白澤 浩

The antiviral effects of chloroquine (CQ) on human coronavirus 229E (HCoV-229E) infection of human fetal lung cell line, L 132 are reported. CQ significantly decreased the viral replication at concentrations lower than in clinical usage. We demonstrated that CQ affects the activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK). Furthermore, p38 MAPK inhibitor, SB203580, inhibits CPE induced by HCoV-229E infection and viral replication. Our findings suggest that CQ affects the activation of MAPKs, involved in the replication of HCoV-229E. (C) 2007 Elsevier B.V. All rights reserved.

The DNA of bovine papillomavirus (BPV) type 8 was extracted from papillomas on cattle kept in Japan, and DNA of bovine papillomavirus BPV-8-EB was extracted from a European bison (Bison bonasus) born in Italy and released into the wild in Slovakia. The DNA genomes of these BPVs were amplified using multiply primed rolling circle amplification and polymerase chain reaction, then characterized by direct sequencing method. The BPV-8 and BPV-8-EB genomes consisted of 7,791 base pairs (bp) and 7,773 bp, respectively (GenBank accession numbers DQ098913 and DQ098917). The nucleotide sequence similarity of these BPVs indicated that BPV-8-EB was a variant of BPV-8. In the genome of BPV-8-EB, one nucleotide substitution was found in the E2 and E5 open reading frame (ORF) and upstream regulatory region (URR), and a short deletion and addition were found in the URR. The high similarity of sequences between the BPV-8 to BPV-5 in total genome (70%) and L1 ORF (75%) as well as a phylogenetic analysis were the bases for classifying BPV-8 in the genus Epsilon papillomavirus. The BPV-8 E6 and E7 ORFs/proteins also showed some characteristic features of genus Epsilon papillomavirus. However, BPV-8 contained E4 ORF, which was not found in BPV-5. In addition, the secondary structure of E5 proteins of BPV-5 and BPV-8 suggested that these proteins may have cell-transforming ability. © 2007 Springer Science+Business Media, LLC.

Six bovine papillonnavirus (BPV) types and 16 putative BPV types have been reported previously. Here, the complete genome sequence of BAPV6, a novel putative BPV type isolated from cattle in Japan, was determined by using multiple-primed rolling-circle amplification. The genome consisted of 7412 bp (G + C content of 46 mol%) that encoded five early (E1, E2, E4, E6 and E7) and two late (L1 and L2) genes, but did not encode the E5 gene. The E6 protein contained a non-consensus CxxC(x)(33)CxxC and a consensus CxxC(x)(29)CxxC zinc-binding domain, and the E7 protein lacked the LxCxE motif. The nucleotide sequence of the L1 open reading frame (ORF) was related most closely (57-58 %) to the L1 ORF of member(s) of the genera Betapapillomavirus, Gammapapillormvirus and Pipapillomavirus. Phylogenetic analysis based on the complete L1 ORF suggests that BAPV6 should be classified in a novel genus in the family Papillomaviridae as BPV-7.

Six bovine papillomavirus (BPV) types, BPV-1 to -6, have been classified in genera Delta-papillomavirus (BPV-1 and -2), Epsilon -papillomavirus (BPV-5) and Xi-papillomavirus (BPV-3, -4 and -6). In addition, 16 unclassified putative BPV types have been reported. In the present study, we characterized genus specific features of E6, E7, E5 (formerly E8) and E8 proteins of seven putative BPV types, BAPV-1 -2, -3, -4 and - 10, BAA-5 and BPV-3c. These putative BPV types were classified in genera Epsilon- or Xi-papillomavirus. The E6 proteins of BPV and putative BPV types in Epsilon-papillomavirus showed high sequence similarities, and contained two typical zinc-binding domains. However, E7 proteins contained atypical zinc-binding domains, and lacked the canonical retinoblastoma tumor suppressor protein (pRB)-binding motif. BPV and putative BPV types in Xi-papillomavirus contained E5 or E8 open reading frame (ORF) in the E6 position. The E5 ORFs encoded proteins consist of 42-amino acid with a hydrophobic transmembrane and a hydrophilic C-terminal domain. But the E8 ORFs encoded protein which have two transmembrane domains. Our results demonstrated that E5, E8, E6, E7 proteins of the putative BPV types, which are presumably classified in genera Epsilon- or Xi-papillomavirus, retained the some genus specific features. (c) 2006 Elsevier B.V. All rights reserved.

