Nuclear localization of activated STAT6 and STAT3 in epidermis of prurigo nodularis.

BACKGROUND: Prurigo nodularis (PN) is a chronic dermatitis characterized by discrete, raised, and firm papulonodules with intense pruritus. The pathogenesis still remains to be elucidated. OBJECTIVES: To clarify the role of Th1 and Th2 cytokines in the pathogenesis of PN. METHODS: We examined the cytokine signatures, such as phosphorylation of STAT1, STAT3 and STAT6, HLA-DR and hyaluronan accumulation, to reveal the Th1 and Th2 cytokine influence on the lesional epidermis of PN. RESULTS: We first optimized antigen retrieval methods to detect these signatures with antibodies for phospho-STAT1 (pSTAT1), phospho-STAT3 (pSTAT3), phospho-STAT6 (pSTAT6), HLA-DR and hyaluronic acid binding protein (HABP) on the formalin-fixed paraffin-embedded sections of psoriasis, lichen planus and atopic dermatitis biopsy samples. Activation of STAT1 and STAT6 in epidermis by Th1 and Th2 cytokines was further confirmed in a cultured skin equivalent model treated with interferon-γ or interleukin (IL)-4/IL-13. With the relevant immunostaining methods, we examined the cytokine signatures in 22 cases of PN. The results revealed that (i) the entire epidermis of 19 cases was stained with anti-pSTAT6 antibody, (ii) 21 cases demonstrated nuclear staining with anti-pSTAT3 antibody, (iii) the entire epidermis of 21 cases was stained with HABP, (iv) the epidermis of eight cases showed scattered staining with anti-pSTAT1 antibody, and (v) six cases were positive for HLA-DR membrane expression. CONCLUSIONS: These data indicated that Th2 cytokines related to STAT6 activation together with some unknown stimuli that activate STAT3 play a principal role in the pathogenesis of PN.

An improved procedure for rapid determination of viral RNA sequences of avian RNA viruses.

A system for rapid determination of viral RNA sequences, RDV, was improved for detection of avian RNA virus in allantoic fluids. We detected avian paramyxovirus nucleotide sequences using RDV method ver 2.0.

Enhancement of cytotoxicity against Vero E6 cells persistently infected with SARS-CoV by Mycoplasma fermentans.

We previously reported that cells with persistent severe acute respiratory syndrome coronavirus (SARS-CoV) infection were established after apoptotic events. In the present study, we investigated the cytopathic effects of dual infection with SARS-CoV and Mycoplasma fermentans on Vero E6 cells. Dual infection completely killed cells and prevented the establishment of persistent SARS-CoV infection. M. fermentans induced inhibition of cell proliferation, but the cells remained alive. Apoptosis was induced easily in M. fermentans-infected cells, indicating that they were primed for apoptosis. These results indicated that M. fermentans enhances apoptosis in surviving cells that have escaped from SARS-CoV-induced apoptosis.

Flurbiprofen does not change the bispectral index and 95% spectral edge frequency during total intravenous anaesthesia with propofol and fentanyl.

BACKGROUND AND OBJECTIVE: Previous studies have shown that general anaesthetic agents modulate the production of hypothalamic prostaglandins (PG) D2 and E2, which are mediators of sleep and wakefulness respectively. Although flurbiprofen, a cyclo-oxygenase inhibitor, is used clinically as a non-steroidal anti-inflammatory agent and postoperative analgesic, it reduces prostaglandin production. Thus, this agent may affect the depth of sedation during general anaesthesia. In this study, we examined if flurbiprofen affects the bispectral index, which correlates with sedation levels. METHODS: Fifteen patients who underwent elective surgery under total intravenous anaesthesia with propofol and fentanyl were studied. The sedation level was monitored using a bispectral index monitor. On attainment of stable haemodynamics and a bispectral index, patients were given flurbiprofen axetil 50 mg intravenously. A bispectral index and 95% spectral edge frequency were recorded before and 5, 10, 15, 20 and 30 min after flurbiprofen axetil intravenously. RESULTS: Bispectral indexes of 51.7 (95% CI: 47.3-56.8), 51.7 (47.1-56.3), 51.3 (46.3-56.3), 50.3 (45.8-54.2), 48.9 (43.6-54.1) and 50.3 (45.5-55.2) at 0, 5, 10, 15, 20, 30 min after flurbiprofen axetil intravenously were observed. There was no change in this or 95% spectral edge frequency. CONCLUSIONS: Clinical dose of flurbiprofen axetil does not alter the bispectral index and 95% spectral edge frequency under total intravenous anaesthesia with propofol and fentanyl.

Molecular characterization of hepatitis C virus genotype 2a from the entire sequences of four isolates.

