methylation at the IL-4 gene locus, and decreased histone hyperacetylation at the IL-5 gene locus but not significantly so at the IL-13/IL-4 gene loci. Thus, GATA3 plays important roles in the maintenance of the Th2 phenotype and continuous chromatin remodeling of the specific Th2 cytokine gene locus through cell division.     

TDAG8 is a proton-sensing and psychosine-sensitive G-protein-coupled receptor

T cell death-associated gene 8 (TDAG8) has been reported to be a receptor for psychosine. Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4, G-protein-coupled receptors (GPCRs) closely related to TDAG8, however, have recently been identified as proton-sensing or extracellular pH-responsive GPCRs that stimulate inositol phosphate and cAMP production, respectively. In the present study, we examined whether TDAG8 senses extracellular pH change. In the several cell types that were transfected with TDAG8 cDNA, cAMP was markedly accumulated in response to neutral to acidic extracellular pH, with a peak response at approximately pH 7.0-6.5. The pH effect was inhibited by copper ions and was reduced or lost in cells expressing mutated TDAG8 in which histidine residues were changed to phenylalanine. In the membrane fractions prepared from TDAG8-transfected cells, guanosine 5'-O-(3-thiotriphosphate) binding activity and adenylyl cyclase activity were remarkably stimulated in response to neutral and acidic pH. The concentration-dependent effect of extracellular protons on cAMP accumulation was shifted to the right in the presence of psychosine. The inhibitory psychosine effect was also observed for pH-dependent actions in OGR1- and GPR4-expressing cells but not for prostaglandin E(2)- and sphingosine 1-phosphate-induced actions in any pH in native and sphingosine 1-phosphate receptor-expressing cells. Glucosylsphingosine and sphingosylphosphorylcholine similarly inhibited the pH-dependent action, although to a lesser extent. Psychosine-sensitive and pH-dependent cAMP accumulation was also observed in mouse thymocytes. We concluded that TDAG8 is one of the proton-sensing GPCRs coupling to adenylyl cyclase and psychosine, and its related lysosphingolipids behave as if they were antagonists against protein-sensing receptors, including TDAG8, GPR4, and OGR1.