Brief communication: mitochondrial DNA variation suggests extensive gene flow from Polynesian ancestors to indigenous Melanesians in the northwestern Bismarck Archipelago

Archaeological, linguistic, and genetic studies show that Austronesian (AN)-speaking Polynesian ancestors came from Asia/Taiwan to the Bismarck Archipelago in Near Oceania more than 3,600 years ago, and then expanded into Remote Oceania. However, it remains unclear whether they extensively mixed with indigenous Melanesians who had populated the Bismarck Archipelago before their arrival. To examine the extent of admixture between Polynesian ancestors and indigenous Melanesians, mitochondrial DNA (mtDNA) variations in the D-loop region and the cytochrome oxidase and lysine transfer RNA (COII/tRNA(Lys)) intergenic 9-bp deletion were analyzed in the following three Oceanian populations: 1) Balopa Islanders as AN-speaking Melanesians living in the northwestern end of the Bismarck Archipelago, 2) Tongans as AN-speaking Polynesians, and 3) Gidra as non-Austronesian-speaking Melanesians in the southwestern lowlands of Papua New Guinea. Phylogenetic analysis of mtDNA sequences revealed that more than 60% of mtDNA sequences in the Balopa Islanders were very similar to those in Tongans, suggesting an extensive gene flow from Polynesian ancestors to indigenous Melanesians. Furthermore, analysis of pairwise difference distributions for the D-loop sequences with the 9-bp deletion and the Polynesian motif (i.e., T16217C, A16247G, and C16261T) suggested that the expansion of Polynesian ancestors possessing these variations occurred approximately 7,000 years ago.     

Association study on chromosome 20q11.21-13.13 locus and its contribution to type 2 diabetes susceptibility in Japanese

Several linkage studies have predicted that human chromosome 20q is closely related to type 2 diabetes, but there is no clear evidence that certain variant(s) or gene(s) have strong effects on the disease within this region. To examine disease susceptibility variant in Japanese, verified SNPs from the databases, with a minor allele frequency larger than 0.15, were selected at 10-kb intervals across a 19.31-Mb region (20q11.21-13.13), which contained 291 genes, including hepatocyte nuclear factor 4α (HNF4α). As a result, a total of 1,147 SNPs were genotyped with TaqMan assay using 1,818 Japanese samples. By searching for HNF4α as a representative disease-susceptible gene, no variants of HNF4α were strongly associated with disease. To identify other genetic variant related with disease, we designed an extensive two-stage association study (725 first and 1,093 second test samples). Although SNP1146 (rs220076) was selected as a landmark within the 19.31 Mb region, the magnitude of the nominal P value (P = 0.0023) was rather weak. Subsequently, a haplotype-based association study showed that two common haplotypes were weakly associated with disease. All of these tests resulted in non-significance after adjusting for Bonferroni’s correction and the false discovery rate to control for the impact of multiple testing. Contrary to the initial expectations, we could not conclude that certain SNPs had a major effect on this promising locus within the framework presented here. As a way to extend our observations, we emphasize the importance of a subsequent association study including replication and/or meta-analysis in multiple populations.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

IL-18 gene polymorphisms affect IL-18 production capability by monocytes

We previously demonstrated a significant association between IL-18 gene polymorphism 105A/C and asthma. In this study, we investigated the relationship of IL-18 gene polymorphism to IL-18 production capability by monocytes. The frequency of gene polymorphisms including IL-18-105A/C and IL-18--137G/C was determined by PCR analyses. The IL-18 production by monocytes stimulated without or with LPS or A23187+PMA for 1day was measured by ELISA. The produced IL-18 spontaneously or in response to A23187+PMA by monocytes was significantly higher for volunteers with 105A/A genotype than with 105A/C genotype. Similarly, the production capability of IL-18 by monocytes from volunteers with -137G/G genotype was significantly higher than that with -137G/C genotype and significant linkage disequilibrium was observed between 105A/C and -137G/C polymorphism. Thus, the genetic capacity to produce more IL-18 in response to stimuli may affect the onset of asthma.     

Evaluations of substrate specificity and inhibition at PR/p3 cleavage site of HTLV-1 protease

Core sequences necessary for substrate recognition and its inhibition at the PR/p3 site of HTLV-1 protease were clarified for the first time. From the cleavage rates of peptides containing a part of the PR/p3 site, a heptapeptide was found to be the minimal sequence required for substrate recognition. The use of synthetic inhibitors containing hydroxyethylamine dipeptide isostere indicated that a tetrapeptide sequence was necessary to achieve potent inhibition.     

Role of Streptococcus sanguinis sortase A in bacterial colonization

Streptococcus sanguinis, a normal inhabitant of the human oral cavity, has low cariogenicity, though colonization on tooth surfaces by this bacterium initiates aggregation by other oral bacteria and maturation of dental plaque. Additionally, S. sanguinis is frequently isolated from infective endocarditis patients. We investigated the functions of sortase A (SrtA), which cleaves LPXTG-containing proteins and anchors them to the bacterial cell wall, as a possible virulence factor of S. sanguinis. We identified the srtA gene of S. sanguinis by searching a homologous gene of Streptococcus mutans in genome databases. Next, we constructed an srtA-deficient mutant strain of S. sanguinis by insertional inactivation and compared it to the wild type strain. In the case of the mutant strain, some surface proteins could not anchor to the cell wall and were partially released into the culture supernatant. Furthermore, adherence to saliva-coated hydroxyapatite beads and polystyrene plates, as well as adherence to and invasion of human epithelial cells were reduced significantly in the srtA-deficient strain when compared to the wild type. In addition, antiopsonization levels and bacterial survival of the srtA-deficient mutant were decreased in human whole blood. This is the first known study to report that SrtA contributes to antiopsonization in streptococci. Our results suggest that SrtA anchors surface adhesins as well as some proteins that function as antiopsonic molecules as a means of evading the human immune system. Furthermore, they demonstrate that SrtA of S. sanguinis plays important roles in bacterial colonization.     