Genotype 2a hepatitis C virus (HCV) has different characteristics from genotype 1b, such as responsiveness to interferon therapy. Such type-specific characteristics appear to be due to differences in the HCV genome sequence. The complete sequences of genotype 2a HCV genome isolated from four patients with chronic hepatitis C were determined, and nucleotide and deduced amino acid sequences were compared within genotype 2a, as well as between genotype 2a and 1b. Whereas the amino acid sequence similarity of the core region was highest within genotype 1b, the NS3 and NS4B regions of exhibited greater similarity than the core region in genotype 2a. The serine protease and helicase motifs in the NS3 region were well conserved in genotype 2a to the same degree as in genotype 1b. However, the putative secondary structure of 2a isolates was significantly different from that of the 1b isolates. Analysis of amino acid similarity between genotypes 2a and 1b revealed the lowest degree of similarity in the E1 region, followed by the NS2 and NS5A region. Sequences of genotype 2a in the interferon-sensitivity determining region (ISDR) located in the NS5A region had a deletion of four amino acids compared with that of genotype 1b. When the ISDR of the genotype 2a was aligned for maximal similarity, it exhibited similarity of only 52.5-55.0% when compared with that of HCV-J, which belongs to genotype 1b. These findings for the entire sequences of genotype 2a isolates will contribute to virological studies of HCV.

Ribosomal protein S5 interacts with the internal ribosomal entry site of hepatitis C virus.

Translational initiation of hepatitis C virus (HCV) genome RNA occurs via its highly structured 5' noncoding region called the internal ribosome entry site (IRES). Recent studies indicate that HCV IRES and 40 S ribosomal subunit form a stable binary complex that is believed to be important for the subsequent assembly of the 48 S initiation complex. Ribosomal protein (rp) S9 has been suggested as the prime candidate protein for binding of the HCV IRES to the 40 S subunit. RpS9 has a molecular mass of approximately 25 kDa in UV cross-linking experiments. In the present study, we examined the approximately 25-kDa proteins of the 40 S ribosome that form complexes with the HCV IRES upon UV cross-linking. Immunoprecipitation with specific antibodies against two 25-kDa 40 S proteins, rpS5 and rpS9, clearly identified rpS5 as the protein bound to the IRES. Thus, our results support rpS5 as the critical element in positioning the HCV RNA on the 40 S ribosomal subunit during translation initiation.

Interaction of poly(rC)-binding protein 2 with the 5'-terminal stem loop of the hepatitis C-virus genome.

The 5' noncoding region (NCR) of hepatitis C virus (HCV) contains an internal ribosome entry site for translation initiation. Cellular proteins (e.g. La, polypyrimidine tract-binding protein, and p25) that interact with HCV 5' NCR have been implicated in facilitating efficient internal initiation. The 5' NCR may also contain RNA structures and specific RNA sequences that interact with cellular proteins to promote RNA replication. UV crosslinking experiments revealed a 43-kDa cellular protein (p43) also interacts with the HCV 5' NCR. Further UV crosslinking experiments with deletion mutants of HCV 5' NCR demonstrated that p43 bound specifically to the 5'-terminal stem-loop of the HCV 5' NCR. Achromobactor proteinase I digests, competition experiments, and immunoprecipitation confirmed that p43 was identical to human poly(rC)-binding protein 2 (PCBP2). We prepared a PCBP2-immunodepleted rabbit reticulocyte lysate with an anti-PCBP2 antibody. Translation activity promoted by the HCV internal ribosome-entry site was the same in PCBP2-depleted lysates as in mock-depleted lysates. In conclusion, PCBP2 specifically interacted with the 5' terminus of HCV genome but had no effect on HCV translation. We speculate that PCBP2's interaction with HCV 5' NCR may be involved in the replication-initiation complex of HCV.

Indirect evidence of TTV replication in bone marrow cells, but not in hepatocytes, of a subacute hepatitis/aplastic anemia patient.

The presence of a new DNA virus (TTV) has been reported in sera from patients with posttransfusion hepatitis of unknown etiology. The precise replication site of TTV, however, has not been established. In this study, the presence of TTV in liver autopsy material, and in bone marrow biopsy and autopsy samples taken from a subacute hepatitis/aplastic anemia patient was determined by PCR and Southern blot analyses. Liver cells were found to contain only TTV DNA and not mRNA. Bone marrow material, especially that taken at biopsy, contained high levels of TTV DNA. It is suggested that the TTV replication site was in the bone marrow rather than in the liver, and that TTV infection was the cause of this patient's aplastic anemia. The precise etiological association of TTV with hepatitis remains to be established.

Specific interaction of a 25-kilodalton cellular protein, a 40S ribosomal subunit protein, with the internal ribosome entry site of hepatitis C virus genome.

