Analysis of the essentiality of ROM2 genes in the pathogenic yeasts Candida glabrata and Candida albicans using temperature-sensitive mutants.

AIMS: To analyse the essentiality of the ROM2 genes originating from the pathogenic yeasts Candida glabrata and Candida albicans by using temperature-sensitive (ts) mutants. METHODS AND RESULTS: Based on the general concepts that ts mutations are generated by virtue of point mutation within essential genes, we have previously established a novel method (termed 'ETS system' for screening and identification of essential genes using ts mutants of C. glabrata). According to this ETS system, the present study successfully identified a putative C. glabrata ROM2 homologue as an essential gene that complements its point mutation (Cys-1275/Tyr substitution). The C. albicans ROM2 mutant (Cys-1281/Tyr), constructed patterned after this point mutation, also displayed ts phenotype. Both ts mutants recovered colony-forming ability, with concomitant suppression of lysis phenotype, at the elevated temperature in the presence of 1 mol l(-1) sorbitol as an osmotic stabilizer. Sequence alignment revealed that human genome possesses relatively low homology against Rom2 homologues, which are highly conserved among yeast species. CONCLUSIONS: ROM2 genes of C. glabrata and C. albicans are essential for viability, probably involved in cell wall integrity. SIGNIFICANCE AND IMPACT OF THE STUDY: ROM2 genes essential for both Candida species may be a potentially useful antifungal targets from chemotherapeutic viewpoint.

The mandibular response to occlusal relief using a flat guidance splint.

BACKGROUND: The mechanism of action of occlusal splints used for the successful treatment of temporomandibular disorders (TMD) remains unclear and controversial. AIM: The aim of this study was to observe the mandibular response during sleep bruxism (SB) on the elimination of occlusal influences by using a flat anterior and lateral guidance splint (FGS). MATERIAL AND METHOD: Any changes in mandibular movement patterns and condylar position with the introduction of this tool were measured. Current SB activity on the natural dentition was evaluated using a Brux Checker® (BC) and compared with the activity after insertion of an FGS in 153 subjects. RESULT: The spatial mandibular position changed individually with a tendency toward forward and downward movement. The insertion of an FGS led to a change in the topographical condyle-fossa relationship and seemed to create an "unloading" condition for the temporomandibular joint. It was found that increased angulation of the maxillar incisors was responsible for altered muscular activity during sleep. CONCLUSION: The masticatory organ appears to self-regulate and to provide an oral behavior modification, which may be more physiological using the FGS as a compensating factor. In this context, it is assumed that sleep bruxism in terms of parafunctional activity is a physiological function of the masticatory organ. The results of this study indicate the importance of controlling anterior guidance in the functional reconstruction of human occlusion.

A lower starting dose of eltrombopag is efficacious in Japanese patients with previously treated chronic immune thrombocytopenia.

BACKGROUND: Eltrombopag is an oral, non-peptide thrombopoietin receptor agonist that has shown efficacy and safety in chronic immune thrombocytopenia (ITP). However, ethnic differences in eltrombopag exposure have been reported: area under the curve exposure to eltrombopag was 87% greater among ITP patients of East Asian descent than among ITP patients of non-East Asian ITP descent. OBJECTIVES: To evaluate the efficacy and safety of eltrombopag by using, in Japanese ITP patients, lower starting (12.5 mg) and maximum (50 mg) doses of eltrombopag than the standard starting (50 mg) and maximum (75 mg) doses approved in the USA and Europe. PATIENTS: We examined 23 Japanese patients with previously treated chronic ITP with a platelet count of < 30,000 µL(-1) in a multicenter study comprising a randomized, double-blind, placebo-controlled phase for 6-week evaluation (15 eltrombopag, and eight placebo) and an open-label phase for 6-month evaluation (23 eltrombopag). RESULTS AND CONCLUSIONS: The response rate (platelet count of ≥ 50,000 µL(-1) ) at week 6 of the 6-week double-blind phase was 60% in eltrombopag-treated patients and 0% in placebo-treated patients. Ten of 23 patients (43.5%) responded for ≥ 75% of predefined assessment visits during the 6-month open-label phase. Notably, 22% (5/23) of patients responded to 12.5 mg of eltrombopag, which was administered within the first 3 weeks of eltrombopag treatment. Bleeding decreased with eltrombopag treatment as compared with baseline. Eltrombopag was generally well tolerated; one patient experienced a transient ischemic attack on day 9. Eltrombopag (12.5-50 mg) is effective for the management of Japanese patients with chronic ITP (NCT00540423).

