[Analysis of the relevance of the mutations of the precore and basic core promoter in HBV genome with the DNA amount of HBV viremia].

In the course of hepatitis B, serum concentrations of the virus usually fall following sero-conversion, characterized by the loss of HBe antigen and appearance of detectable anti-HBe. However, hepatitis B viremia may persist even after sero-conversion. We assessed the association of continuous viremia with the precore (PC) (G1896A) mutation, basic core promoter (BCP) (A1762T, G1764A) mutations, the viral genotype and the quantity of viral DNA. Neither PC nor BCP mutations were detected in the viral DNA isolated from cases in which HBV became negative during the course of infection. The virus quantity increased after sero-conversion in all of the cases of persistent viremia with HBV genotype C harboring BCP mutations, indicating that the BCP mutation in genotype C is a determinant of continuous viremia. This feature was not evident in infections with HBV genotype B. Although the PC mutation was detected in the viral DNA from both genotypes B and C in continuous viremia, the mutation was not relevant to the quantity of virus. Our data suggest that HBV genotype C has a predisposition to acquire BCP mutations and these mutations activate virus replication after sero-conversion. This mechanism may be a cause of the poor prognosis of hepatitis with HBV genotype C. In conclusion, analyses of HBV genotype and BCP mutations are imperative to determine the prognosis of hepatitis B.

[Evaluation of the anti-TP antibody latex agglutination immunoassay in routine testing and a clinical viewpoint].

We evaluated TP-LAIA (anti-TP latex agglutination immunoassay) and compared the results with those obtained using Serological Tests for Syphilis (STS), namely Venereal Disease Research Laboratory (VDRL) method and Rapid Plasma Reagin (RPR) test. We also examined early-stage antibody reaction using rabbits infected with active pathogen, and analyzed early-stage syphilis patient serum with IgM class anti-TP antibody. Based on routine test results and case history reviews for possible syphilis infection, TP-LAIA showed high specificity, 0.64% false positive results in comparison with 13.5% by VDRL method. Sensitivity was also significantly higher than TP-Hemagglutination Assay(TPHA). In the examination of TP early-stage infection, the fastest positive antibody reaction was observed with TP-LAIA, indicating its significance in the diagnosis of early-stage syphilis. TP-LAIA was confirmed to give a reliable reaction with IgM class anti-TP antibody. TP-LAIA results coincided with VDRL method results in the decrease in anti-treponemal antibody titers following medical treatment, suggesting that TP-LAIA will be a valuable tool for monitoring the effect of medical treatments. We concluded that not only the high sensitivity and specificity of TP-LAIA assay and its suitability for automation make it an ideal screening test, but also the assay performs sufficiently and satisfactorily for its use in monitoring medical treatments.

[Isolation of beta-lactamase non-producing ampicillin-resistant Haemophilus influenzae (BLNAR) and analysis of its PBP3 gene].

We analyzed the incidence of beta-lactamase non-producing ampicillin-resistance (BLNAR) Haemophilus influenzae in Showa University Hospital from June 2004 to March 2005. The ratio of BLNAR in total H. influenzae isolates was 44.5%. BLNAR accounted for 89.6% of the ampicillin non-susceptible strains displaying a minimal inhibitory concentration(MIC) of ampicillin of more than 2 microg/ml, which is the break point advocated by the Clinical and Laboratory Standards Institute. Of the total BLNARs, 71.4% were isolated from children of six and under. We analyzed mutations of the SSN and KTG motif of penicillin binding protein 3 encoded by the ftsI gene, since mutations in these regions are considered to be responsible for the drug-resistance of BLNAR. Mutations in SSN and KTG motifs were identified in all BLNAR isolates. We also detected mutations in the ampicillin susceptible strains displaying an ampicillin MIC of 0.5 or microg/ml. When a noticeable MIC increase of ABPC or other beta-lactams in the routine clinical laboratory practice, gene analysis of ftsI gene of the isolates displaying increased MIC is required to measure the spreading of BLNAR.

Megakaryopoiesis and platelet function in polycythemia vera and essential thrombocythemia patients with JAK2 V617F mutation.

