Translational control by the ER transmembrane kinase/ribonuclease IRE1 under ER stress.

Under conditions of endoplasmic reticulum (ER) stress, mammalian cells induce both translational repression and the unfolded protein response that transcriptionally activates genes encoding ER-resident molecular chaperones. To date, the only known pathway for translational repression in response to ER stress has been the phosphorylation of eIF-2alpha by the double-stranded RNA-activated protein kinase (PKR) or the transmembrane PKR-like ER kinase (PERK). Here we report another pathway in which the ER transmembrane kinase/ribonuclease IRE1beta induces translational repression through 28S ribosomal RNA cleavage in response to ER stress. The evidence suggests that both pathways are important for efficient translational repression during the ER stress response.

Identification of a novel mammalian endoplasmic reticulum-resident KDEL protein using an EST database motif search.

Several endoplasmic reticulum (ER)-resident proteins contain a unique C-terminal sequence (KDEL) which is required for the retention of these proteins in the ER. By searching a mouse EST database for records containing the nucleotide sequence encoding the KDEL motif, we extracted cDNAs encoding putative novel ER-resident proteins in addition to all of the known ER proteins bearing the KDEL motif. Using the sequence information obtained by this database search, we cloned the cDNA encoding a novel KDEL motif-bearing protein, ER protein 58 (EP58), sharing no significant homology to any of the known ER-resident proteins. Subcellular localization of EP58 in the ER was confirmed by cytoimmunofluorescence studies using epitope-tagged EP58. The EP58 gene was primarily expressed in embryo, placenta, and adult heart. Neither heat shock nor ER stress as tested here was sufficient to induce expression of the EP58 gene. A putative role of the N-terminal half of EP58 in protein-protein interaction is suggested by its similarity to the filamin rod domain. Similarity of the EP58 sequence with bacterial and fungus proteins suggests a possible role for EP58 in polysaccharide biosynthesis.

Detection of GB virus-C/hepatitis G virus genome in peripheral blood mononuclear cells and liver tissue.

The replication site for the GB virus-C/hepatitis G virus (GBV-C/HGV) was investigated by using polymerase chain reaction (PCR)-based assays and in situ hybridisation. A total of 28 patients with consecutive GBV-C/HGV infection were enrolled in this study: Nine patients were being treated with immunosuppressive therapy after liver transplantation, and the remaining 19 patients were not receiving such treatment. GBV-C/HGV RNA was detected by using reverse transcriptase-polymerase chain reaction (RT-PCR) and was quantitated by using competitive RT-PCR in all patients. Positive and negative strands of GBV-C/HGV RNA in liver tissue were detected with in situ hybridisation by using RNA probes that were specific for the GBV-C/HGV genome. Concentrations of GBV-C/HGV RNA in serum were significantly higher (P=0.003) in the nine patients who were receiving immunosuppression (median, 10(7) copy/ml; range, 10(5)-10(7)) than in the 19 patients who were not receiving immunosuppressive therapy (median, 10(4) copy/ml; range, 10(2)-10(7)). In situ hybridisation of GBV-C/ HGV RNA was performed on paraffin-embedded liver tissue that was obtained from six patients with GBV-C/HGV infection. Two of those six patients were receiving immunosuppressive therapy, and four were not. Significant positive signals were observed in the samples from two of the six patients who were infected with GBV-C/HGV, but such signals were not observed in any of the six patients who were without the infection. The two patients with positive signals (both were undergoing immunosuppressive therapy) showed both positive and negative strands of GBV-C/HGV RNA in mononuclear cells that infiltrated into portal areas, but neither of the strands was observed in hepatocytes. Moreover, the GBV-C/HGV replication was analysed in peripheral blood mononuclear cells by using strand-specific PCR (conventional RT-PCR and rTth method). Two of the six patients were positive for negative-strand GBV-C/HGV RNA by using conventional RT-PCR. In conclusion, GBV-C/HGV replication was active under an immunosuppressive state, and it is suggested that GBV-C/HGV replicates in mononuclear cells.

A comprehensive system to explore p53 mutations.

To establish an effective and reliable system for the detection of p53 mutations, we evaluated the detection efficiencies of nonisotopic polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), fluorescence in situ hybridization (FISH), and immunohistochemistry. Ten cell lines (AsPc1, BxPc3, Miapaca2, Panc1, Colo320-011, Lovo, MCF7, LNCaP, HL-60, and Daudi), a peripheral blood sample from a patient with a p53 germline mutation (p53GML), and a normal peripheral blood sample were used for examination. Direct nucleotide sequencing identified p53 mutations in 7 of 12 samples (AsPc1, BxPc3, Miapaca2, Panc1, Colo320-011, HL-60, and p53GML). The nonisotopic PCR-SSCP detected anomalies of the PCR fragments in 5 cell lines. In the FISH analysis, 2 cell lines exhibited loss of heterozygosity of the p53 locus. Immunohistochemistry detected an accumulation of the abnormal p53 in 4 cell lines. The combination of these 3 methods produced no false-negative or false-positive results. This combination may be an excellent and beneficial system for the clinical diagnosis of the various human cancers.

