Ubiquitous Presence in Mammalian Cells of Enzymatic Activity Specifically Cleaving 8-Hydroxyguanine-containing DNA

Here we report the finding of enzymatic activity that specifically cleaves DNA containing 8-hydroxyguanine (oh⁸Gua) residues in various mammalian cells. To detect this activity, we used a synthetic double-stranded DNA containing a single oh⁸Gua at a defined position as the substrate, and analyzed the products of enzymatic digestion by polyacrylamide gel electrophoresis. Two cleavage sites near the oh⁸Gua residue were detected with partially purified fractions from cow brain and rat liver, and also with preparations from all mammalian tissues examined. These results suggest that enzymatic activity for the removal of oh⁸Gua from DNA is widely distributed in mammalian cells.     

CCR1 acts downstream of NFAT2 in osteoclastogenesis and enhances cell migration.

We found that a chemokine receptor gene, CCR1, acts downstream of NFAT2 in RANKL-stimulated RAW264 and bone marrow cells. The upstream regulatory region of CCR1 showed RANKL-dependent and CsA-suppressible promoter activity. Downregulation of the expression and function of CCR1 suppressed cell migration. INTRODUCTION: We previously reported that the expression of NFAT2 induced by RANKL is a key process for progression to multinucleated cells in an in vitro osteoclastogenesis system. Identifying the target genes of NFAT2 would thus be informative about the differentiation process. We focused here on chemokine and chemokine receptor genes that act downstream of NFAT2 in RAW264 cells as well as osteoclast precursors prepared from bone marrow cells. MATERIALS AND METHODS: RAW264 mouse monocyte/macrophage line cells were cultured with or without cyclosporin A (CsA) in the presence of RANKL or glutathione S-transferase (GST). Osteoclast precursors were prepared from bone marrow cells. RANKL-inducible and CsA-suppressible genes were searched for by microarray analysis, and expression was confirmed by quantitative RT-PCR. Promoter activity was measured by luciferase gene reporter assay. Short interfering (si)RNA for CCR1 was introduced in RAW264 cells. Cell migration activity was examined using a Boyden chamber assay. RESULTS AND CONCLUSIONS: We identified the chemokine receptor gene CCR1 as a gene showing significant differential expression profiles in osteoclastogenesis in the presence versus the absence of CsA, an inhibitor of NFAT. This property was unique to CCR1 among the chemokine and chemokine receptor genes examined in both RAW264 and bone marrow cells. The upstream regulatory region was isolated from CCR1, and its RANKL-dependent and CsA-suppressible promoter activity was confirmed. The functional significance of CCR1 was assessed by monitoring the migration of cells in a transwell migration assay, and this activity was abolished when either CsA- or CCR1 siRNA-treated cells were used. Moreover, treatment with a Galpha inhibitor pertussis toxin (PTX) or methiolynated-regulated on activation, normal T cells expressed and secreted (Met-RANTES), an antagonist of CCR1, suppressed multinucleated cell formation in the bone marrow cell system. Together, these results suggest that the CCR1 signaling cascade is under the control of NFAT2 and seems to enhance the migration of differentiating osteoclasts.     

Genetic changes in JC virus possibly associated with progressive multifocal leukoencephalopathy]

Progressive multifocal leukoencephalopathy (PML) is a fetal demyelinating disease in the central nervous system. PML was once a rare disease, but it is now relatively common among AIDS (acquired immunodeficiency syndrome) patients. The immunological state of patients mainly contributes to the pathogenesis of PML. Genetic changes of the etiological agent, however, may also be involved in the development and progression of the disease. The major genetic changes possibly associated with PML include the regulatory region rearrangement and the VP1 loop mutation. Both changes have been identified as genetic changes usually occurring in JCV (JCvirus) DNAs from the brain and cerebrospinal fluid of PML patients. Although it remained to be clarified how these changes are involved in the pathogenesis of PML, accumulating evidence suggests that the VP1 loop mutation is associated with the progression of PML. Here we overview studies (mainly those performed by ourselves) on these genetic changes.     