Development of thrombopoietin receptor agonists for clinical use.

Thrombopoietin (TPO) is an essential hematopoietic cytokine for megakaryopoiesis. In 2002, we demonstrated that pegylated-recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) increased platelet counts in patients with chronic immune thrombocytopenic purpura (ITP) in a Phase I/II clinical trial. After the cessation of clinical trials of PEG-rHuMGDF because of severe thrombocytopenia or pancytopenia due to the development of the neutralizing antibody cross-reacting with endogenous TPO, second generation non-immunogenic TPO receptor agonists have been developed. A small molecule eltrombopag and Romiplostim were approved for clinical use by FDA in 2008 to treat patients with chronic ITP who are refractory to the prior therapy. Although the efficacy of both TPO receptor agonists is convincing for the refractory ITP, further investigation is necessary to assess the potential long-term side effects and clinical applications of these therapies for other thrombocytopenic conditions.

Establishment of a screening system for essential genes from the pathogenic yeast Candida glabrata: identification of a putative TEM1 homologue.

AIMS: To establish a system for screening and identification of essential genes from the pathogenic haploid yeast Candida glabrata by using temperature-sensitive (ts) mutants. METHODS AND RESULTS: Based on the general concepts that ts mutations are generated within essential genes in the genome by virtue of point mutation, we attempted to establish a system where essential genes were screened and identified from the C. glabrata genomic DNA library by the complementation of ts point mutations. By using this system, we successfully identified a putative TEM1 homologue as an essential gene by the complementation of a point mutation (-GAT-/-AAT- corresponding to Asp-143/Asn substitution) within its coding region in a ts mutant, T-3. CONCLUSIONS: We were able to establish a system for screening and identification of the essential genes, such as the TEM1 homologue, from the pathogenic yeast C. glabrata, as the gene that complements ts mutation. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of essential genes, by using the present system, may provide novel potential antifungal targets.

Age-specific antibody to hepatitis E virus has remained constant during the past 20 years in Japan.

We investigated the presence of antibodies to hepatitis E virus (anti-HEV) and hepatitis A virus (anti-HAV) by enzyme immunoassays in sera from 1015 individuals collected in 1974, 1984 and 1994. Age-specific profiles of anti-HEV remained unchanged with a peak at 40-49 years, while those of anti-HAV started to increase in individuals aged 20-29 years in 1974, 30-39 years in 1984 and 40-49 years in 1994. These results suggest that a silent HEV infection has been taking place in the last 20 years or so in Japan, while HAV infection has been terminated at least since 1974.

Association of interleukin-18 gene single-nucleotide polymorphisms with susceptibility to inflammatory bowel disease.

Interleukin-18 (IL-18) is believed to be one of the most important cytokines in the pathogenesis of inflammatory bowel disease (IBD). The aim of the study was to clarify the significance of single-nucleotide polymorphisms (SNPs) at the 5'-end of the IL-18 gene in the development of IBD. DNA was obtained from peripheral blood of 99 patients with ulcerative colitis (UC), 79 patients with Crohn's disease (CD), and 102 healthy controls. All participants were Japanese. SNPs at -656G/T, -607C/A, -137G/C, +113T/G, and +127C/T were determined by means of direct sequencing, and a genetic association with IBD was examined. The frequencies of the G allele at +113 and the T allele at +127 were significantly higher in patients with CD and UC compared with controls. The differences in allelic frequencies were more striking in patients with CD than in patients with UC, and at position +127 than at position +113. The haplotype estimation, according to the E-M algorithm, suggested that TACGT is closely associated with IBD, especially with CD. It was concluded that SNPs at the 5'-end of IL-18 gene might be closely related to the etiology of IBD.