[Clinical evaluation of RT-PCR method for detection of HCV-RNA: second report availability of RT-PCR system using PCR internal control for detection of HCV-RNA and the influence of interfering substances on the detection].

The COBAS Amplicor system is an automated diagnostic PCR system which contains a PCR internal control (P. I. C.) template to monitor the amplification. The applicability of COBAS Amplicor HCV was examined using sera of patients with hepatitis. Furthermore, the effects of possible interfering substance (total protein, triglyceride, hemoglobin, glucose, total bilirubin, heparin, lysis reagent including guanidium) on HCV-RNA detection were investigated. The sensitivity of COBAS Amplicor HCV was equivalent to the manual method of Amplicor HCV, moreover all of the results in 54 clinical samples analyzed on both COBAS Amplicor HCV and Amplicor HCV were in agreement. Detection sensitivity of HCV-RNA decreased in the presence of total bilirubin and heparin. Ten and 25mg/dl of total bilirubin affected HCV-RNA detection but did not affect P. I. C. This result suggested that total bilirubin interfered with the protein denature caused by the lysis reagent. Fifteen U/ml of heparin in the sample completely inhibited amplification both of the HCV-RNA and P. I. C. One U/ml of heparin did not affect amplification, but heparinized blood samples should not be used for the detection of HCV-RNA. To examine the effect of possible carry over contamination on the lysis reagent which contains guanidium, various concentrations of lysis reagent in P. I. C. were tested. RT-PCR was inhibited by 1/500 volume contamination of lysis reagent in specimen diluent. Other substances did not affect the sensitivity. Our results indicate that the carryover contamination of lysis reagent cause more "false negative" results than interfering substances in sera. In conclusion, HCV-RNA detection system containing P. I. C., such as COBAS Amplicor HCV, will become a very useful to differentiate "false negative" and "true negative" result.

[Clinical evaluation of RT-PCR method for detection of HCV-RNA--with special reference to Amplicor HCV].

Detection of the viral genome in serum is the most reliable way to analyze HCV viremia. In the present study, we evaluated the availability of a new detection kit for HCV-RNA, Amplicor HCV, which is based on the RT-PCR microplate hybridization protocol. The procedure of Amplicor HCV is simple, unlike the conventional RT-nested-PCR method. Although Amplicor HCV assay exhibited the lower sensitivity than the the conventional RT-nested-PCR method (10 x for HCV type 1a, 1b and 2b; 10(3) x for type 2a), Amplicor HCV assay could detect the HCV-RNA in all HCV-RNA positive cases by conventional RT-nested PCR method, except for one case who contained low concentration of HCV-RNA (10 copies/ml). The coincidence rate was 99.2% in 120 clinical samples between two assays. Amplicor HCV assay, moreover, could efficiently evaluate the viremia regardless of anti-HCV-2 antibody titer. This assay was useful for monitoring the effect of interferon therapy during and after administration. These results suggest that Amplicor HCV has an excellent availability for the clinical laboratories.

Histone H3 messenger RNA in situ hybridization for identifying proliferating cells in formalin-fixed rat gastric mucosa.

To devise a more sensitive method for identifying proliferative cells in routinely formalin-fixed, paraffin-embedded tissues, we applied an in situ hybridization (ISH) technique for the detection of histone H3 mRNA in rat gastric mucosa and amplified the signal by a silver intensification method. ISH was performed using a Fluorescein-labelled, single-stranded DNA probe for the human histone H3 gene. To determine the optimal conditions for detecting H3 mRNA in rat gastric mucosa, we tested the effect of changing conditions, such as fixation time and digestion time, by a proteinase before hybridization. Next, the proliferation indices obtained using H3 ISH were compared with those obtained using bromodeoxyuridine (BrdU) immunohistochemistry. In normal rat gastric mucosa, H3 ISH- and BrdU-positive cells were confined to the neck region of both fundic and pyloric mucosa. The two labelling indices were almost the same. In all the serial sections studied, H3 ISH-positive cells were almost always BrdU-positive too. Taken together, these results indicate that the H3 ISH technique is useful for the evaluation of proliferative activity in gastric epithelial cells by virtue of its detection of S-phase cells.

