An improved method for recovery of F-defective Sendai virus expressing foreign genes from cloned cDNA

An improved system is described to recover non-transmissible Sendai virus that lack the envelope fusion (F) gene from cloned cDNA. The system (1) used plasmids that expressed the F and the HN viral envelope proteins, as well as the plasmids that expressed the viral NP, P, and L proteins as helper plasmids for recovery, and (2) overlaid packaging cells that expressed the F protein. With this improved system, we have succeeded in recovery of F-defective Sendai virus expressing two foreign proteins, and expression vectors that do not contain the EGFP reporter gene. This system may provide the basis for constructing recombinant F-defective Sendai virus for preventing and treating human diseases in the form of vaccines and vectors for gene therapy.     

Effects of repeated subculturing and prolonged storage at room temperature of enterohemorrhagic Escherichia coli O157:H7 on pulsed-field gel electrophoresis profiles

Three clinical strains of enterohemorrhagic Escherichia coli O157:H7 which were subcultured repeatedly or stored at room temperature over a 25-week period showed appreciable variations in their pulsed-field gel electrophoresis fragment patterns. The variations could be explained by a couple of spontaneous genetic events at most and thus did not invalidate the genetic lineage of the strains.     

Cytomegalovirus infections following umbilical cord blood transplantation using reduced intensity conditioning regimens for adult patients.

Cytomegalovirus (CMV) infection is a major complication after allogeneic hematopoietic stem cell transplantation (Allo-HSCT); however, we have little information on the clinical features of CMV reactivation after cord blood transplantation using reduced-intensity regimens (RI-CBT) for adults. We reviewed medical records of 140 patients who underwent RI-CBT at Toranomon Hospital between January 2002 and March 2005. All the patients were monitored for CMV-antigenemia weekly, and, if turned positive, received preemptive foscarnet or ganciclovir. Seventy-seven patients developed positive antigenemia at a median onset of day 35 (range, 4-92) after transplant. Median of the maximal number of CMV pp65-positive cells per 50,000 cells was 22 (range, 1-1806). CMV disease developed in 22 patients on a median of day 35 (range, 15-106); 21 had enterocolitis and 1 had adrenalitis. CMV antigenemia had not been detected in 2 patients, when CMV disease was diagnosed. CMV disease was successfully treated using ganciclovir or foscarnet in 14 patients. The other 8 patients died without improvement of CMV disease. In multivariate analysis, grade II-IV acute graft-versus-host disease was a risk factor of CMV disease (relative risk 3.48, 95% confidential interval 1.47-8.23). CMV reactivation and disease develop early after RI-CBT. CMV enterocolitis may be a common complication after RI-CBT.     

Evaluation of endocrine disrupting activity of plasticizers in polyvinyl chloride tubes by estrogen receptor alpha binding assay

Polyvinyl chloride (PVC) tubing is an indispensable medical material for extracorporeal circulation therapy. However, di(2-ethylhexyl)phthalate (DEHP), a suspected endocrine disruptor, can be eluted from PVC, suggesting that an alternative material that does not contain DEHP is needed for clinical applications. First, we evaluated the endocrine disrupting risks of the plasticizers contained in PVC tubes by investigating their binding affinities for the human estrogen receptor alpha (ERα). Our results revealed that, while DEHP has some binding affinity for ERα, neither epoxidized soybean oil nor tris(2-ethylhexyl)trimellitate (an alternative to DEHP) has any affinity for ERα. Second, we evaluated the endocrine disrupting risks of a tube made of newly developed plasticizer-free (PF) materials. We confirmed the presence of DEHP and detected several unidentified substances in plasma stored within the PVC tube. This plasma's competitive binding affinity for ERα was significantly higher than that of control plasma (P < 0.01). In contrast, the profile of plasma stored in the PF tube was similar to that of the control, both in terms of high-performance liquid chromatography chromatograms and competitive binding capacity for ERα, suggesting that the PF tube is biocompatible and is useful for reducing the elution of substances capable of binding to ERα. Presented in part at the 42nd Congress of the Japanese Society for Artificial Organs, October 5–7, 2004, Tokyo, Japan     

Single nucleotide polymorphisms in TNFSF15 confer susceptibility to Crohn's disease

The inflammatory bowel diseases (IBDs), Crohn's disease (CD) and ulcerative colitis, are chronic inflammatory disorders of the digestive tract. The pathogenesis of IBD is complicated, and it is widely accepted that immunologic, environmental and genetic components contribute to its etiology. To identify genetic susceptibility factors in CD, we performed a genome-wide association study in Japanese patients and controls using nearly 80,000 gene-based single nucleotide polymorphism (SNP) markers and investigated the haplotype structure of the candidate locus in Japanese and European patients. We identified highly significant associations (P = 1.71 x 10(-14) with odds ratio of 2.17) of SNPs and haplotypes within the TNFSF15 (the gene encoding tumor necrosis factor superfamily, member 15) genes in Japanese CD patients. The association was confirmed in the study of two European IBD cohorts. Interestingly, a core TNFSF15 haplotype showing association with increased risk to the disease was common in the two ethnic groups. Our results suggest that the genetic variations in the TNFSF15 gene contribute to the susceptibility to IBD in the Japanese and European populations.     

