Chronic hepatitis E: a review of the literature.

In 1978, the first case of hepatitis E was identified as non-A, non-B hepatitis. Hepatitis E virus (HEV) infection is believed to be one of the common causes of enterically transmitted acute hepatitis in developing countries and is rare in developed countries, except in patients with a history of travel. However, an increasing number of chronic HEV infection cases have recently been reported in developed countries. In these countries, immunosuppressed patients with HEV infection, such as organ transplant recipients, human immunodeficiency virus (HIV)-infected patients or patients with haematological malignancies, could develop chronic hepatitis E (CHE) infection. Approximately 60% of HEV infections in immunocompromised patients after solid organ transplantation evolve to CHE without antiviral treatment. Clinical manifestations of CHE are often nonspecific symptoms. Many patients with CHE infection are asymptomatic, but some have jaundice, fatigue, abdominal pain, fever and asthenia. Several extrahepatic manifestations have also been reported. Although chronic HEV infection can result in progressive severe liver failure and cirrhosis, diagnosis is often controversial because of the lack of specific diagnostic criteria. Many CHE cases are diagnosed by HEV RNA-positive serum or stool for >6 months. Immunosuppressive drugs, interferon-alpha and ribavirin have been used for treatment. Diagnostic reverse-transcription polymerase chain reaction is useful for estimating treatment efficacy. Preventive measures for HEV infection have been discussed, while systematic guidelines have not yet been reported.

Transcription factor AP-2beta inhibits expression and secretion of leptin, an insulin-sensitizing hormone, in 3T3-L1 adipocytes.

BACKGROUND: We have previously reported an association between the activator protein-2beta (AP-2beta) transcription factor gene and type 2 diabetes. This gene is preferentially expressed in adipose tissue, and subjects with a disease-susceptible allele of AP-2beta showed stronger AP-2beta expression in adipose tissue than those without the susceptible allele. Furthermore, overexpression of AP-2beta led to lipid accumulation and induced insulin resistance in 3T3-L1 adipocytes. RESULT: We found that overexpression of AP-2beta in 3T3-L1 adipocytes decreased the promoter activity of leptin, and subsequently decreased both messenger RNA (mRNA) and protein expression and secretion. Furthermore, knockdown of endogenous AP-2beta by RNA-interference increased mRNA and protein expression of leptin. Electrophoretic mobility shift and chromatin immunoprecipitation assays revealed specific binding of AP-2beta to leptin promoter regions in vitro and in vivo. In addition, site-directed mutagenesis of the AP-2-binding site located between position +34 and +42 relative to the transcription start site abolished the inhibitory effect of AP-2beta. Our results clearly showed that AP-2beta directly inhibited insulin-sensitizing hormone leptin expression by binding to its promoter. CONCLUSION: AP-2beta modulated the expression of leptin through direct interaction with its promoter region.

Antibody fusions with fluorescent proteins: a versatile reagent for profiling protein expression.

We developed a system by which antibodies, fused to fluorescent proteins with different wavelengths, can be prepared within a month against various antigens. An antibody library composed of a large number of single-chain Fv-CL fragment was constructed by means of a phage-display system. The constructs were designed to facilitate changing of the protein forms by simple enzyme manipulation. In the present study, we adopted a molecular form of antibody in which a single-chain Fv-CL fragment is fused with a green fluorescent protein (GFP) or red fluorescent protein (RFP). In addition, a His-tag was inserted between CL and GFP (or RFP). We describe the utility of this system using Caenorhabditis elegans embryo as a model.

Determination of hydrogen sulfide and acid-labile sulfur in animal tissues by gas chromatography and ion chromatography.

A sensitive and reliable method was developed for the determination of hydrogen sulfide and acid-labile sulfur (ALS) in animal tissues using gas chromatography with flame photometric detector (GC-FPD) and ion chromatography (IC). Hydrogen sulfide trapped in alkaline solution was determined by GC-FPD as hydrogen sulfide or by IC as sulfate after oxidation with hydrogen peroxide. Sodium sulfide used as a source of hydrogen sulfide was standardized by IC. Fresh rat liver and heart tissues contained 112.2+/-23.0 and 274.1+/-34.6 nmol/g of ALS respectively. Free hydrogen sulfide was not detected.