Germline mutations of the APC gene in two Japanese adenomatous polyposis patients.

Germline mutations of the adenomatous polyposis coli (APC) gene have been reported in patients with familial adenomatous polyposis (FAP) and are believed to be an early event in colorectal carcinoma. We report the results of screening for germline mutations of the APC gene in 4 cases of 2 kindreds using non-radioactive PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) analysis. The mutation in kindred 1 was a 4 bp deletion at codon 849 in exon 15, resulting in a frameshift leading to truncation of the APC gene product. In kindred 2, a transversion of C to G at codon 2038 was observed, resulting in an amino acid change from leucine to valine. In this case, it is possible to screen presymptomatic diagnosis easily and quickly by digestion with restriction enzyme EcoNI.

[Differentiation of selected abnormal hemoglobin by PCR-RFLP method].

Abnormal hemoglobin is one of the most frequent genetic disorder, of which about 700 variants have been reported in the world. Some abnormal hemoglobins such as HbS and HbC are endemic among limited human populations. We have found five subjects with abnormal hemoglobin in routine hematology analysis using an automated system Sysmex NE-7000. We isolated genomic DNA from the peripheral blood leukocyte of each subject, and amplified a 558 bp fragment of DNA including the entire exons 1 and 2 as well as intron 1 of the beta-globin gene. The amplified DNA fragments were subjected to direct sequencing by the dideoxy termination method on an automated DNA sequencer. The sequence analysis showed the abnormalities including HbA/C, HbS/C, HbE/E, and HbA/G-Szuhu. In the present study, we report a simple method for the mutual differentiation of these abnormal hemoglobins using restriction fragment length polymorphism (PCR-RFLP) method. Digestion of the amplified fragments described above with a restriction enzyme MnlI gave different RFLP patterns for HbA, HbC or S, HbE, and for HbG-Szuhu. RFLP using another enzyme, DdeI, could distinguish for HbS from HbC.

[Detection and identification of mycobacteria by PCR-RFLP method].

The frequency of isolation of Mycobacterium tuberculosis has reported to be decreased, however, Mycobacterium species including MOTT (Mycobacteria other than M. tuberculosis complex) still remain to be the important bacteria causing opportunistic infection. Two clinical procedures have long been adopted to date for the detection of acid fast bacteria belonging to the family Mycobacteriaceae. One is the direct microscopic examination of the specimen stained by Ziehl-Neelsen method, which is rapid, but is relatively insensitive and less specific. And another is a time- consuming culturing technique on slants of Ogawa's media, which takes 4 to 8 weeks of incubation. Recently, various methods to detect mycobacterial DNA have been reported. However, they require a complicated and tedious processes. We represent here a simple method for the detection and identification of Mycobacteria (M. tuberculosis, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium chelonae) using PCR-RFLP method for the 65kDa antigen with high sensitivity and specificity. This includes the amplification of Mycobacterial gene encoding a part of the 65kDa antigen. Subsequently, we sequenced the region amplified by PCR from the seven standard bacterial strains. As the results, we found that HaeIII restriction enzyme is suitable for the prompt and easy discrimination among the seven strains examined. These findings led us to conclude that this method is time-saving and possibly applicable for the rapid detection of Mycobacterial species including MOTT from clinical specimens in routine clinical laboratories.

[Studies on the fragments of FDP in 3 patients with DIC associated with acute promyelocytic leukemia].

We previously reported a study on fibrinolysis and fibrinogenolysis that analyzed fragments of fibrin/fibrinogen degradation products (FDP) using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. In this report, we characterized the fragments of FDP in three patients with disseminated intravascular coagulation (DIC) associating with acute promyelocytic leukemia (APL). D, Y, DD, DY/X and high molecular weight fragments were observed in sera of all the patients obtained at the onset of APL. These results showed that various degrees of fibrinogenolysis, concomitant with fibrinolysis, was occurring in APL patients presenting DIC. However, changes in the patterns of the FDP fragments during anticoagulation therapy were apparently different among three patients. Namely, fibrinogenolysis was dominant in case 1, while fibrinolysis was dominant in case 2. Interestingly, fibrinogenolysis and fibrinolysis were almost equivalent from the onset to the end of DIC in case 3. In case 3, FDP and FDP-D dimer were remarkably elevated about two months before the onset of APL, although their elevation was not complicated with DIC but with bone marrow necrosis. At that time, serum LDH levels and plasma polymorphonuclear elastase (PMN-Ela) were increased presumably due to the release of these enzymes from necrotic bone marrow, and the levels of CRP and plasma fibrinogen were increased probably due to an infectious complication. In non-DIC period of case 3, FDP and FDP-D dimer were spontaneously decreased without reduction of PMN-Ela levels, after three weeks of chemotherapy for microbial agents. Taken together, characteristics of FDP fragments were unique to each case of APL-DIC, probably because many factors differently affected the degradation of fibrin and/or fibrinogen.

