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Biomedical subjects

H B Lim

Publications and source records attributed to H B Lim.

15 recordsLinked to original sources
PubMed1998

Inhalation of diesel exhaust enhances allergen-related eosinophil recruitment and airway hyperresponsiveness in mice.

We have previously shown that intratracheal instillation of suspension of diesel exhaust particles enhances allergen-related eosinophilic airway inflammation, airway hyperresponsiveness, and local expression of interleukin (IL)-5 and granulocyte macrophage-colony stimulating factor (GM-CSF) in mice. The present study was designed to elucidate the effects of daily inhalation of diesel exhaust (DE) on the allergen-related respiratory disease. ICR mice were exposed for 40 weeks to clean air or DE at a soot concentration of 0.3, 1.0, or 3.0 mg/m3 with aerosol allergen challenges (1% ovalbumin in isotonic saline for 6 min) at 3-week intervals during the last 24 weeks of exposures. Exposure to DE enhanced allergen-related eosinophil recruitment to the submucosal layers of the airways and to the bronchoalveolar space, and increased protein levels of GM-CSF and IL-5 in the lung in a dose-dependent manner compared to exposure to clean air. There were strong correlations between the number of eosinophils in bronchoalveolar lavage (BAL) fluid and IL-5 concentrations in BAL supernatants and lung tissue supernatants. In addition, the increases in eosinophil recruitment and local cytokine expression were accompanied by goblet cell proliferation in the bronchial epithelium and airway hyperresponsiveness to inhaled acetylcholine. In contrast, the control mice exposed for 40 weeks to clean air or DE at a soot concentration of 0.3, 1.0, or 3.0 mg/m3 without allergen provocation showed no eosinophil recruitment to the submucosal layers of the airways nor to the bronchoalveolar space and few goblet cells in the bronchial epithelium. The present study provides experimental evidence that daily inhalation of DE can enhance allergen-related respiratory diseases such as allergic asthma. This effect may be mediated by the enhanced local expression of IL-5 and GM-CSF. Increased ambient levels of DE may be implicated in the increasing prevalence of bronchial asthma in recent years.

Administration, Inhalation↗
PubMed1998

Differential requirements for conserved E2 binding sites in the life cycle of oncogenic human papillomavirus type 31.

Human papillomavirus (HPV) E2 proteins regulate viral replication by binding to sites in the upstream regulatory region (URR) and by complex formation with the E1 origin recognition protein. In the genital HPV types, the distribution and location of four E2 binding sites (BS1 to BS4) which flank a single E1 binding site are highly conserved. We have examined the roles of these four E2 sites in the viral life cycle of HPV type 31 (HPV31) by using recently developed methods for the biosynthesis of papillomaviruses from transfected DNA templates (M. G. Frattini et al., Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). In transient assays, no single site was found to be necessary for replication, and mutation of the early promoter-proximal site (BS4) led to a fourfold increase in replication. Cotransfection of the HPV31 wild-type (HPV-wt) and mutant genomes with expression vectors revealed that E1 stimulated replication of HPV31-wt as well as the HPV31-BS1, -BS2, and -BS3 mutants. In contrast, increased expression of E2 decreased replication of these genomes. Replication of the HPV31-BS4 mutant genome was not further increased by cotransfection of E1 expression vectors but was stimulated by E2 coexpression. In stably transfected normal human keratinocytes, mutation of either BS1, BS3, or BS4 resulted in integration of viral genomes into host chromosomes. In contrast, mutation of BS2 had no effect on stable maintenance of episomes or copy number. Following growth of stably transfected lines in organotypic raft cultures, the differentiation-dependent induction of late gene expression and amplification of viral DNA of the BS2 mutant was found to be similar to that of HPV31-wt. We were unable to find a role for BS2 in our assays for viral functions. We conclude that at least three of the four E2 binding sites in the URRs of HPVs are essential for the productive viral life cycle. The specific arrangement of E2 binding sites within the URR appears to be more important for viral replication than merely the number of sites.

Adenovirus E2 Proteins↗
PubMed1998

Diesel exhaust enhances allergic airway inflammation and hyperresponsiveness in mice.

We previously reported that the intratracheal instillation of diesel exhaust particles enhances allergic airway inflammation and hyperresponsiveness in mice. However, it is not known whether the effects of such instillation differ from those obtained with the daily inhalation of diesel exhaust. We therefore examined whether the inhalation of diesel exhaust would also enhance allergic reactions. Mice were exposed to diesel exhaust or clean air for 5 wk. After the first week, the animals were sensitized to ovalbumin by intraperitoneal injection. At the end of the exposure period, they underwent an ovalbumin challenge. Control animals received saline instead of ovalbumin. Independently of ovalbumin sensitization, diesel exhaust caused an increase in the numbers of neutrophils and macrophages in bronchoalveolar lavage fluid, whereas a significant increase in eosinophil numbers occurred only after antigen challenge combined with diesel exhaust exposure. Furthermore, ovalbumin alone caused an increase in eosinophil numbers in lung tissue, and this was enhanced by diesel exhaust. Exposure to diesel exhaust combined with ovalbumin sensitization, but not diesel exhaust inhalation alone, enhanced the number of goblet cells in lung tissue, respiratory resistance, production of ovalbumin-specific immunoglobulin E and G1 in the serum, and expression of interleukin-5 in lung tissue.