Patients with Ph chromosome negative myeloproliferative disease (Ph-MPD) have an increased risk of vascular complications. It remains controversial whether patients with the JAK2 V617F mutation (V617F) exhibit increased risk, while recent growing evidence has shown a critical role for V617F in clonal erythropoiesis in Ph-MPD. We studied 53 patients with Ph-MPD especially in relation to megakaryopoiesis, the thrombotic complications and the presence of V617F. Using novel mutation-specific PCR which is a highly sensitive PCR-based assay for detection of JAK2 mutated allele(s), we identified V617F in 38 Ph-MPD, which include 13 polycythemia vera (PV), 23 essential thrombocythemia (ET) and 2 chronic idiopatic myelofibrosis. The numbers of megakaryocytes were significantly increased in PV and ET patients with V617F, but the platelet counts were slightly lower. Although statistically not significant, the incidence of thrombotic events was higher in the group with V617F compared to in those without the mutation. Agonist-induced in vitro platelet aggregation and platelet adhesion were not affected by the presence of this mutation. Nonetheless, we found a hypercoagulable state in Ph-CMPD with V617F by employing whole blood thromboelastography. It suggests pre-thrombotic tendencies in CMPD are complex and JAK2 V617F mutation might have a role in vivo blood coagulation by altering not only the number, but function(s) of all three myeloid cells, including red blood cells, white blood cells and platelets in Ph-CMPD.

[Analysis of the macrolide resistance gene in penicillin-resistant Streptococcus pneumoniae].

We examined the penicillin and macrolide resistance of 496 strains of Streptococcus pneumoniae (S. pneumoniae) isolated at Showa University Hospital from November 2004 to May 2005. According to the classification established by the Clinical and Laboratory Standards Institute, the ratio of penicillin susceptible S. pneumoniae (PSSP) was 25.8%, penicillin intermediate S. pneumoniae was 35.9% and penicillin resistant S. pneumoniae (PRSP) was 38.3%. The ratios of macrolide resistant S. pneumoniae were 85.3% for erythromycin and 76.2% for clarithromycin. S. pneumoniae strains were isolated mainly from pediatric patients, and the ratios of PRSP were similar between outpatients (39.8%) and inpatients (45.6%). We screened for mutations in pbp1a, pbp2b and pbp2x, and the retention rate of the macrolide resistance genes, ermB and mefA in 90 strains isolated in the same period. Seventy two strains had at least one mutation in the pbp genes. Interestingly, some of the penicillin susceptible strains had one or two pbp mutations, suggesting a progressive genetic acquirement of penicillin resistance. In screening for retention of the macrolide resistance genes, we found that 42 strains(46.7%) had ermB, 19 strains (21.1%) had mefA and 13 strains (14.4%) had both ermB and mefA. The possession of resistance genes and the minimal inhibitory concentration indicate that the resistance to erythromycin and clarithromycin were induced by ermB or mefA, and the resistance to clindamycin was induced only by ermB. Among the 72 strains with pbps mutations, 65 strains (90.3%) had ermB or mefA or both. Together, these results show that the strains with pbp mutations were being selected and, after acquiring the macrolide resistance gene, transform to multidrug resistant S. pneumoniae.

[Analysis of the metallo-beta-lactamase gene in multidrug-resistant Pseudomonas aeruginosa isolated at Showa University Hospital].

We conducted a molecular epidemiological analysis of multidrug-resistant Pseudomonas aeruginosa (MDRP) isolated at Showa University Hospital. The ratio of MDRP to total P. aeruginosa was 0.8% (1/132 strains) from September to December 1993. This ratio was increased to 1.8% (18/1000 strains) from October 2003 to October 2004. We analyzed the genome type and drug-resistant gene of 18 MDRPs isolated from October 2003 to October 2004, and 6 MDRPs isolated from January to April 2005 in a follow-up survey. Genome-type analysis using pulsed-field gel electrophoresis after SpeI restriction enzyme digestion revealed that 12 of the 24 MDRP strains had an identical genome type, indicating a possible nosocomial infection. In order to analyze the drug resistance mechanism, the 2-mercaptopropionic acid inhibition test was performed. Twenty of 24 MDRP strains were positive for metallo-beta-lactamase. Of all the metallo-beta-lactamase-positive strains, IMP type beta-lactamase was detected by PCR. Sequence analysis of the PCR product of IMP type betalactamase identified that 2 strains were IMP-1 and 18 were IMP-10. Of the 12 strains having an identical genome type, IMP-1 was detected in 1 strain and IMP-10 was detected in 11 strains. From our results, we conclude that P. aeruginosa with the same genome type was continuously colonized in the hospital and independently acquired a drug-resistant gene.