Multiplex PCR assay for rapid identification of oculopathogenic adenoviruses by amplification of the fiber and hexon genes

Eye infections caused by adenovirus (Ad) often result in nosocomial infections and community epidemics with significant rates of morbidity. No antiviral agent effective against Ad is yet available for clinical use. Therefore, early diagnosis is still the mainstay for patient management and the prevention of epidemics. A multiplex PCR assay based on amplification of a combination of the fiber and hexon genes which can identify the six important oculopathogenic serotypes of Ads (Ad serotype 3[Ad3], Ad4, Ad7, Ad8, Ad19, and Ad37) in a single-tube amplification reaction was developed. Ad serotypes could be distinguished by the different amplicon sizes. The assay correctly identified prototype strains as well as isolates in clinical specimens. In comparison with a previously described PCR-restriction fragment polymorphism method, our assay gave unequivocal results for clinical specimens. Our multiplex PCR has the potential to serve as a rapid and cost-effective tool for the typing of important ocular Ads.     

Development of enrofloxacin ELISA using a monoclonal antibody tolerating an organic solvent with broad cross-reactivity to other newquinolones

Antibacterial reagents, especially quinolones, are widely used in animals and humans, and have caused serious problems to human health because of their residual contaminants in food. In order to screen for different kinds of newquinolones at the same time, a sensitive and specific enzyme-linked immunoassay (ELISA) has been developed. The anti-enrofloxacin monoclonal antibody was selected because of its ability to react with structurally related newquinolones in organic solvent. The antibody has 100% cross-reactivity with norfloxacin, ciprofloxacin and other newquinolones at 50% inhibition of control values IC₅₀, but not with nitroflazone, sulphadimethoxine. The lowest detection limit of this ELISA was 0.7 ng/ml (ppb) when enrofloxacin was used as the calibrator. Eel extracts were spiked with enrofloxacin and the average recoveries at 10, 50, 100 ng/ml were 98, 102 and 91%, respectively. The proposed ELISA is a useful method for the practical microquantitation of various newquinolones in biological and environmental specimens.     

Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene

Infection of human cells with the human immunodeficiency virus type I (HIV-1) can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 together with the appropriate human chemokine receptors. In this study, a T-tropic HIV cell-cell fusion assay was established that utilized CD4, human CXCR4 and HIV NL4-3 gp160 as fusion components and a T7 polymerase-activated luciferase as a reporter system. The HeLa T4 cells used, expressed CD4 and CXCR4, and the applied HeLa KS386 cells expressed HIV NL4-3 gp160. By combining HeLa T4 cells with HeLa KS386 cells, an approximately about 100- to 300-fold increase in luciferase activity could be elicited relative to the control. The addition of anti-CD4 monoclonal antibody (Mab) (RPA-T4) or anti-CXCR4 Mab (12G5) in the assay significantly inhibited the fusion event; in contrast, an anti-CCR5 Mab (2D7) had no effect, indicating that the fusion assay was CD4 and CXCR4 dependent. In this report, fusion events could be monitored by both the luciferase reporter system and syncytia formation. Fusion events were monitored and compared using these two approaches. The luciferase reporter system was found to be more sensitive than syncytia formation. Moreover, compared with previous HIV fusion models, such as using recombinant vaccinia viruses, this system has several advantages, including simplicity and sensitivity. Finally, the system provides a powerful tool to study fusion mechanisms mediated by T-tropic HIV gp160, as well as to screen for fusion-blocking antibodies and antiviral agents.     

Parent-to-child transmission is relatively common in the spread of the human polyomavirus JC virus.

JC polyomavirus (JCV), the causative agent of progressive multifocal leukoencephalopathy, is ubiquitous in the human population, infecting children asymptomatically and then persisting in renal tissue. In most adults, renal JCV replicates and the progeny are excreted in urine. We used this urinary JCV to elucidate the routes of JCV transmission. A 610-bp JCV DNA region (IG region) encompassing the 3'-terminal sequences of both T-antigen and VP1 (major capsid protein) genes was amplified by means of PCR from urine specimens collected from all members of seven families. JCV strains were then unequivocally identified by the nucleotide sequences of the amplified IG regions. We could identify 18 distinctive JCV strains from 27 individuals. Different JCV strains were detected from all unrelated persons. However, the same viral strain was detected from one (four families), two (one family), or three offspring (one family) as well as from the fathers (three families) or from the mothers (three families). In total, the JCV strains detected in half of the JCV-positive children were identified in their parents. Since most humans are infected during childhood, these findings indicated that JCV is transmitted frequently from parents to children. We roughly estimated that 50% of JCV transmission occurs by this route and that the other 50% occurs outside the family.     