Wild-type precore and core promoter sequences in patients with acute self-limited or chronic hepatitis B.

BACKGROUND: Mutations in the precore region and core promoter were compared between patients with acute and chronic hepatitis B. METHODS: There were 69 patients with acute self-limited hepatitis B and 210 with chronic hepatitis B who had been followed for > 15 years. The hepatitis B virus (HBV) of genotypes A, B and C was detected in 14 (23%), 8 (13%) and 28 (45%) of the patients with acute self-limited hepatitis, respectively, in contrast to 11 (5%), 25 (12%) and 167 (80%) of those with chronic hepatitis. RESULTS: At presentation, hepatitis B e antigen (HBeAg) in serum was the more common (82% versus 65%, P < 0.05), and the wild-type sequences of the precore region (100% versus 74%, P < 0.001) and core promoter (88% versus 36%, P < 0.00001) were more frequent in the 50 patients with acute self-limited hepatitis than the 203 patients with chronic hepatitis B who were infected with HBV of genotype A, B or C. Wild-types of both the precore region and core promoter persisted in acute self-limited hepatitis, while they decreased from 28% to 10% in chronic hepatitis over the course of > 15 years. CONCLUSION: HBV with the wild-type sequences of the precore region and core promoter prevails in patients with acute self-limited hepatitis, unlike in patients with chronic hepatitis.

The roles of phosphatidylinositol 3-kinase and protein kinase Czeta for thrombopoietin-induced mitogen-activated protein kinase activation in primary murine megakaryocytes.

Thrombopoietin (TPO) stimulates a network of intracellular signaling pathways that displays extensive cross-talk. We have demonstrated previously that the ERK/mitogen-activated protein kinase pathway is important for TPO-induced endomitosis in primary megakaryocytes (MKs). One known pathway by which TPO induces ERK activation is through the association of Shc with the penultimate phosphotyrosine within the TPO receptor, Mpl. However, several investigators found that the membrane-proximal half of the cytoplasmic domain of Mpl is sufficient to activate ERK in vitro and support base-line megakaryopoiesis in vivo. Using BaF3 cells expressing a truncated Mpl (T69Mpl) as a tool to identify non-Shc/Ras-dependent signaling pathways, we describe here novel mechanisms of TPO-induced ERK activation mediated, in part, by phosphoinositide 3-kinase (PI3K). Similar to cells expressing full-length receptor, PI3K was activated by its incorporation into a complex with IRS2 or Gab2. Furthermore, the MEK-phosphorylating activity of protein kinase Czeta (PKCzeta) was also enhanced after TPO stimulation of T69Mpl, contributing to ERK activity. PKCzeta and PI3K also contribute to TPO-induced ERK activation in MKs, confirming their physiological relevance. Like in BaF3 cells, a TPO-induced signaling complex containing p85PI3K is detectable in MKs expressing T61Mpl and is probably responsible for PI3K activation. These data demonstrate a novel role of PI3K and PKCzeta in steady-state megakaryopoiesis.

Determination of hepatitis B virus genotype G by polymerase chain reaction with hemi-nested primers.

Hepatitis B virus (HBV) has been classified into six genotypes designated A-F by sequence divergence in the entire genome exceeding 8%. Very recently, the seventh genotype was reported and named genotype G. HBV genotype G is distinct from genomes of the other six genotypes in that it possesses an insertion of 36 nucleotides in the core gene, and has been found so far in France and the United States. A method for determining HBV genotype G was developed by polymerase chain reaction (PCR) with primers deduced from the 36-nucleotide (nt) insertion in five isolates of HBV genotype G the sequences of which have been deposited in DNA databases. The validity of this method, for specifically detecting HBV genotype G, was verified on a panel consisting of 142 HBV isolates of six major genotypes and four of genotype G. A total of 540 sera containing HBV in Japan covering symptom free carriers and patients with a spectrum of chronic liver disease were tested by this method, but not a single HBV genotype G sample was found. A possible method for serological determination of hepatitis B surface antigen of genotype G is suggested, without amplification or sequencing nucleotides, which would expand epidemiological and clinical researches on HBV genotype G.