Five European bison (Bison bonasus) from three European zoos were shipped to the Bukovské Vrchy Hills (Slovakia) in June 2004 and kept together in an acclimatization enclosure. The European bison were released into the wild in December 2004. At that time, papillomas were found at the medial canthus of the left eye of a 12-yr-old female bison. Cutaneous papillomatosis was confirmed histologically. Negative stain transmission electron microscopic examination revealed papillomavirus in the papillomas, and papillomavirus DNA also was detected using the polymerase chain reaction with FAP59 and FAP64 primers. The amplified 413 bp DNA sequence was identical to that of BAPV2 bovine papillomavirus. This paper is the first report of papillomatosis in European bison. © Wildlife Disease Association 2006.

Background. Viral vectors have gained widespread use as vehicles for somatic gene transfer, and the targeted expression of foreign proteins by these vectors offers advantages over the systemic administration of the drugs in some therapeutic situations. Selective virus-mediated gene transfer to the peripheral nervous system (PNS), however, remains to be established. There are no data showing efficiency of protein transduction in the PNS, which consists of a variety of cell types, many of which are postmitotic. Methods. We prepared the first-generation replication-deficient recombinant adenovirus vectors engineered to express LacZ. Eight-week-old Wister rats were used in this study. Adenovirus vector (5 μl) containing the LacZ gene (5 × 108 pfu) was injected into rat sciatic nerves or the dorsal root ganglia at the level of L5. The sciatic nerves, the dorsal root ganglia, and the spinal cords were obtained 7, 14, 21, and 28 days after injection. Expression of LacZ was assessed by X-gal histochemistry and β-gal immunohistochemistry. Results. Following injection of the adenovirus carrying the LacZ gene into the sciatic nerve, LacZ expression was seen mainly in the Schwann cells and the small neurons in the dorsal root ganglion. In contrast, expression was observed in the primary nerve terminals of the spinal dorsal horn and the small to large dorsal root ganglion neurons and the Schwann cells after injection of the vectors into the L5 dorsal root ganglion. There were no side effects in rats with injection in the dorsal root ganglia or the sciatic nerve. Conclusions. The present study shows efficient protein transduction by adenovirus vectors in the PNS. It is noted that injection of the virus into the dorsal root ganglia leads to extensive expression of LacZ in the spinal cord, the dorsal root ganglia, and the sciatic nerves. © The Japanese Orthopaedic Association.

Conclusion. The results of this study demonstrate that suppression of inflammation by dexamethasone attenuates the host immune response against adenoviral-mediated gene transfection and thereby prolongs transgene expression in murine nasal mucosa. Objectives. Gene transfer using a recombinant adenovirus is a good tool for research and clinical applications, but the immune response to adenoviral vectors can induce inflammation and loss of transgene expression in transfected tissues. In this study we investigated the effects of dexamethasone-induced immunosuppression on adenovirus gene transfer in the nasal mucosa of the mouse. Material and methods. We administered the recombinant adenovirus Ax1CAlacZ, which encodes Escherichia coli β-galactosidase (lacZ gene), to the nasal mucosa of mice treated with or without i.p. dexamethasone and evaluated the expression of the lacZ gene on Days 2, 4, 7, 14 and 28. The nasal mucosa was dissected out, and the mRNA level was measured using a LightCycler. The expression of the exogenous β-galactosidase was detected by means of histochemistry. Results. Dexamethasone treatment significantly increased the mRNA level compared with that in the controls at Days 4, 7 and 14. Histochemistry showed that the expression of β-galactosidase protein persisted in the dexamethasone-treated mice at Days 7 and 14 but had diminished almost to nothing in the control group. © 2005 Taylor &amp Francis.