Administration, Inhalation↗
PubMed1998

Murine strain differences in airway inflammation caused by diesel exhaust particles.

To elucidate whether immunoglobulin (Ig) E or IgG are involved in the murine asthma model, we compared the pathogenic features of mice that were high IgG responders (C3H/He) with mice that were high IgE responders (BALB/c) after intratracheal instillation of diesel exhaust particles (DEP) and ovalbumin sensitization. Both mouse strains received DEP intratracheally once a week for 5 weeks. After the second injection of DEP, ovalbumin and aluminium hydroxide were injected intraperitoneally. After the last DEP administration, the mice were challenged by exposure to an aerosol of ovalbumin. DEP caused increased IgG1 production and airway hyperresponsiveness after ovalbumin sensitization in C3H/He mice, although IgE production did not change in either strain. Furthermore, in C3H/He mice, the number of eosinophils and goblet cells in the bronchial epithelium, and the expression of interleukin-5 and interleukin-2 were increased by DEP and ovalbumin treatments. In contrast, the pathogenic changes in BALB/c mice were weak, even though the same protocol was used. In conclusion, murine strain differences in response to air pollutants and allergens seem to be related to antigen-specific immunoglobulin G1 production and cytokine expression in the lungs.

Aerosols↗
PubMed1998

Oral administration of L-arginine potentiates allergen-induced airway inflammation and expression of interleukin-5 in mice.

The role of nitric oxide in the airway hyperresponsiveness and inflammation of bronchial asthma has not yet been established. However, L-arginine, the substrate for nitric oxide synthases, reportedly alleviates airway hyperresponsiveness caused by parainfluenza virus and reduces granulocytic inflammation induced by ischemia-reperfusion. We investigated the effects of L-arginine on a murine model of allergic asthma that included airway hyperresponsiveness, eosinophilic inflammation and expression of interleukin (IL)-5 in the lung. The mice received drinking water with or without L-arginine for 9 weeks. Histologic evaluation and cellular profiles in bronchoalveolar lavage fluid showed that p.o. administration of L-arginine (72 micromol/kg/day) significantly enhanced eosinophilic airway inflammation and goblet cell proliferation that were associated with intratracheal instillation of ovalbumin. L-Arginine also increased protein levels of IL-5 and IL-2 in supernatants from the lung exposed to ovalbumin. The number of eosinophils in bronchoalveolar lavage fluid correlated significantly with the expression of IL-5. L-Arginine did not reverse ovalbumin-associated airway hyperresponsiveness to inhaled ACh. These results suggest that p.o. administration of L-arginine aggravates allergen-induced eosinophilic airway inflammation via expression of IL-5, and in this model it does not show therapeutic efficacy against airway hyperresponsiveness associated with allergen exposure. Oral administration of L-arginine, the precursor of nitric oxide, may not be an effective intervention in allergic asthma.

Animals↗
PubMed1997

Abrogation of a mitotic checkpoint by E2 proteins from oncogenic human papillomaviruses correlates with increased turnover of the p53 tumor suppressor protein.

Human papillomavirus (HPV) E2 and E1 proteins are required for the replication of viral genomes in vivo. We have examined the effects of increasing the level of E2 on viral and cellular replication using recombinant adenoviruses. Infection of cells which maintain HPV 31 DNA episomally with E2 recombinant adenoviruses resulted in a 5-fold increase in genome copy number as well as an S phase arrest allowing for the continued replication of cellular DNA. Similar effects on cell cycle progression were seen following infection of normal human foreskin keratinocytes, the natural host cell. The DNA content of these cells increased beyond 4N indicating that multiple rounds of replication had occurred without an intervening mitotic event. In addition, increased cyclin A and E associated kinase activity was observed, while no change was detected in cyclin B associated kinase activity or in the activation state of cdc2 kinase. Interestingly, the levels of the p53 tumor suppresser protein were dramatically reduced through a post-transcriptional mechanism following infection. These data suggest a role for E2 in regulating viral and cellular replication by abrogation of a mitotic checkpoint, which is, at least in part, controlled by p53.

Adenoviridae↗
PubMed1997

Induction of human papillomavirus type 18 late gene expression and genomic amplification in organotypic cultures from transfected DNA templates.