Computer assisted analysis of S-potentials

S-potentials from the channel catfish were analyzed using a high speed biological data processing system developed at this Institute. The analysis has shown that, when moderately dark adapted, the relation between the (maximal) amplitude of S-potential and log intensity of flash was the hyperbolic tangent throughout the whole visible spectrum. This is what one could expect if the S-potential in the fish was generated by signals from a single class of pigment and each signal had discrete action on the S-potential generating mechanism. The maximal absorption of the pigment involved was at around 625 nm.     

Lipopolysaccharide signaling induces serum amyloid A (SAA) synthesis in human hepatocytes in vitro

To investigate the role of lipopolysaccharide (LPS) in hepatocyte activation, we examined the expression of Toll-like receptor 4 (TLR4), the putative receptor for LPS in human hepatocytes. TLR4 mRNA and protein expression was confirmed in human hepatocytes. Stimulation of human hepatocytes with LPS results in rapid degradation of IkappaB-alpha and mitogen activated protein kinase activation. Human hepatocytes stimulated by LPS produced serum amyloid A protein. Our data suggest that human hepatocytes utilize components of TLR4 signal transduction pathways in response to LPS and these direct LPS-mediated effects on hepatocytes may contribute to liver inflammation and injury.     

Establishment of hepatitis C virus replicon cell lines possessing interferon-resistant phenotype

To clarify the mechanism underlying resistance to interferon (IFN) by the hepatitis C virus (HCV) in patients with chronic hepatitis, we attempted to develop an IFN-resistant HCV replicon from the IFN-sensitive 50-1 replicon established previously. By treating 50-1 replicon cells with a prolonged low-dose treatment of IFN-alpha and then transfecting the total RNA derived from the IFN-alpha-treated replicon cells, we successfully obtained four clones (named 1, 3, 4, and 5) of HCV replicon cells that survived against IFN-alpha (200 IU/ml). These cloned cells were further treated with IFN-alpha or IFN-beta (increased gradually to 2000 or 1000 IU/ml, respectively). This led to four replicon cell lines (alphaR series) possessing the IFN-alpha-resistant phenotype and four replicon cell lines (betaR series) possessing the IFN-beta-resistant phenotype. Furthermore, we obtained an additional replicon cell line (alphaRmix) possessing the IFN-alpha-resistant phenotype by two rounds of prolonged treatment with IFN-alpha and RNA transfection as mentioned above. Characterization of these obtained HCV replicon cell lines revealed that the betaR series were highly resistant to both IFN-alpha and IFN-beta, although the alphaR series containing alphaRmix were only partially resistant to both IFN-alpha and IFN-beta. Genetic analysis of these HCV replicons found one common amino acid substitution in the NS4B and several additional amino acid substitutions in the NS5A of the betaR series, suggesting that these genetic alterations are involved in the IFN resistance of these HCV replicons. These newly established HCV replicon cell lines possessing IFN-resistant phenotypes are the first useful tools for understanding the mechanisms by which HCV acquires IFN resistance in vivo.     

Stress-induced premature senescence in hTERT-expressing ataxia telangiectasia fibroblasts

In addition to replicative senescence, normal diploid fibroblasts undergo stress-induced premature senescence (SIPS) in response to DNA damage caused by oxidative stress or ionizing radiation (IR). SIPS is not prevented by telomere elongation, indicating that, unlike replicative senescence, it is triggered by nonspecific genome-wide DNA damage rather than by telomere shortening. ATM, the product of the gene mutated in individuals with ataxia telangiectasia (AT), plays a central role in cell cycle arrest in response to DNA damage. Whether ATM also mediates signaling that leads to SIPS was investigated with the use of normal and AT fibroblasts stably transfected with an expression vector for the catalytic subunit of human telomerase (hTERT). Expression of hTERT in AT fibroblasts resulted in telomere elongation and prevented premature replicative senescence, but it did not rescue the defect in G(1) checkpoint activation or the hypersensitivity of the cells to IR. Despite these remaining defects in the DNA damage response, hTERT-expressing AT fibroblasts exhibited characteristics of senescence on exposure to IR or H(2)O(2) in such a manner that triggers SIPS in normal fibroblasts. These characteristics included the adoption of an enlarged and flattened morphology, positive staining for senescence-associated beta-galactosidase activity, termination of DNA synthesis, and accumulation of p53, p21(WAF1), and p16(INK4A). The phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK), which mediates signaling that leads to senescence, was also detected in both IR- or H(2)O(2)-treated AT and normal fibroblasts expressing hTERT. These results suggest that the ATM-dependent signaling pathway triggered by DNA damage is dispensable for activation of p38 MAPK and SIPS in response to IR or oxidative stress.