[A case of repeated seroconversion to HBe antibody due to co-infection with Mutant- and wild-type hepatitis B virus clones].

It is reported that co-infection with different hepatitis B virus (HBV) clones in a patient with chronic hepatitis B induces rare serotypes (adywr) or abnormal laboratory data such as negative HBs antigen, in the presence of positive HBV DNA. In this study, we experienced a case of repeated seroconversion to HBe antibody in a patient with chronic hepatitis B. Since seroconversion is considered to be related to genetic mutations, we investigated the HBV genes in this male patient in his 30's. We amplified and cloned parts of the HBV genes by the polymerase chain reaction (PCR), and sequenced the PCR products. As a result, mutated HBV genes were found in the serum of each specified period. By DNA sequencing we confirmed the coexistence of different HBV clones (wild-type clone and pre-S deletion mutant) and that both clones had the same genotype C. These clones took turns to be dominant; when the wild-type clone was dominant, HBe antigen was positive, and when the mutant clone was dominant, HBe antibody was positive. These findings demonstrated that repeated seroconversion of HBe antigen to HBe antibody was induced by co infection with mutant- and wild-type HBV clones. It is interesting that increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was noted at the time of the change from positive wild-type HBV clone to positive mutant clone.

Cytoplasmic Localization of Cyclin Kinase Inhibitor p21 Delays the Progression of Apoptosis.

The different functions of the cyclin kinase inhibitor, p21, rely on its localization to either the cytoplasm or nucleus. Phosphorylation at Thr-145 and/or Ser-146 was reported to target p21 to the cytoplasm. To clarify the function of cytoplasmic p21, we constructed non-phosphorylatable mutants, Thr-145 to Ala (T145A) and Ser-146 to Ala (S146A), and phosphorylation mimic mutants, Thr-145 to Asp (T145D) and Ser-146 to Asp (S146D), and the cells stably expressing those mutants were identified. The association of all four mutants with either CyclinA or CDK2 was increased by Á-irradiation, indicating that the mutants functioned as cyclin kinase inhibitors. PCNA binding was detected in T145A and S146A, but not in T145D and S146D. In the stably-expressing cells, T145D and S146D binding was observed in the cytoplasm, while T145A and S146A in the nucleus. Further, lactacystin treatment enhanced T145A and S146A, but not T145D and S146D, which is consistent with the degradation of p21 by proteasome in the nucleus. Apoptosis induced by Á-irradiation was delayed in the cells expressing either T145D or S146D. The activities of caspase 3 were not reduced in mutant-expressing cells. These results suggest that the PCNA-unbound form of the full length p21 in the cytoplasm delays apoptosis through the interaction with caspase 3 or downstream components.

[The relation between virus genotypes and coexisting surface antigen and antibody in patients with hepatitis B, and the development of preS region deletions].

In recent years, there has been renewed interest in hepatitis B virus (HBV) genotypes. Our previous data have shown the importance of in-frame deletions in the preS region in cases of coexisting hepatitis B surface antigen (HBsAg) and anti-hepatitis B surface antibody (HBsAb). The aim of the present study was to investigate the relation between HBV genotypes and coexisting HBsAg and HBsAb, preS deletion mutants. We investigated the HBV genotypes in 9 patients with coexisting HBsAg and HBsAb. Viral DNA was extracted from the patients' sera and the HBV S gene region was amplified by polymerase chain reaction (PCR). HBV genotypes were then investigated by restriction fragment length polymorphism(RFLP) analysis. All 9 cases were found to have genotype C. This result clearly indicates that the unique finding of coexisting HBsAg and HBsAb depends on the HBV genotype. After genotypic screening was performed for HBV-positive samples from randomly selected 60 cases. The results of the 60 cases we investigated showed 26 cases of genotype B (43.3%), 31 cases of genotype C (51.7%), 1 case of coexisting genotype B and C (1.7%), and 2 cases of other genotypes (3.3%). Of the 60 cases, 45 cases consisting of 21 with genotype B and 24 with genotype C were subject to direct DNA sequencing of PCR products in the preS region to determine the presence or absence of preS deletion mutants. PreS deletion mutants were found in a total of 7 of the 45 HBV cases that underwent sequencing(7/45; 15.6%), and 6 of these had genotype C (6/24 cases, 25.0%), whereas only 1 had genotype B (1/21 cases, 4.8%). These results demonstrate a greater frequency of preS deletion mutants with genotype C. Interestingly, many preS deletion mutants showed deletions at the same point, namely the amino terminal side of the preS2 region. These results indicate that the HBV genotype is involved in the molecular pathogenesis of hepatitis B.