The genetic analysis of human papillomavirus (HPV) functions during the vegetative viral life cycle is dependent upon the ability to generate human keratinocyte cell lines which maintain episomal copies of transfected viral genomes. We have previously demonstrated that lipofection of normal human foreskin keratinocytes with recircularized cloned HPV-31 genomic sequences resulted in a high frequency of cell lines which maintained viral genomes as extrachromosomal elements (M.G. Frattini, H. Lim, and L.A. Laimins, Proc. Natl. Acad. Sci. USA 93:3062-3067, 1996). Following the growth of these cell lines in organotypic (raft) cultures, the differentiation-dependent expression of viral late genes, the amplification of viral genomes, and virion biosynthesis were observed. In the present study, we demonstrate that these methodologies are not restricted to HPV-31 but are applicable to other HPV types, including the oncogenic HPV-18. HPV-18 genomes were purified from bacterial vector sequences, religated, and transfected into normal human foreskin keratinocytes together with a neomycin-selectable marker. Following drug selection, resistant cells were expanded and examined for the state of the viral DNA. All cell lines examined were found to contain approximately 100 to 200 episomal copies of HPV-18 DNA per cell. Growth of these cell lines in raft cultures resulted in the differentiation-dependent expression of the E1 [symbol: see text] E4 and L1 capsid genes. In addition, viral genome amplification was observed in suprabasal cells following DNA in situ hybridization analysis of differentiated raft cultures. The induction of these late viral functions has previously been shown to be directly associated with differentiation-dependent virion biosynthesis. Our studies indicate the ability to perform a detailed genetic analysis of the various phases of the viral life cycle, including control of the differentiation-dependent late viral functions, using a second oncogenic HPV type.

Capsid↗
PubMed1996

In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.

Human papillomavirus (HPV) types 16, 18, 31, and 51 are the etiologic agents of many anogenital cancers including those of the cervix. These "high risk" HPVs specifically target genital squamous epithelia, and their lytic life cycle is closely linked to epithelial differentiation. We have developed a genetic assay for HPV functions during pathogenesis using recircularized cloned HPV 31 genomes that were transfected together with a drug resistance marker into monolayer cultures of normal human foreskin keratinocytes, the natural host cell. After drug selection, cell lines were isolated that stably maintained HPV 31 DNA as episomes and underwent terminal differentiation when grown in organotypic raft cultures. In differentiated rafts, the expression of late viral genes, amplification of viral DNA, and production of viral particles were detected in suprabasal cells. This demonstrated the ability to synthesize HPV 31 virions from transfected DNA templates and allowed an examination of HPV functions during the vegetative viral life cycle. We then used this system to investigate whether an episomal genome was required for the induction of late viral gene expression. When an HPV 31 genome (31E1*) containing a missense mutation in the E1 open reading frame was transfected into normal human keratinocytes, the mutant viral sequences were found to integrate into the host cell chromosomal DNA with both early and late regions intact. While high levels of early viral gene transcription were observed, no late gene expression was detected in rafts of cell lines containing the mutant viral genome despite evidence of terminal differentiation. Therefore, the induction of late viral gene expression required that the viral genomes be maintained as extrachromosomal elements, and terminal differentiation alone was not sufficient. These studies provide the basis for a detailed examination of HPV functions during viral pathogenesis.

Blotting, Northern↗
PubMed1995

Simple and sensitive laser-induced fluorescence detection for capillary electrophoresis and its application to protein separation.

A low-cost and highly sensitive laser-induced fluorescence detector for on-column detection systems was constructed and its applications were demonstrated in protein separation by capillary electrophoresis. The limit of detection (LOD) at a signal-to-noise ratio of 2 for Lissamine 20 was about 1.4 x 10(-21) mole at 10 nL sample injection and the relative standard deviation (RSD) for 1.5 x 10(-11) M of Lissamine 20 solution was about 15%. These values are comparable to or even better than those reported using a similar on-column detection system. This sensitive LIF detection was applied for the study of tryptic digestion of insulin and for urinary protein profiling.

Electrophoresis↗
PubMed1995

Human papillomavirus type 31b late gene expression is regulated through protein kinase C-mediated changes in RNA processing.

