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.     

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.     

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.     

Chromosome 1q terminal deletion resulting fromde novo translocation with an acrocentric chromosome

Summary Distal deletion of chromosome 1q has been reported in nearly 30 patients, all being associated with a deletion ranging from the 1q42 or q43 band to 1qter region. Here, we describe a girl with 1q terminal deletion resulting from an unbalancedde novo translocation t(1;D or G)(q44; p11), as revealed by the presence of a satellited feature and an NOR-stained region at the tip of 1q. We suggest that most of the phenotypic abnormalities seen in patients with 1q distal deletion are attributable to the monosomy for band 1q44.     

Histopathological grading and clinical features of patients with mucoepidermoid carcinoma of the salivary glands]

In the 22 years between March 1979 and February 2001, we treated 16 patients--10 men and 6 women aged 10-80 years (mean: 44 years)--with mucoepidermoid carcinoma (MEC) of the salivary gland, evaluating them clinically and histopathologically. Tumor sites included 12 at the parotid gland, 3 at the submandibular gland, and 1 at the minor salivary gland. All tumors were graded histopathologically based on the criteria of Goode et al. as follows: low grade (n = 10), intermediate grade (n = 1), and high grade (n = 5). Female gender was associated with low grade MEC and male gender with high grade MEC (P < 0.05). The age at onset in high grade MEC was older than that in low grade MEC (P < 0.005). Lymph-node metastasis was detected in 7 out of the 16 patients (44%) associated significantly with high grade MEC (P < 0.05). Distant metastasis was detected in 4 of 16 patients (25%). Distant metastasis was significantly associated with high grade MEC (P < 0.05). Local recurrence was detected in 3 of 15 patients undergoing surgery (20%). No difference was seen in local recurrence frequency between low and high grade MEC. Survival was calculated with Kaplan-Meier's method. In all 16, 5-year survival was 86% and 10-year survival 75%. Five-year survival in low grade MEC was 100%, whereas that in high grade MEC was 67% (P < 0.05). In MEC of the salivary gland, it was suggested that the histopathological MEC grade evaluated by Goode's criteria significantly correlated with gender, age, lymph-node metastasis, distant metastasis, and 5-year survival.     

Long-term outcome of 17 cases of large-giant posterior fossa aneurysm

Long-term outcome of 17 patients who harbored a large or giant aneurysm of posterior fossa was summarized. The anatomical distribution of aneurysms included eight cases of basilar artery (BA) bifurcation aneurysms, three cases of BA trunk aneurysms, and six cases of vertebral artery (VA) aneurysms. Eight patients received surgical or endovascular treatment for their lesion. The clinical outcome was good recovery in six, moderate disability in one, and vegetative state in one case, respectively. The other nine patients were followed conservatively. Four of them had fatal aneurysmal rupture, and another two patients suffered from aggravation of pre-existing symptoms related to their aneurysm. Only three patients remain intact. Comparison of the radiographic parameters between those who bled and those who did not bleed revealed that those with subsequent rupture had significantly higher rate of aneurysmal thrombus and had a trend for larger diameter of the aneurysm. Although more aggressive and multidisciplinary measure should be taken to these patients to improve their long-term outcome, our results showed the limitation of treatment for these patients in the present era at the same time. The patients with broad neck BA bifurcation aneurysm in which efferent vessels were incorporated into aneurysmal dome, and those with fusiform, giant BA trunk aneurysm with thrombus were the least amenable to treatment in our series.     

Suppression of IFN-gamma production in atopic group at the acute phase of RSV infection

Several studies have suggested that respiratory syncytial virus (RSV) bronchiolitis induced the change of cytokine production profile in childhood. We sought to determine whether the RSV-induced cytokine production was affected by the patient's atopic background. We quantified interferon-gamma (IFN-gamma) and interleukin (IL)-4 in the supernatant of peripheral blood mononuclear cells (PBMCs) cultured for 24 h and in the presence of phytohemaglutinin (PHA), IL-12, or IL-18, from 14 infants who were divided into two groups, those who are non-atopic and an atopic group. In RSV-infected infants with atopic diseases, IFN-gamma production from IL-12- or especially IL-18-stimulated PBMCs was subtotally suppressed in the acute phase, whereas in RSV-infected infants without atopic diseases IFN-gamma production was not suppressed on acute phase. The IFN-gamma suppression observed in the atopic group is not caused by the immaturity of an infant's immune system since reduced IFN-gamma production to RSV is not observed in the infants of non-atopic group. IFN-gamma suppression in regard to RSV infection might be caused by some genetic factor involved in the development of atopic disease such as IL-18 signal cascade.