Insulin-induced c-Jun N-terminal kinase activation is negatively regulated by protein kinase C delta.

We investigated the role of protein kinase C (PKC) in insulin-induced c-Jun N-terminal kinase (JNK) activation in rat 1 fibroblasts expressing human insulin receptors. Insulin treatment led to increased SAPK/ERK kinase 1 (SEK1) phosphorylation, and then stimulated JNK activity in a dose- and time-dependent manner, as measured either by a solid-phase kinase assay using glutathione S-transferase (GST)-c-Jun fusion protein as a substrate, or by quantitation of the levels of phosphorylated JNK by Western blotting using anti-phospho-JNK antibody. Insulin-induced JNK activation was potentiated by either preincubating cells with 2 nM GF109203X (PKC inhibitor) or down-regulation of PKC by overnight treatment with 100 nM tetradecanoyl phorbol acetate. In contrast, brief preincubation with 100 nM tetradecanoyl phorbol acetate inhibited the insulin- induced JNK activation. Furthermore, we found that 5 microM rottlerin, a PKCdelta inhibitor, enhanced insulin-induced JNK activation, but a PKCbeta inhibitor, LY333531, had no effect. Consistent with these findings, overexpression of PKCdelta led to decreased insulin-induced JNK activation, whereas overexpression of PKCbeta had no effect. Although overexpression of wild-type PKCdelta attenuated insulin-induced JNK activation, a kinase-dead PKCdelta mutant did not cause such attenuation. Finally, we found that the magnitude of insulin-induced JNK activation was inversely correlated with the expression level of PKCdelta among different cell lines. In conclusion, the expression of PKCdelta may negatively regulate insulin-induced JNK activation.

Protein-tyrosine phosphatase-1B negatively regulates insulin signaling in l6 myocytes and Fao hepatoma cells.

Insulin signaling is regulated by tyrosine phosphorylation of the signaling molecules, such as the insulin receptor and insulin receptor substrates (IRSs). Therefore, the balance between protein-tyrosine kinases and protein-tyrosine phosphatase activities is thought to be important in the modulation of insulin signaling in insulin-resistant states. We thus employed the adenovirus-mediated gene transfer technique, and we analyzed the effect of overexpression of a wild-type protein-tyrosine phosphatase-1B (PTP1B) on insulin signaling in both L6 myocytes and Fao cells. In both cells, PTP1B overexpression blocked insulin-stimulated tyrosine phosphorylation of the insulin receptor and IRS-1 by more than 70% and resulted in a significant inhibition of the association between IRS-1 and the p85 subunit of phosphatidylinositol 3-kinase and Akt phosphorylation as well as mitogen-activated protein kinase phosphorylation. Moreover, insulin-stimulated glycogen synthesis was also inhibited by PTP1B overexpression in both cells. These effects were specific for insulin signaling, because platelet-derived growth factor (PDGF)-stimulated PDGF receptor tyrosine phosphorylation and Akt phosphorylation were not inhibited by PTP1B overexpression. The present findings demonstrate that PTP1B negatively regulates insulin signaling in L6 and Fao cells, suggesting that PTP1B plays an important role in insulin resistance in muscle and liver.

Crescentic glomerulonephritis with positive antineutrophil cytoplasmic autoantibody specific for myeloperoxidase associated with autoimmune hemolytic anemia and thrombocytopenic purpura.