Translation initiation of hepatitis C virus (HCV) RNA is controlled by an internal ribosome entry site (IRES) contained in 5' noncoding region (NCR) and in several nucleotides of the coding region. The ability of a 25-kilodalton cellular protein (p25) to bind the HCV 5' NCR is correlated with the efficiency of translation initiation of HCV RNA, indicating that this protein plays a critical role in HCV translation (S. Fukushi, C. Kurihara, N. Ishiyama, F. B. Hoshino, A. Oya, and K. Katayama, J Virol 71, 1662-1666, 1997). We have extended the study for identification of the IRES region required for p25 binding. For this purpose, we have performed UV cross-linking competition analyses using 5'- or 3'- deleted mutants of the HCV 5' NCR as competitor RNAs for binding of p25 to wild-type HCV 5' NCR. Competitor RNAs lacking nucleotides (nt) 47-74 or nt 279-331 did not inhibit p25 binding to the HCV IRES, indicating that these regions are necessary for interaction of the p25 and HCV IRES. Since p25 binding was not observed in the IRES elements of encephalomyocarditis virus and poliovirus in UV cross-linking competition analyses, the p25 binding may be specific for the HCV IRES. p25 bound to the HCV IRES was detected when a purified 40S ribosomal subunit was used for UV cross-linking experiment, indicating that p25 is one of 40S ribosomal subunit proteins. These results reveal an unique interaction between the 40S ribosomal subunit and HCV IRES to contribute to translation initiation of the HCV genome.

Slow evolutionary rate of GB virus C/hepatitis G virus.

With the aim of elucidating evolutionary features of GB virus C/hepatitis G virus (GBV-C/HGV), molecular evolutionary analyses were conducted using the entire coding region of this virus. In particular, the rate of nucleotide substitution for this virus was estimated to be less than 9.0 x 10(-6) per site per year, which was much slower than those for other RNA viruses. The phylogenetic tree reconstructed for GBV-C/HGV, by using GB virus A (GBV-A) as outgroup, indicated that there were three major clusters (the HG, GB, and Asian types) in GBV-C/HGV, and the divergence between the ancestor of GB- and Asian-type strains and that of HG-type strains first took place more than 7000-10,000 years ago. The slow evolutionary rate for GBV-C/HGV suggested that this virus cannot escape from the immune response of the host by means of producing escape mutants, implying that it may have evolved other systems for persistent infection.

Multicyclic reverse transcription-polymerase chain reaction assay system for quantification of GB virus-C/hepatitis G virus RNA in serum.

A new quantitative reverse transcription-polymerase chain reaction (RT-PCR) method is described for analyzing the amount of GB virus-C (GBV-C)/hepatitis G virus (HGV) RNA in serum. This multicyclic RT-PCR (MRT-PCR) method used oligonucleotide primers deduced from the 3' noncoding region (3'NCR) that is highly conserved among GBV-C/HGV isolates. Quantitation of GBV-C/HGV RNA using MRT-PCR ranged between 10(2) and 10(10) copies/ml when PCR cycle number was regulated at exponential amplification of the products. Competitive RT-PCR (CRT-PCR) was carried out with mutant RNA and sample that had been measured by MRT-PCR. Quantitation of GBV-C/HGV RNA using both methods agreed. MRT-PCR detected viral RNA in a single step PCR, and demonstrated a high degree of sensitivity that was equal to that of the RT-PCR procedure, which used nested primers deduced from the non-structural (NS) 3 region. The MRT-PCR method for quantitation of GBV-C/HGV RNA in serum may prove useful for diagnosis.

Full-length GBV-C/HGV genomes from nine Japanese isolates: characterization by comparative analyses.

The genomes of nine GBV-C/HGV isolates from Japanese chronic hepatitis patients were fully sequenced and characterized. They shared 85% nucleotide sequence homology with previously characterized isolates from the US and West Africa. Homology studies and phylogenetic analyses showed that the Japanese isolates formed a third group distinct from the established groups 1 and 2. The genetic distances between the three groups of GBV-C/HGV were very similar to the distances between the two classical swine fever virus (CSFV) serotypes, which suggested that they might belong to a separate GBV-C/HGV serotype. Plot similarity analysis comparing the three groups exposed relatively conserved terminal non-coding regions. Hairpin structures predicted in the Japanese isolates are probably involved in viral replication. The region coding E1-E2-NS-2 showed the least similarity (80%); in HCV the similarity here is only 50% due to its hypervariability. NS-3 and NS-5b that respectively encode the helicase/protease and RNA-dependent RNA polymerase, had a high degree of amino acid homology, suggesting a high degree of functional constraint in this region. The NS-5b nucleotide sequence was highly conserved perhaps because of constraints from RNA secondary structure and/or an open reading frame in the negative strand.

[Preanesthetic meals in elective surgical patients].