Critical role for chicken Rad17 and Rad9 in the cellular response to DNA damage and stalled DNA replication

The Rad17-replication factor C (Rad17-RFC) and Rad9-Rad1-Hus1 complexes are thought to function in the early phase of cell-cycle checkpoint control as sensors for genome damage and genome replication errors. However, genetic analysis of the functions of these complexes in vertebrates is complicated by the lethality of these gene disruptions in embryonic mouse cells. We disrupted the Rad17 and Rad9 loci by gene targeting in the chicken B lymphocyte line DT40. Rad17-/- and Rad9-/- DT40 cells are viable, and are highly sensitive to UV irradiation, alkylating agents, and DNA replication inhibitors, such as hydroxyurea. We further found that Rad17-/- and Rad9-/- but not ATM-/- cells are defective in S-phase DNA damage checkpoint controls and in the cellular response to stalled DNA replication. These results indicate a critical role for chicken Rad17 and Rad9 in the cellular response to stalled DNA replication and DNA damage.     

Significant association between nonsyndromic oral clefts and arylhydrocarbon receptor nuclear translocator (ARNT)

The etiology of nonsyndromic oral clefts (cleft lip, cleft palate, or cleft lip and palate) is still controversial, but is considered to involve both genetic and environmental factors. One of suspected environmental factors is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) found in tobacco, herbicides, contaminated soil, and food. TCDD administered during organogenesis in mice causes a high incidence of CP in fetuses. There is ample evidence that aryl hydrocarbon receptor (AHR), AHR nuclear translocator (ARNT), and cytochrome P450 1A1 (CYP1A1) are involved in TCDD metabolism. We assessed whether there is any association in the Japanese population of nonsyndromic oral clefts with single nucleotide polymorphisms (SNPs) in the AHR, ARNT, and CYP1A1 genes using transmission disequilibrium test (TDT) and case-control study. We identified and investigated three SNPs in ARNT; 567G/C (V189V), IVS12-19T/G, and 2117C/T (P706L). Two amino acid substitutions, R554L in AHR and I462V in CYP1A1, were also investigated. In the TDT, the C allele of ARNT 567G/C was preferentially transmitted to patients (P = 0.033). When a haplotype consisting of 567G/C and IVS12-19T/G in ARNT was considered, the preferential transmission of the CT (567C-IVS12-19T) haplotype was observed (P = 0.0012). In a case-control study, a significant association of IVS12-19T/G in ARNT was observed (P = 0.021). The SNPs studied in AHR and CYP1A1 were not associated with the disease. Our results suggest that ARNT is involved in the development of nonsyndromic oral clefts in the Japanese population.     

Changes in the somatosensory N250 and P300 by the variation of reaction time

We investigated the relationship between somatosensory event-related potentials (ERP) and the variation of reaction time (RT). For this purpose, we recorded the ERPs (N250 and P300) in fast- and slow-reaction trials during a somatosensory discrimination task. Strong, standard, and weak target electrical stimuli were randomly delivered to the left median nerve at the wrist with a random interstimulus interval (900–1,100 ms). All the subjects were instructed to respond by pressing a button with their right thumb as fast as possible whenever a target stimulus was presented. We divided all the trials into fast- and slow-RT trials and averaged the data. N250 latency tended to be delayed when the RT was slow, but not significantly. P300 latency was delayed significantly when the RT was slow, but to a much lesser extent than the RT delay, so we concluded that the change of RT was not fully determined by the processes reflected by the somatosensory N250 or P300. Furthermore, the larger and earlier P300 in the fast-RT trials implied that when larger amounts of attentional resources were allocated to a given task, the speed of stimulus evaluation somewhat increased and RT was shortened to a great extent. N250 amplitude did not significantly vary in the two RT clusters. In conclusion, the somatosensory N250 reflects active target detection, which is relatively independent of the modulation of the response speed, whereas the somatosensory P300 could change without manipulation of either the stimulus or the response processing demand. Electronic Publication     

Caenorhabditis elegans RBX1 is essential for meiosis, mitotic chromosomal condensation and segregation, and cytokinesis

BACKGROUND: The RING-H2 finger protein RBX1 (ROC1/HRT1) is a common subunit of SKP1-CDC53/CUL1-F-box (SCF), other cullins and von Hippel-Lindau (VHL) tumour suppressor E3 ubiquitin ligase complexes. RBX1 protein sequences are highly conserved in various species, including yeasts, Drosophila melanogaster, mice and humans. In Saccharomyces cerevisiae, RBX1 is essential for the G1/S transition. RESULTS: Caenorhabditis elegans RBX1 is strongly expressed in early embryos and in the gonad, including meiotic cells. Depletion of RBX1 by RNA-mediated interference (RNAi) caused pronounced defects in the first meiotic division. Several irregular phenotypes were identified in embryos that escaped from meiotic arrest: defects in mitotic chromosomal condensation and segregation, abnormal chromosome bridges, giant nuclei, abnormal cortical protrusion, multinucleate cells and defects in germ cell proliferation. Moreover, histone H3 phosphorylation at Ser10 and Ser28 was significantly reduced in these embryos. The histone H3 phosphorylation defect of embryos was rescued by the additional depletion of protein phosphatase 1 (GLC7alpha/beta) by RNAi. CONCLUSION: These results indicate that the RBX1 protein participates in diverse functions relevant to chromosome metabolism and cell cycle control.