A 41-year-old woman was admitted to the hospital with severe uremia, hemolytic anemia, and thrombocytopenic purpura. Emergency hemodialysis with plasmapheresis was started in view of consideration of hemolytic uremic syndrome (HUS), which resulted in improvement of renal function and platelet count. Positive antineutrophil cytoplasmic autoantibody specific for myeloperoxidase (MPO-ANCA) suggested crescentic glomerulonephritis, which was pathologically evidenced by renal biopsy. The diagnosis of MPO-ANCA associated crescentic glomerulonephritis with autoimmune hemolytic anemia (AIHA) and thrombocytopenic purpura were confirmed. Three courses of steroid pulse therapy with heparin were successfully performed, followed by oral prednisolone and warfarin. Such a case has not been previously reported to our knowledge.

Expression of a dominant negative SHP-2 in transgenic mice induces insulin resistance.

To elucidate the roles of SHP-2, we generated transgenic (Tg) mice expressing a dominant negative mutant lacking protein tyrosine phosphatase domain (DeltaPTP). On examining two lines of Tg mice identified by Southern blot, the transgene product was expressed in skeletal muscle, liver, and adipose tissues, and insulin-induced association of insulin receptor substrate 1 with endogenous SHP-2 was inhibited, confirming that DeltaPTP has a dominant negative property. The intraperitoneal glucose loading test demonstrated an increase in blood glucose levels in Tg mice. Plasma insulin levels in Tg mice after 4 h fasting were 3 times greater with comparable blood glucose levels. To estimate insulin sensitivity by a constant glucose, insulin, and somatostatin infusion, steady state blood glucose levels were higher, suggesting the presence of insulin resistance. Furthermore, we observed the impairment of insulin-stimulated glucose uptake in muscle and adipocytes in the presence of physiological concentrations of insulin. Moreover, tyrosine phosphorylation of insulin receptor substrate-1 and stimulation of phosphatidylinositol 3-kinase and Akt kinase activities by insulin were attenuated in muscle and liver. These results indicate that the inhibition of endogenous SHP-2 function by the overexpression of a dominant negative mutant may lead to impaired insulin sensitivity of glucose metabolism, and thus SHP-2 may function to modulate insulin signaling in target tissues.

Silencing of an aleurone-specific gene in transgenic rice is caused by a rearranged transgene.

In rice, silencing of the aleurone-specific Ltp2-gus transgene, causing easily detectable staining patterns on the grain surface, offers a convenient tool to study quantitative aspects of gene silencing in monocots. In this paper we analyzed phenotypes, occurrence, inheritance and environmental effects on the silencing. We also report on the cloning of transgenes, determination of their structure and analysis of transcripts from the transgene loci. The results show that various patterns of silencing appeared in the R2 generation at which most of the transgenes became homozygous and that they were inherited for five generations. In addition, silencing independently occurred in three generations and reversion to full expression was also found. Cloning of transgenes from a silenced L3.3 line demonstrated that this line carried two transgene loci: one carried an intact Ltp2-gus gene and the other carried a rearranged transgene in which part of the gus gene was in the antisense orientation. Analysis of gus transcripts indicated that partial antisense RNA derived from the rearranged transgene was present in silenced lines and was polyadenylated but that it was absent in non-silenced lines. RNA analyses suggested that the Ltp2-gus silencing in the aleurone layer was post-transcriptional and that it may be caused by interaction of partial antisense gus transcripts with normal sense transcripts. Possible involvement of antisense transcripts in post-transcriptional silencing is discussed.

A new antidiabetic agent (JTT-501) rapidly stimulates glucose disposal rates by enhancing insulin signal transduction in skeletal muscle.