This study investigated the effect of preanesthetic meals on the volume and pH of gastric contents in forty elective surgical patients ranging in ages from 20 to 60 years. Twenty patients who were given either isotonic beverage 250 ml or apple juice 250 ml on the morning of the operative day were subjected as control group and twenty patients of the breakfast group took two slices of bread with the above drink. About seven hours following drinking and feeding, the mean values of gastric volume were 20.9 +/- 18.3 ml in the control group and 19.2 +/- 16.3 ml in the breakfast group. The mean values of gastric pH were 4.3 +/- 2.3 in the drink group and 4.6 +/- 2.3 in the breakfast group. There were no significant differences in the gastric volume and pH between the two groups. However, very small amount of the bread was detected in the gastric fluid of three patients in the breakfast group. As preanesthetic drinking and feeding are advantageous for reducing the anxieties of preoperative patients and also for their nutrition during operation, it is encouraging that eating two slices of bread did not induce a significant effect of gastric volume or pH. The minute fragment of bread seems to have no clinically significant effect.

The sequence element of the internal ribosome entry site and a 25-kilodalton cellular protein contribute to efficient internal initiation of translation of hepatitis C virus RNA.

Translation of hepatitis C virus (HCV) RNA is initiated by internal entry of ribosomes into the 5' noncoding region (NCR). This process depends on genomic elements within the 5' NCR called the internal ribosome entry site (IRES) and may involve host factors. The alpha-branch structure (nucleotides 47 to 67) of the HCV IRES is considered a cis-acting element critical for translation initiation because it is indispensable for translation in vitro (S. Fukushi, K. Katayama, C. Kurihara, N. Ishiyama, F. B. Hoshino, T. Ando, and A. Oya, Biochem. Biophys. Res. Commun. 199:425-432, 1994). In order to further characterize the function of the alpha-branch, we determined whether sequence exchange within the alpha-branch had any effect on translation initiation. An in vitro translation study revealed that the stem sequences of this region played an important role in efficient IRES function. In addition to several HeLa cell proteins, which had a binding affinity for the 5' NCR, a novel 25-kDa protein that specifically interacted with the HCV IRES was discovered. The binding affinity of the 25-kDa protein for the 5' NCR was correlated with the efficiency of translation initiation of HCV RNA, indicating a critical role for the 25-kDa protein in HCV translation.

[Calculation of left ventricular volume and ejection fraction from ECG-gated myocardial SPECT: automatic detection of endocardial borders by threshold method].

A new method which calculate end-diastolic volume (EDV), end-systolic volume (ESV) and ejection fraction (LVEF) of the left ventricle from myocardial short axis images of ECG-gated SPECT using 99mTc myocardial perfusion tracer has been designed. Eight frames per cardiac cycle ECG-gated 180 degrees SPECT was performed. Threshold method was used to detect myocardial borders automatically. The optimal threshold was 45% by myocardial SPECT phantom. To determine if EDV, ESV and LVEF can also be calculated by this method, 12 patients were correlated ventriculography (LVG) for 10 days each. The correlation coefficient with LVG was 0.918 (EDV), 0.935 (ESV) and 0.900 (LVEF). This method is excellent at objectivity and reproductivity because of the automatic detection of myocardial borders. It also provides useful information on heart function in addition to myocardial perfusion.

New variant groups identified from HGV isolates.

We have determined the primary sequence of the 5' noncoding region (5' NCR) and putative helicase regions (NS-3) of hepatitis G virus (HGV) and GB virus C (GBV-C) that were isolated in Japan from suspected cases of nonA-nonB and/or nonA-nonB-nonC viral hepatitis by using RT-PCR, and we compared the newly isolated sequences with three established isolates. The addition of a "G" residue was found at the 5' terminus of all 8 Japanese isolates. These isolates were more clearly distinguished from the prototype viruses by comparison with the 5' NCR sequence than by comparison with the NS-3 region. Our results suggested that at least three distinct genomic variants of HGV exist. Genotyping of HGV by using RT-PCR based on the sequence of the 5' NCR seems highly feasible.

Nucleotide sequence of the 5' noncoding region of hepatitis G virus isolated from Japanese patients: comparison with reported isolates.

The nucleotide sequences of the 5' noncoding region (NCR) of hepatitis G virus (HGV) from sera of Japanese patients were determined. Among these isolates, there was a high degree (> 96.9%) of sequence identity, whereas identity with previously reported GB virus-C or HGV strains was low (> 87.0%). Phylogenetic analyses showed that the HGV strains from Japanese patients clustered in groups distantly separated from previously reported strains. Among the Japanese HGV isolates, the genetic distances corresponded to subtype differences observed within hepatitis C virus (HCV) isolates, whereas the differences between the Japanese isolates and the prototypes corresponded to genetic distances observed between HCV genotypes. The Japanese HGV isolates found in this study should be placed in a new genotype distinct from previously described isolates.