Rapid downregulation of cyclin D1 mRNA and protein levels by ultraviolet irradiation in murine macrophage cells.

DNA damage causes G1 cell cycle arrest through stabilization of p53 and its induction. As this process requires transcription, it takes several hours to achieve cell cycle arrest. We observed that ultraviolet (UV) light induces an immediate G1 arrest by rapid clearance of cyclin D1 in the murine macrophage cell line Bac1.2F5. The rapid disappearance of the cyclin D1 protein after exposure to UV was caused by at least two different mechanisms. In the first mechanism, cyclin D1 mRNA promptly disappeared within 1 min after UV irradiation, although cdk4 mRNA levels were unchanged. In the second mechanism, UV irradiation accelerated the degradation of cyclin D1 protein through the proteasome pathway. The half-life of the cyclin D1 protein was measured by pulse chase analysis and was shortened by UV light. These findings suggest that in the UV-irradiated Bac1.2F5 cells the amount of cyclin D1 protein is regulated at both the mRNA and protein levels. These two clearance mechanisms were also observed in murine bone-marrow-derived macrophages from wild type and p53 -/- mice, indicating that cyclin D1 mRNA and protein levels are independent of p53 function. This machinery might contribute to G1 cell cycle arrest and prevent cells from accumulating further DNA damage.

Thrombopoietin induces phosphoinositol 3-kinase activation through SHP2, Gab, and insulin receptor substrate proteins in BAF3 cells and primary murine megakaryocytes.

Thrombopoietin (TPO) is a recently characterized member of the hematopoietic growth factor family that serves as the primary regulator of megakaryocyte (MK) and platelet production. The hormone acts by binding to the Mpl receptor, the product of the cellular proto-oncogene c-mpl. Although many downstream signaling targets of TPO have been identified in cell lines, primary MKs, and platelets, the molecular mechanism(s) by which many of these molecules are activated remains uncertain. In this report we demonstrate that the TPO-induced activation of phosphoinositol 3-kinase (PI3K), a signaling intermediate vital for cellular survival and proliferation, occurs through its association with inducible signaling complexes in both BaF3 cells engineered to express Mpl (BaF3/Mpl) and in primary murine MKs. Although a direct association between PI3K and Mpl could not be demonstrated, we found that several proteins, including SHP2, Gab2, and IRS2, undergo phosphorylation and association in BaF3/Mpl cells in response to TPO stimulation, complexes that recruit and enhance the enzymatic activity of PI3K. To verify the physiological relevance of the complex, SHP2-Gab2 association was disrupted by overexpressing a dominant negative SHP2 construct. TPO-induced Akt phosphorylation was significantly decreased in transfected cells suggesting an important role of SHP2 in the complex to enhance PI3K activity. In primary murine MKs, TPO also induced phosphorylation of SHP2, its association with p85 and enhanced PI3K activity, but in contrast to the results in cell lines, neither Gab2 nor IRS2 are phosphorylated in MKs. Instead, a 100-kDa tyrosine-phosphorylated protein (pp100) co-immunoprecipitated with the regulatory subunit of PI3K. These findings support a model where PI3K activity is dependent on its recruitment into TPO-induced multiphosphoprotein complexes, implicate the existence of a scaffolding protein in primary MKs distinct from the known Gab and IRS proteins, and suggest that, in contrast to erythroid progenitor cells that employ Gab1 in PI3K signaling complexes, utilization of an alternate member of the Gab/IRS family could be responsible for specificity in TPO signaling.

Expression of prolactin gene in spontaneous mammary tumors in aging Fischer 344 rats.