Expression of the human papillomavirus (HPV) capsid genes, L1 and L2, as well as amplification of viral DNA and virion assembly occur in the terminally differentiated layers of infected stratified squamous epithelium in vivo. These processes can be duplicated in the laboratory through the use of organotypic or raft cultures. When CIN612 cells, which contain episomal copies of the high-risk HPV type 31b, are allowed to differentiate in raft cultures, the expression of transcripts encoding the early genes E1--E4 and E5 is induced. These transcripts are initiated at the differentiation-dependent P742 promoter located in the middle of the E7 open reading frame. Exposure of raft cultures to activators of protein kinase C, such as phorbol esters, results in the further induction of late gene expression as well as virion assembly. In this study, we have investigated the mechanism by which activators of protein kinase C induce late gene expression. The major L1 transcript was found to be encoded by a bicistronic E1--E4, L1 RNA which initiated at the differentiation-dependent promoter P742. Additional low-level expression of L1-containing RNAs was also observed from the early-region promoter, P97. The major L2 transcripts were found to be encoded by E1--E4, E5, L2, L1 RNAs which were also initiated in the early region, probably at the differentiation-specific promoter P742. While early and late RNAs were found to be expressed from the same promoter, they differed in utilization of splicing and polyadenylation sites. Raft cultures treated with activators of protein kinase C induced expression of late genes, but no change in the abundance of early RNAs initiated at the P742 promoter was observed. Thus, the increase in late gene expression was likely due to changes in RNA processing or stabilization rather than an increase in the rate of transcription from P742. Regulation of HPV late gene expression therefore occurs at two levels: differentiation-dependent induction of the P742 promoter, which can be mimicked in vitro by growth in raft cultures, and posttranscriptional changes that can be induced by activation of protein kinase C. These posttranscriptional changes may occur through inactivation or down-regulation of splicing factors which inhibit use of the late region polyadenylation site, resulting in increased stability of late region transcripts.

Cells, Cultured↗
PubMed1994

Modulation of cytochrome P-450 induction by long-term food restriction in male rats.

The influence of food restriction (FR) on the induction of liver microsomal cytochrome P-450 (P-450) was examined in rats, FR by 40% for 6 weeks caused increase in the levels of P-450, cytochrome b5 (b5), NADPH P-450 reductase, NADH b5 reductase, P-450 dependent testosterone 6 beta-, 16 alpha- and 16 beta-hydroxylases (P < 0.05). Treatment of 3-methylcholanthrene (MC) to food restricted rats (FR rats) induced more P-450, but phenobarbital (PB) induced it less. MC-inducible P-450 from FR rats did not increase its specific ethoxycoumarin O-deethylase activity but it increased testosterone 2 alpha-hydroxylase activity, and the maximum absorption of its CO complex was shifted from 448 nm to 450 nm. PB-inducible P-450 showed less increase in the activities of its specific benzphetamine N-demethylase and it rather significantly decreased testosterone 16 alpha-hydroxylase activity (P < 0.05). These results indicate that the drugs did not induce their unique types of P-450 in FR rats. Such alterations in P-450 induction in FR rats seem to be closely related to the level of P-450 and its related components enhanced by FR, and it could result in modulation of mixed function oxidase system.

Animals↗
PubMed1993

Effect of subchronic administration of antioxidants against cigarette smoke exposure in rats.

Effects of subchronic administration of antioxidants against pulmonary damage mediated by cigarette smoke were investigated in rats. Rats were continuously received ascorbic acid, N-acetylcysteine and ginseng extract together drinking water from day 25 after birth. After 30 days of antioxidant supplementation, rats were exposed to cigarette smoke generated from six cigarettes (11 mg tar) for 20 min per day throughout 30 days, and then several biochemical markers related to the redox status in vivo were analyzed in the respiratory system. The cigarette smoke induced mild histological changes in trachea and lungs. The activity of superoxide dismutase (SOD) in the lung was significantly increased, and catalase and glutathione peroxidase activities were increased less than SOD, but total sulfhydryl compounds (Total-SH) content was decreased by cigarette smoking. In spite of the increase in activities of antioxidant enzymes, the inhibitory capacity of lung preparations on in vitro lipid peroxidation using ox brain homogenates was decreased and the change in the capacity was not related to the changes of these intracellular enzymes activities, but with the content of Total-SH. On the other hand, the content of thiobarbituric acid reactive substances and the ratio of elastase to anti-protease in the lung homogenates were significantly increased. Supplementation of antioxidants, however, effectively attenuated all of such alterations induced by cigarette smoke. These results indicate that although cigarette smoking induces antioxidant enzymes in the lung as a self defense mechanism, it seems to be not sufficient to protect the pulmonary system, and that chronic antioxidant feeding could be effective to reduce pulmonary damage induced by free radicals.

Animals↗
PubMed1992

Metabolism of 16 alpha-hydroxytestosterone by cytochrome P-450 in rat liver.

The metabolism of 16 alpha-hydroxytestosterone by cytochrome P-450 system was studied with two different inductions. At least 5 different metabolites with higher polarity were produced from 16 alpha-hydroxytestosterone. MT-B, one of their products, appeared to be specifically increased by 3-methylcholanthrene- and phenobarbital-inducible P-450, respectively. This suggests that monohydroxylated testosterones are further metabolized by the P-450 system and that the reactions are isozyme specific. This can contribute at least in part to the diversity of steroid metabolism by P-450.

Animals↗