[The cause of failure of eradication treatment of Helicobacter pylori].

The drug treatment, the combination of lansoprazole + amoxicillin + clarithromycin, for Helicobacter pylori infection with gastroduodenal ulcer was approved for the national heath insurance November 2000 in JAPAN, and has been widely applied. However, failures of eradication have been counted in 10-20% of the cases. The major reason of the failure has been reported as the drug resistance of the H. pylori. Here, we surveyed the antimicrobial resistance of 70 clinical isolates in a Showa University Hospital 2001, 1 to 2002, 1. As a result, the ratio of primary resistance to amoxicillin was about 1.4%, and clarithromycin was about 11.4%. Among 70 H. pylori positive cases, 14 cases were treated with eradication 3 drug combination therapy. In 5 cases, H. pylori were detected after eradication treatment and these five strains acquired the second resistance to neither amoxicillin nor clarithromycin. To distinguish the cause of H. pylori culture-positive after eradication treatment is whether the failure of eradication itself or re-infection, we attempted the analysis of the restriction pattern of H. pylori genome (genome type) using pulsed-field gel electrophoresis. In all 5 cases, genome types of before and after treatment were identical, suggesting the failure of eradication treatment. Three of 5 cases, isolates before and after treatment were susceptible to both of amoxicillin and clarithromycin. Thus, the reason of failure of eradication is considered to ingestion compliance, not antimicrobial agent resistance nor reinfection. The rest of 2 cases, the primary resistance to clarithromycin may result the failure of eradication. Test for drug susceptibility and genome type analysis of H. pylori are significant in certification of an authenticity of an eradication treatment.

[Detection of micro mutation in dystrophin gene of DMD female carrier].

We attempted to identify a mutation in dystrophin gene in a female patient who was suspected a Duchenne muscular dystrophy (DMD) carrier with muscle weakness of upper limbs and congestive heart failure. We examined the mutation hot spots in DMD gene, exon 3, 6, 8, 13, 17, 19, 43, 44, 45, 47, 48, 49, 50, 52, 60 by multiplex PCR which had been a diagnostic screening strategy, and detected an extra band in exon 43 product. We also detected an extra band in exon 43 products by SSCP analysis for detection of small mutations which could not be detected by multiplex PCR. As a result of sequencing a PCR product of an exon 43, we confirmed an allele having the insertion of a 2 base of AT in Intron42, which is described for the first time. Although we can not conclude that this insertion is responsible for DMD, but it may cause abnormal splicing. In carrier detection of DMD without genetic information of proband, it is difficult to detect mutations by multiplex PCR solely. Therefore, SSCP of PCR products are recommended to detect mutation in DMD carrier.

The association of cyclin A and cyclin kinase inhibitor p21 in response to gamma-irradiation requires the CDK2 binding region, but not the Cy motif.

The cyclin kinase inhibitor p21 associates with and inhibits cyclin-CDKs to retard the progress of the cell cycle in response to DNA damage. The recognition sites for cyclin binding on the various cell cycle-related molecules have been identified as RXL motifs. In the case of p21, the dependence of the Cy1 (18CRRL) or Cy2 (154KRRL) motifs on cyclin E, but not on cyclin A has been demonstrated by in vitro experiments. In this study, to clarify the mechanism of p21 association with cyclin A, we constructed a p21 expression system in mammalian cells. After transfection with an expression vector containing cDNA of various p21-mutants, cells were irradiated with 10 Gy of gamma-rays to introduce DNA damage, followed by quantification of the p21-cyclin A association. The p21-mutant constructs were single or multiple deletions in Cy1, Cy2, and the CDK2 binding region, and a nonphosphorylatable alanine mutant of the C-terminal phosphorylation site. We demonstrated that the association of p21 and cyclin A in response to gamma-irradiation requires the CDK binding region, 49-71 aa, but not the Cy motifs. We believe the mechanism by which p21 inhibits cyclin-CDKs is distinct in each phase of the cell cycle. Furthermore, the increase in the association of p21 and cyclin A was not correlated with the levels of p21. This suggests that DNA damage triggers a signal to the p21 region between 21 and 96 aa to allow cyclin A association.