The expression of adenoviral vector (Ad)-mediated lacZ and brain-derived neurotrophic factor (BDNF) in mouse olfactory epithelium (OE) was examined, and the effect of BDNF on the survival of the bulbectomized OE was evaluated. A recombinant adenovirus, Ax1CAlacZ, was administrated into the mouse OE after bulbectomy, and the expression of a transferred E. coli beta-galactosidase (beta-gal) gene was confirmed by X-gal staining. The expression and effects of exogenous BDNF in the OE after bulbectomy were examined using immunohistochemistry and the TUNEL method. The adenoviral vector-mediated expression of beta-gal in the mouse OE was detectable for up to 14 days after bulbectomy in vivo. The Ad-mediated expression of BDNF was also observed in the OE after bulbectomy. Exogenously induced BDNF suppressed the degenerative changes of bulbectomized OE. TUNEL staining indicated that the exogenous BDNF enhanced the survival of the bulbectomized OE by inhibiting apoptosis. Ad-mediated expression of BDNF in the mouse nasal mucosa alleviated degenerative changes in bulbectomized OE. Ad-mediated transfer of neurotrophic factors might be applicable in the treatment of olfactory disorders. (C) 2004 Elsevier B.V. All rights reserved.

To investigate the prevalence of bovine papillomavirus (BPV) in bovine papilloma and healthy skin, DNA extracted from teat papillomas and healthy teat skin swabs was analysed by PCR using the primer pairs FAP59/FAP64 and MY09/MY11. Papillomavirus (PV) DNA was detected in all 15 papilloma specimens using FAP59/FAP64 and in 8 of the 15 papilloma specimens using MY09/MY11. In swab samples, 21 and 8 of the 122 samples were PV DNA positive using FAP59/FAP64 and MY09/MY11, respectively. Four BPV types (BPV-1, -3, -5 and -6), two previously identified putative BPV types (BAA1 and -5) and 11 putative new PV types (designated BAPV1 to -10 and BAPV11MY) were found in the 39 PV DNA-positive samples. Amino acid sequence alignments of the putative new PV types with reported BPVs and phylogenetic analyses of the putative new PV types with human and animal PV types showed that BAPV1 to -10 and BAPV11MY are putative new BPV types. These results also showed the genomic diversity and extent of subclinical infection of BPV. © 2004 SGM.

Neurotrophins have been shown to promote axonal regeneration, but the techniques available for delivering neurotrophins have limited effectiveness. The aim of this study was to evaluate the effect of adenovirus vector mediated gene transfer of brain-derived neurotrophic factor (BDNF) on axonal regeneration after spinal cord injury. We prepared adenovirus vectors encoding either beta-galactosidase (AxCALacZ) or BDNF (AxCABDNF). AxCALacZ was used to assess infection levels of the adenovirus BDNF produced by AxCABDNF was detected by Western blotting and its bioactivity was confirmed by bioassay. As a model of spinal cord injury, the rat spinal cord was completely transected at the T8 level. Immediately after transection, the vectors were injected into both stumps of the spinal cord. Axonal regeneration after transection was assessed by retrograde and anterograde tracing. In AxCALacZ-injected rats, adenovirus-infected cells were observed not only at the injected site but also in brainstem nuclei, as shown by LacZ expression. After the injection of the retrograde tracer fluorogold (FG) distal portion to the transection, AxCABDNF-injected rats showed FG-labeled neurons in the red nucleus. The anterograde tracer biotinylated dextran amine (BDA) injected into the red nucleus was also found in regenerating rubrospinal fibers distal to the transection. These tracing experiments demonstrated the regeneration of descending axons. In addition, rats of the AxCABDNF group showed significant locomotor recovery of hindlimb function, which was completely abolished by re-transection. These results indicate that the recovery was caused by regeneration of rubrospinal axons, not by simple enhancement of the central pattern generator.