Familial lateral semicircular canal malformation with external and middle ear abnormalities

We report a family with inner ear lateral semicircular canal (LSC) malformation and external and middle ear abnormalities. The family had no history of known syndromes or toxic exposures. Distinct phenotypic manifestations were found in three family members. A young girl exhibited bilateral LSC malformation with a right-sided preauricular tag, a mildly deformed auricle, a stenotic external auditory canal, and a constricted middle ear cavity. She had moderate conductive hearing loss in the right ear but normal hearing in the left ear. Her younger brother exhibited right-sided LSC malformation, microtia, external auditory canal atresia, a malformed middle ear cavity, and abnormal auditory ossicles. He had severe mixed hearing loss in his right ear. Their mother exhibited left-sided LSC malformation without external and middle ear abnormalities, and the hearing was normal in her left ear. None of the three cases had vestibular symptoms, and their results of balance tests were appropriate for the corresponding ages. In contrast, significantly decreased LSC function was revealed by caloric tests in an ear with LSC malformation. Previously, LSC malformation may have been underdiagnosed in patients presenting with external and middle ear abnormalities and their relatives, since this malformation is frequently associated with normal hearing and balance or conductive hearing loss only. To our knowledge, this condition has not been described previously. This condition supports a genetic basis for the combination of LSC malformation and external and middle ear abnormalities and may represent an autosomal dominant condition with variable expressivity.     

JC virus genotyping using formalin-fixed, paraffin-embedded renal tissues

Recently genotyping of JC virus (JCV) DNA in renal tissue was reported to be useful to identify the geographic origin of unidentified cadavers. In the above study, autopsied tissue samples without storage or stored in a frozen state were used. This study examined JCV DNA sequence modifications caused by formalin-fixation, in an attempt to elucidate whether formalin-fixed, paraffin-embedded tissue samples can also be used to determine the genotypes of JCV DNA in the kidney. In four cases, a 610 bp typing region of the JCV genome was PCR-amplified from renal tissues stored for 1 year in three different states: frozen at -80 degrees C [Amaker, B.H., Chandler, F.W., Huey, L.O., Colwell, R.M., 1997. Molecular detection of JC virus in embalmed, formalin-fixed, paraffin-embedded brain tissue. J. Forensic Sci., 1157-1159], formalin-fixed, paraffin-embedded [Ault, G.S., Stoner, G.L., 1992. Two major types of JC virus defined in progressive multifocal leukoencephalopathy brain by early and late coding region DNA sequences. J. Gen. Virol. 73, 2669-2678], and soaked in 5% formalin [Baksh, F.K., Finkelstein, S.D., Swalskey, P.A., Stoner, G.L., Ryschkewitsch, C.F., Randhawa, P.R., 2001. Molecular genotyping of BK and JC virus in human polyomavirus-associated interstitial nephritis after renal transplantation. Am. J. Kidney Dis. 38 (2), 354-365]. The amplified fragments were cloned, and the resultant clones were sequenced. In frozen samples, single sequences ('original' sequences) were detected in all cases. In formalin-fixed, paraffin-embedded samples, not only the original sequences but also those with 1-6 base substitutions were detected. From formalin-soaked samples, the original sequences and those with 1-5 and 10-13 substitutions were detected. The genotyping of JCV DNA was not hampered by the presence of 1-6 substitutions, but a shift in JCV genotypes was observed in sequences with 10-13 substitutions. Thus, it was concluded that the genotypes of JCV DNA in the kidney can be determined only with specimens stored in a frozen state or formalin-fixed for a short time.