[Compliance with the core curriculum of laboratory medicine (private school)].

A model core curriculum for medical education was proposed in March 2001. Medical schools have to revise their curriculum according to the guideline. Showa University School of Medicine revised its curriculum of lectures and clinical practice for laboratory medicine according to the guideline. The GIO of the lecture at 4th grade was for the clinical practice at 5th grade. The GIO of the clinical practice at 5th grade was for the post-graduate clinical training. This revision is expected to improve education and result in better doctors.

[Detection of novel nucleotide substitutions resulting amino acid substitutions in TEM type beta-lactamase gene isolated from a clinically isolated extended spectrum beta-lactamases (ESBLs) productive Escherichia coli].

We screened Escherichia coli (E. coli) from specimens submitted to the Showa University hospital clinical laboratory that showed an advanced resistance to third generation cephems (Cefotaxime or Ceftazidime), as candidate extended spectrum beta-lactamases(ESBLs) producing strains. Among the candidates, two strains showed the characteristics of class A ESBLs in their susceptibility to Cefmetazole, a second generation cephem, and their resistance was inhibited by the addition of clavulanic acid. Further, we detected the TEM-type gene in one of the two E. coli strains. By determining the nucleotide sequence of the whole coding region of the TEM gene, two nucleotide substitutions with amino acid substitutions 82Val-->Ile, and 182Ala-->Val were identified.

Identification of the regulatory region required for ubiquitination of the cyclin kinase inhibitor, p21.

The expression of cyclin kinase inhibitor p21 is regulated by the ubiquitin-proteasome protein degradation system, as well as by transcriptional regulation. Generally, ubiquitination is regulated by the phosphorylation of the substrate. In this study, we identified the region of p21 responsible for the regulation of ubiquitination. Since the phosphorylation sites of p21 are distributed in the C-terminal region, we constructed sequential C-terminal truncated fragments and examined their ubiquitination in eukaryotic cells. The ubiquitination was observed in the 1-164 (full length) and 1-157 fragments with the same efficiency, but not in the 1-147 fragment. The lack of ubiquitination in the 1-147 fragment was unlikely due to the removal of a Lys residue at position 154, since the p21 K154R mutant was ubiquitinated as efficiently as the full-length p21. Furthermore, the 148-157 deleted form of p21 was not ubiquitinated, just like the 1-147 fragment. Thus, the C-terminal 148-157 region, not a ubiquitination site by itself, should contain an essential regulatory region for the efficient ubiquitination of p21.

[Arbekacin resistant gene, aacA/aphD, in Staphylococcus aureus is lost during in vitro passage].

We analyzed for the presence of the aacA/aphD gene in arbekacin resistant-Staphylococcus aureus. Tested strains were 50 clinically isolated Staphylococcus aureus that had an MIC of more than 2 micrograms/ml for arbekacin. Among the primary cultures, aacA/aphD genes were detected in 42 of 50 strains(84%) overall, including 34 of 41 Methicillin resistant Staphylococcus aureus(MRSA) strains, and 8 of 9 strains of Methicillin sensitive Staphylococcus aureus(MSSA). After 10 or 20 passages, the aacA/aphD gene was lost in 5 strains of MRSA and 2 strains of MSSA. In the strains that lost the gene, the MIC of arbekacin was reduced remarkably in 3 strains of MRSA and 2 strains of MSSA. Further, MICs of arbekacin decreased along with passages even in the strains that did not loose the aacA/aphD gene, as well as in the strains that did not possess the aacA/aphD gene originally. The fact that MIC values are easily reduced with in vitro passage is important to consider when performing the arbekacin-resistance test for Staphylococcus aureus.