A newly synthesized antidiabetic agent, JTT-501 is an isoxazolidinedione rather than a thiazolidinedione. An oral dose of JTT-501 (100 mg x kg(-1) x day(-1)) given to 12-week-old male Zucker fatty rats for 7 days led to the amelioration of both hyperinsulinaemia (40% of non-treated) and hypertriglyceridaemia (23% of non-treated) as well as a 2.4-fold increased insulin sensitivity as determined by a euglycaemic insulin clamp. In our study, we further evaluated the acute effect of JTT-501 on both the glucose infusion rates (GIR) and insulin signalling in skeletal muscle. Male Sprague-Dawley (SD) rats aged 10 weeks were injected intravenously with JTT-501 (5 mg/kg) and then a euglycaemic insulin clamp was initiated and glucose infusion rates monitored for 150 min. We found that this treatment increased the glucose infusion rate by 33% during the last 30 min in SD rats. After the clamp had been initiated for 30 min, the insulin-stimulated phosphatidylinositol 3-kinase (PI3-kinase) activities co-immunoprecipitated with insulin receptor substrate 1 (IRS-1) were also enhanced, resulting in increased glycogen synthase activities in the soleus muscles. Treatment with JTT-501 also enhanced the phosphorylation of insulin receptors and insulin receptor-substrate 1 rapidly as well as the phosphatidylinositol 3-kinase activities, which were stimulated by a bolus injection of insulin. Similarly, JTT-501 stimulated the glucose infusion rate by 30% and enhanced insulin signalling in Zucker fatty rats. In conclusion, a newly developed isoxazolidinedione, JTT-501, rapidly potentiates the insulin sensitivity of skeletal muscle by enhancing insulin signalling and could be useful for the treatment of insulin-resistant diabetic subjects.

A naturally occurring functional allele of the rice waxy locus has a GT to TT mutation at the 5' splice site of the first intron.

In cultivated rice two wild-type alleles, Wxa and Wxb, predominate at the waxy locus, which encodes granule-bound starch synthase. The activity of Wxa is 10-fold higher than that of Wxb at the level of both protein and mRNA. Wxb has a +1G to T mutation at the 5' splice site of the first intron. Sequence analysis of Wxb transcripts revealed that splicing occurs at the mutant AG/UU site and at two cryptic sites: the first is A/GUU, one base upstream of the original site and the second is AG/GU found approximately 100 bases upstream of the mutant splice site. We introduced single base mutations to the 5' splice sites of both Wxa and Wxb, fused with the gus reporter gene and introduced them into rice protoplasts. Analysis of GUS activities and transcripts indicated that a G to T mutation in Wxa reduced GUS activity and the level of spliced RNA. Conversely, a T to G mutation of Wxb restored GUS activity and the level of spliced RNA to that of wild-type Wxa. These results demonstrated that the low level expression of Wxb results from a single base mutation at the 5' splice site of the first intron. It is of interest that the Wxb allele of rice carrying the G to T mutation of intron 1 has been conserved in the history of rice cultivation because there is a low amylose content of the seed caused by this mutation.

Molecular cloning of a human protein that binds to the retinoblastoma protein and chromosomal mapping.

We have isolated distinct clones for cellular proteins that bind to the retinoblastoma protein by direct screening of cDNA expression libraries using purified pRB as a probe. The total nucleotide sequence of one of these clones, RBQ-3, was determined and found to encode a protein of 66 kDa localized in the nucleus. The RBQ-3 preferentially binds to underphosphorylated pRB. The region used for binding to this protein was mapped to the E1A-binding pocket B of pRB, which has sequence similarity to the general transcription factor TFIIB. We have mapped the gene to 1q32 using polymerase chain reaction analysis on a human-hamster hybrid cell panel and chromosomal fluorescence in situ hybridization.

General pharmacology of the novel angiotensin converting enzyme inhibitor benazepril hydrochloride. Effects on central nervous and sensory systems and other functions.

The effects of benazepril hydrochloride (CGS 14824 A, CAS 86541-74-4), a novel angiotension I converting enzyme inhibitor, on the central nervous systems, were studied in experimental animals. Benazepril hydrochloride (3 or 10 mg/kg/d, p.o. for 14 days) dose-dependently inhibited the increase in the blood pressure caused by continuous norepinephrine (NE) infusion in spontaneously hypertensive rats (SHR) and suppressed in seizures induced by a monoamine oxidase inhibitor, tranylcypromine in NE infused SHR. Benazepril hydrochloride transiently increased spontaneous motor activity in mice, tended to inhibit acetic acid-induced writhing in mice and decreased fast wave sleep and slow wave deep sleep on EEG in cats at a high dose of 100 mg/kg p.o. However, benazepril hydrochloride at the same dose showed no effect on other central nervous and sensory systems in experimental animals.