Expression of extra pituitary prolactin (PRL) has been recently reported in the mammary gland. However, spontaneous mammary tumors occurring in aging rats have not been investigated for PRL production. The present study was undertaken to examine the expression of PRL gene in rat mammary tumors spontaneously arisen in rats with pituitary prolactinomas among 130 female Fischer-344 (F-344) rats. The tumors examined were fibroadenoma (adenomatous type) in the 18-month old rat and adenocarcinoma (alveolar/tubular type) in the 21-month old rat. PRL mRNA was examined by solution and in situ reverse transcription-polymerase chain reaction (RT-PCR) method. The predicted amplified products for PRL mRNA were identified in both tumors, and its expression was confirmed to be in the cytoplasm of epithelial cells. The results of the present study showed that PRL gene is expressed in spontaneously arising mammary tumors.

Species-specific TT viruses in humans and nonhuman primates and their phylogenetic relatedness.

By means of polymerase chain reaction with a primer pair (NG133-NG147) deduced from the untranslated region (UTR) of TT virus (TTV), TTVs with markedly distinct genomic lengths were recovered from sera of humans and nonhuman primates, and their entire nucleotide sequences were determined. A human TTV [TGP96 of 2908 nucleotides (nt)] was obtained that was about 900 nt shorter than heretofore reported TTVs (3787-3853 nt). Likewise, TTVs of chimpanzee occurred in two distinct genomic sizes [Pt-TTV6 (3690 nt) and Pt-TTV8-II (2785 nt)]. Two TTVs of Japanese macaque [Mf-TTV3 (3798 nt) and Mf-TTV9 (3763 nt)] were comparable in genomic length, but only 55% similar in sequence. These five human and nonhuman primate TTVs, along with TTVs of tamarin [So-TTV2 (3371 nt)] and douroucouli [At-TTV3 (3718 nt)], were compared over the entire nucleotide sequence. Although the seven TTVs were only < or = 55% similar, they share a common genomic organization with two open reading frames (ORFs), designated ORF1 (654-735 amino acids) and ORF2 (91-152 amino acids). The N-terminal sequences of ORF1 proteins were rich in arginine, and sequence motifs necessary for transcription and replication were conserved among them all. Like the human prototype TTV (TA278), all seven TTVs from various animals possessed in common two 15-nt sequences (CGAATGGCTGAGTTT and AGGGGCAATTCGGGC) in the UTR that were covered by NG133 and NG147, respectively. These primers would be instrumental in research on TTVs in previously unexamined species for defining their virological characteristics and evolutionary relationships.

Sequestration of TT virus of restricted genotypes in peripheral blood mononuclear cells.

Peripheral blood mononuclear cells (PBMC) harbored TT virus (TTV) of genotypes (3 and 4) different from those (1 and 2) of free virions in plasma of the same individuals. PBMC may act as a reservoir, and TTV of particular genotypes might have tropism for hematopoietic cells.

The entire nucleotide sequences of two distinct TT virus (TTV) isolates (TJN01 and TJN02) remotely related to the original TTV isolates.

TT virus (TTV) has a wide range of sequence divergence by which it is classified into at least 16 genotypes. A TTV isolate of genotype 12 (TJNO1) and another of genotype 13 (TJN02) were sequenced in the entire genome, and compared with the reported TTV isolates. TJN01 and TJN02 had genomic lengths of 3787 and 3794 nucleotides (nt), respectively, which were shorter by 66 and 59 nt than the prototype TTV isolate of genotype 1 (TA278). TJN01 and TJN02 shared the nucleotide sequence with TA278 merely in 53.9% and 55.2%, respectively. They possessed two major open reading frames (ORFs) and the noncoding region with a GC-rich region forming stem-loop structures, which are characteristic of TTV. However, their amino acid sequences in ORF1 were similar to that of TA278 in only 35.4 and 34.0%, respectively; TJN01 was 45.4% similar to TJN02. Comparison with TTV isolates of the same genotype identified hypervariable regions in ORF1 of TJN01 and TJN02, as in the prototype TTV of genotype 1. However, quasispecies were barely observed in them. Furthermore, sequences of hypervariable regions scarcely changed during 2-5.5 years in both TJN01 and TJN02. These results indicate that TTV of genotypes 12 and 13 are much different from the prototype TTV of genotype 1.