Purpose: To describe the histopathological findings and successful treatment with 5-fluorouracil (5-FU) of a conjunctival intraepithelial neoplasia with limbal stem cell deficiency that was refractive to topical mitomycin-C (MMC). Design: Interventional case report. Intervention: A 64-year-old male patient presented with a diffuse conjunctival intraepithelial neoplasia (CIN) that was excised with concurrent keratoepithelioplasty. Because of a recurrence and the presence of limbal stem cell deficiency, he was placed on topical MMC. Despite two courses of MMC, the tumor size did not decrease, and topical 5-FU was started 1 year after MMC therapy began. Limbal autograft transplantation was performed thereafter. Main Outcome Measures: The clinical and histopathologic findings including impression cytology and biomicroscopic observations. Results: After 5-FU treatment, the patient was free of the tumor clinically and cytologically, and the corneal surface had cleared. No recurrence was observed during the 30 months after the 5-FU therapy. Serious complications have not been observed. Conclusions: Topical 5-FU may be a therapeutic option for the treatment of patients with MMC-resistant CINs. The success of 5-FU is believed to be the result of a difference in the mechanism of cytotoxicity of MMC and 5-FU or the additive effects of the two agents. (C) 2002 by the American Academy of Ophthalmology.

To clarify the molecular mechanism of phencyclidine (PCP)-induced schizophreniform psychosis in humans and of behavioral abnormalities in experimental animals, we used differential screening of a cDNA library from the cerebral cortex of rats treated with PCP. We identified a PCP-induced cDNA clone as the gene encoding glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism. GDH mRNA levels significantly increased as early as 15 min following PCP administration in both the cerebral cortex and the cerebellum. This effect was observed even in the presence of a protein synthesis inhibitor, cycloheximide. In contrast to a transient increase in c-fos expression, the elevation of GDH mRNA levels lasted up to 8 days after a single PCP injection. These results suggest that GDH mRNA induction may be involved in the pathology of PCP-induced psychosis, and that GDH may be one of the candidate genes that are vulnerable in subjects with schizophrenia. (C) 1997 Elsevier Science B.V.

The presence of human papillomavirus (HPV) DNA in oral, sinus, pharynx, and larynx lesions of Japanese patients was studied by Southern blot hybridization under less stringent (25% formamide, 42-degrees-C) and stringent (50% formamide, 42-degrees-C) conditions. Three samples from 10 benign tumors, and 3 of 30 malignant tumors, contained HPV DNA or HPV-related sequences. The HPV DNAs harbored in three laryngeal papillomas were HPV-11, -6, and -6 or -11, respectively. The HPV DNA and viral capsid antigens were easily detected by in situ hybridization, Western blotting, and peroxidase-antiperoxidase staining. However, neither the typical restriction pattern of HPV DNA nor viral antigen was identified in the malignant tumors, suggesting that subgenomic fragments remained integrated in the host cell DNA.

The presence of human papillomavirus (HPV) DNA in oral, sinus, pharynx, and larynx lesions of Japanese patients was studied by Southern blot hybridization under less stringent (25% formamide, 42°C) and stringent (50% formamide, 42°C) conditions. Three samples from 10 benign tumors, and 3 of 30 malignant tumors, contained HPV DNA or HPV-related sequences. The HPV DNAs harbored in three laryngeal papillomas were HPV-11, -6, and -6 or -11, respectively. The HPV DNA and viral capsid antigens were easily detected by in situ hybridization, Western blotting, and peroxidase-antiperoxidase staining. However, neither the typical restriction pattern of HPV DNA nor viral antigen was identified in the malignant tumors, suggesting that subgenomic fragments remained integrated in the host cell DNA. © 1991.