[Reproductive and developmental toxicity study of mofezolac (N-22) (2)--Study by oral administration of N-22 during the period of fetal organogenesis in rats].

The teratogenicity of mofezolac (N-22), a new developed analgesic and anti-inflammatory agent, was investigated in rats. N-22 was given orally to pregnant rats of the Jcl: Wistar strain (30 rats per group) at dose levels of 10, 50, 100 and 150 mg/kg/day from days 7 to 17 of gestation. Caesarean sections were performed on 20 dams per group on day 20 of gestation and their fetuses were examined for external, visceral and skeletal abnormalities. The remaining 10 dams per group were allowed to deliver and their offspring were examined for growth and reproductive performance. Results were as follows. 1. Effects on F0 generation At 150 mg/kg, eleven out of the 30 dams exhibited decreased motor activity, pale eyes, unkempt fur, urine-smeared lower abdomen, weakness and emaciation. At autopsy, twelve dams revealed gastrointestinal ulcers, peritonitic lesions, hypertrophy of the spleen, adrenal and mesenteric lymph node, atrophy of the submaxillary gland, thymus and liver and discoloration of the liver and kidney. Death, sacrificing in extremis, premature or delayed delivery and poor nursing occurred in one to two dams each. Food consumption was significantly decreased and body weight gain was significantly retarded in this dose level group. At 100 mg/kg, urine-smeared lower abdomen, hypertrophy of the spleen and poor nursing were observed in one dam each. 2. Effects on F1 generation At 150 mg/kg, significantly decreased fetal weight, increased number of immature fetuses and significantly retarded ossification of the 5th and 6th sternebrae and coccygeal vertebrae as well as significantly depressed body weight gain of female offspring were observed. No abnormalities were observed in each treated group in terms of development, behavior, learning ability and reproductive performance of offspring. 3. Effects on F2 generation No abnormalities were observed in fetuses and newborn young in each treated group. Based on these results, the maximum non-effective doses of N-22 in this study were considered to be 50 mg/kg/day for dams and offspring and 100 mg/kg/day for fetuses.

Rapid growth of a pseudosarcoma of the esophagus.

The growth of a pseudosarcoma of the esophagus was observed chronologically by serial esophagography. Esophagograms taken 12 and 6 months prior to diagnosis showed no abnormalities. At diagnosis, the tumor in the lower esophagus had a polypoid and nodular surface with a stalk, and it was approximately 3 cm in diameter. Four months later, the tumor had increased in size to 4 cm in diameter. The tumor was 5 cm in length and incarcerated into the stomach with an elongated stalk at operation. The growth curve was linear on semilogarithmic paper; its doubling time was calculated as 2.2 months. The surgical specimen showed squamous cell carcinoma and spindle cell sarcoma-like elements that comprised the greater part of the tumor. The sarcoma-like elements had metastasized to the abdominal lymph nodes. These findings confirm that this polypoid tumor grows rapidly.

[Effects of cadralazine on the central nervous system].