[Studies on the fragments of FDP in 3 non-DIC patients with increased FDP levels in the sera].

We previously studied fibrinolysis and fibrinogenolysis by analyzing fragments of fibrin/fibrinogen degradation products (FDP) employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. In this report, we characterized the fragments of FDP in three patients with increased serum FDP, that were caused by various diseases. In the patient suffering from tuberculous constrictive pericarditis (case 1), the most part of the FDP fragments were DD and D. In the patient suffering from infection in addition to liver cirrhosis (case 2), the most part of the FDP fragments were high molecular weight (HMW) and D. In case 1 and 2, serum FDP levels were increased in parallel with the elevations of CRP levels. Although DD and HMW fragments were remarkably increased in case 1 and 2 with our immunoblotting analysis, DD levels assayed with LPIA system were much lower than FDP levels. The reason this discrepancy was explained by the observation that affinities of the monoclonal antibody used in LPIA system with DD and HMW fragment were markedly lower than that to DD-E fragment. In the patient suffering from deep vein thrombosis probably caused by steroid therapy of nephrotic syndrome (case 3), the most part of detected FDP fragments were DD and HMW in the period when APTT was shorter than normal, whereas D was mainly observed in the period when APTT was normal. In case 3, FDP and DD levels were increased in parallel with the shortening of APTT. In these non-DIC patients, increased serum FDP levels were induced by the presence of ascites and/or pleural effusion plus infection.(ABSTRACT TRUNCATED AT 250 WORDS)

[Studies on the fragments of FDP in 4 patients with DIC].

We previously studied fibrinolysis and fibrinogenolysis by analyzing fragments of fibrin/fibrinogen degradation products (FDP) employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. In this report, we characterized the fragments of FDP in four patients with disseminated intravascular coagulation (DIC), that were caused by various diseases. In the patients suffering from acute lymphoblastic leukemia (case 1) and acute suppurative cholangitis (case 3), DD and DY/X fragments resulting from fibrinolysis accounted for the most part of the FDP fragments. In case 3, D fragments resulting from fibrinogenolysis were also observed to much less extent. In a DIC associated with acute myeloblastic leukemia (case 2), both fibrinolysis and fibrinogenolysis were increased and resulted in high levels of D, Y and DY/X fragments, concomitant with moderate levels of DD and high molecular weight (HMW) fragments in the patient's sera. The increased fibrinogenolysis in this case was attributed to accelerated activation of plasmin. In a DIC patient of case 4, who underwent an operation due to hepatocellular carcinoma, marked increase in DY/X and HMW fragments and slight increase in DD fragment were observed on the day of operation. Hyperfibrinolysis documented in case 4 was explained by both increased production of thrombin and moderately accelerated activation of plasmin. Both qualitative and quantitative changes in the fragments of FDP during the courses of treatment in two cases of DIC were also noted. In summary, each underlying disease expresses characteristic pattern of FDP fragments in DIC.

[Differentiation of fibrinolysis and fibrinogenolysis by analysis of FDP fragments].

We previously analysed the fragments of fibrin/fibrinogen degradation products (FDP) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) combined with immunoblotting. In this report, we studied the semi-quantitative analysis of fibrinolysis (degradation of cross-linked fibrin) and fibrinogenolysis (degradation of fibrinogen and/or unstable fibrin) of patients' samples by our method. In vitro study of FDP made it clear that an appearance of D fragment confirmed fibrinogenolysis and an appearance of DD fragment and/or high molecular weight fragments which have higher molecular weight than DY or X fragment confirmed fibrinolysis. In addition, a study with mixtures of various concentrations of fibrin degradation products (FbDP) and fibrinogen degradation products (FgDP) demonstrated a dose dependent intensity of band by immunoblot method. These results show that our method is favorable for the semi-quantitative analysis of fibrinolysis and fibrinogenolysis. We applied the method to 6 samples from patients with disseminated intravascular coagulation (DIC). Consequently, fibrinogenolysis was observed in all of 6 samples, in which fibrinogenolysis was more enhanced than fibrinolysis in one sample, and an equivalent degree of fibrinolysis and fibrinogenolysis were observed in 3 of 6 samples. Although our method was probably devoid of the ability to distinguish FgDP from degradation products of unstable fibrin, these findings indicate that fibrinogenolysis is, at any rate, enhanced in the majority of patients with DIC, besides fibrinolysis.