The effect of cadralazine, a new antihypertensive agent, were studied on the central nervous systems in experimental animals. Oral administration of 0.5 mg/kg or more of cadralazine depressed spontaneous motor activity and enhanced electroshock-induced convulsions in mice. The drug produced flush on the tail or ears at 0.5 mg/kg, p.o. or more and enhanced respiratory movement at 5.0 mg/kg, p.o. or more in rats. At 2.5 mg/kg, p.o., cadralazine prolonged the thiopental-sleeping time and inhibited methamphetamine-induced hypermotility as well as acetic acid-induced writhing in mice. Pretreatment of naloxone, however, failed to antagonize this inhibitory effect on acetic acid-induced writhing. Cadralazine at 5.0 mg/kg, p.o., lowered body temperature in rats. This same dose antagonized tremorine-induced behaviors in mice. Cadralazine at a dose of 1.0 or 5.0 mg/kg, i.v., had no effect on the spontaneous EEG pattern and the threshold of arousal EEG response induced by electrical stimulation to the midbrain reticular formation in rabbits. Even at a dose as large as 100 mg/kg, p.o., the drug showed no significant effect on the following effects: conditioned avoidance response in rats, spinal reflex in cats, tail pinch-induced pain in mice, and somatic function in the inclined screen or in the traction test in mice. In conclusion, cadralazine, having no passage through the blood-brain barrier, showed several pharmacological actions on behaviors. These actions are considered to be derived from its vasodilative properties and were qualitatively similar to those of hydralazine.

Cloning and sequencing of Schizosaccharomyces pombe DNA topoisomerase I gene, and effect of gene disruption.

We cloned the structural gene topl+ for Schizosaccharomyces pombe DNA topoisomerase I (topo I) by hybridization. An eight-fold increase of topo I relaxing activity was obtained in S. pombe cells transformed with multicopy plasmid with topl+ insert. Nucleotide sequence determination showed a hypothetical coding frame interrupted by two short introns, encoding a 812 residue polypeptide (M.W. 94,000), 43 residues longer than and 47% homologous to Saccharomyces cerevisiae topo I. We show that the topl (null) strain made by gene disruption is viable, although its generation time is 20% longer than that of wild type. The topl locus is mapped in the long arm of chromosome II, using the Leu+ marker integrated with the cloned topl+ sequence. We constructed a double mutant topl (null) top2 (ts) and found its defective phenotype similar to that of previously obtained topl (heat sensitive) top2 (ts). The other double mutant topl (null) top2 (cs), however, was lethal. Our results suggest that topl+ gene of S. pombe is dispensable only if topo II activity is abundant.

DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe.

We show that DNA topoisomerase II (topo II) is continuously required for mitotic chromosome changes in Schizosaccharomyces pombe. We constructed cold-sensitive (cs) or temperature-sensitive (ts) strains mutated in the genes coding for topo II (top2) and beta-tubulin (nda3). The ATP-dependent activity of the top2cs gene product is cs in vitro. The cloned top2cs gene sequence predicts an amino acid substitution. A cs top2-cs nda3 double mutant at 20 degrees C shows long, entangled chromosomes, which condense and separate upon the shift to permissive temperatures. If spindle formation is prevented at permissive temperatures, the chromosomes condense but do not separate. Thus topo II is required for final chromosome condensation; moreover, pulse-shift experiments show that topo II is required for chromatid disjuction. Experiments with ts top2-cs nda3 cells show that topo II is also required for chromosome separation in anaphase: inactivation of topo II and activation of beta-tubulin allow normal spindle formation but result in "streaked" chromosomes.

Tumor promoting activity of teleocidin in skin and forestomach of mice initiated transplacentally with 7,12-dimethylbenz(a)anthracene.

Experiments on the effect of transplacental initiation with 7,12-dimethylbenz(a)anthracene (DMBA) and postnatal promotion with teleocidin were carried out in mice. The percentage of tumor-bearing mice among females treated with DMBA transplacentally on day 17 of gestation and postnatally by topical application of teleocidin to the skin of the back was 73.3% in week 30, whereas that among females treated with DMBA on day 10 of gestation and postnatally by topical application of teleocidin was 20.0%. This indicates that teleocidin shows potent tumor promoting activity on mouse skin in a transplacental initiation and postnatal promotion protocol. Furthermore, in the males treated with DMBA transplacentally on day 17 of gestation and given diet containing 0.01% teleocidin postnatally five tumors of the forestomach were found in 5 of 19 effective mice (26.3%) in week 52. One of these five tumors was a squamous cell carcinoma, and the others were papillomas. This indicates that teleocidin also has tumor promoting activity in the forestomach of mice.