PARP inhibition by olaparib or gene knockout blocks asthma-like manifestation in mice by modulating CD4(+) T cell function.

BACKGROUND: An important portion of asthmatics do not respond to current therapies. Thus, the need for new therapeutic drugs is urgent. We have demonstrated a critical role for PARP in experimental asthma. Olaparib, a PARP inhibitor, was recently introduced in clinical trials against cancer. The objective of the present study was to examine the efficacy of olaparib in blocking established allergic airway inflammation and hyperresponsiveness similar to those observed in human asthma in animal models of the disease. METHODS: We used ovalbumin (OVA)-based mouse models of asthma and primary CD4(+) T cells. C57BL/6J WT or PARP-1(-/-) mice were subjected to OVA sensitization followed by a single or multiple challenges to aerosolized OVA or left unchallenged. WT mice were administered, i.p., 1 mg/kg, 5 or 10 mg/kg of olaparib or saline 30 min after each OVA challenge. RESULTS: Administration of olaparib in mice 30 min post-challenge promoted a robust reduction in airway eosinophilia, mucus production and hyperresponsiveness even after repeated challenges with ovalbumin. The protective effects of olaparib were linked to a suppression of Th2 cytokines eotaxin, IL-4, IL-5, IL-6, IL-13, and M-CSF, and ovalbumin-specific IgE with an increase in the Th1 cytokine IFN-γ. These traits were associated with a decrease in splenic CD4(+) T cells and concomitant increase in T-regulatory cells. The aforementioned traits conferred by olaparib administration were consistent with those observed in OVA-challenged PARP-1(-/-) mice. Adoptive transfer of Th2-skewed OT-II-WT CD4(+) T cells reversed the Th2 cytokines IL-4, IL-5, and IL-10, the chemokine GM-CSF, the Th1 cytokines IL-2 and IFN-γ, and ovalbumin-specific IgE production in ovalbumin-challenged PARP-1(-/-)mice suggesting a role for PARP-1 in CD4(+) T but not B cells. In ex vivo studies, PARP inhibition by olaparib or PARP-1 gene knockout markedly reduced CD3/CD28-stimulated gata-3 and il4 expression in Th2-skewed CD4(+) T cells while causing a moderate elevation in t-bet and ifn-γ expression in Th1-skewed CD4(+) T cells. CONCLUSIONS: Our findings show the potential of PARP inhibition as a viable therapeutic strategy and olaparib as a likely candidate to be tested in human asthma clinical trials.

Unconventional activation of PRKDC by TNF-α: deciphering its crucial role in Th1-mediated inflammation beyond DNA repair as part of the DNA-PK complex.

BACKGROUND: The DNA-dependent protein kinase (DNA-PK) complex comprises a catalytic (PRKDC) and two requisite DNA-binding (Ku70/Ku80) subunits. The role of the complex in repairing double-stranded DNA breaks (DSBs) is established, but its role in inflammation, as a complex or individual subunits, remains elusive. While only ~ 1% of PRKDC is necessary for DNA repair, we reported that partial inhibition blocks asthma in mice without causing SCID. METHODS: We investigated the central role of PRKDC in inflammation and its potential association with DNA repair. We also elucidated the relationship between inflammatory cytokines (e.g., TNF-α) and PRKDC by analyzing its connections to inflammatory kinases. Human cell lines, primary human endothelial cells, and mouse fibroblasts were used to conduct the in vitro studies. For animal studies, LPS- and oxazolone-induced mouse models of acute lung injury (ALI) and delayed-type hypersensitivity (DHT) were used. Wild-type, PRKDC+/-, or Ku70+/- mice used in this study. RESULTS: A ~ 50% reduction in PRKDC markedly blocked TNF-α-induced expression of inflammatory factors (e.g., ICAM-1/VCAM-1). PRKDC regulates Th1-mediated inflammation, such as DHT and ALI, and its role is highly sensitive to inhibition achieved by gene heterozygosity or pharmacologically. In endothelial or epithelial cells, TNF-α promoted rapid PRKDC phosphorylation in a fashion resembling that induced by, but independent of, DSBs. Ku70 heterozygosity exerted little to no effect on ALI in mice, and whatever effect it had was associated with a specific increase in MCP-1 in the lungs and systemically. While Ku70 knockout blocked VP-16-induced PRKDC phosphorylation, it did not prevent TNF-α - induced phosphorylation of the kinase, suggesting Ku70 dispensability. Immunoprecipitation studies revealed that PRKDC transiently interacts with p38MAPK. Inhibition of p38MAPK blocked TNF-α-induced PRKDC phosphorylation. Direct phosphorylation of PRKDC by p38MAPK was demonstrated using a cell-free system. CONCLUSIONS: This study presents compelling evidence that PRKDC functions independently of the DNA-PK complex, emphasizing its central role in Th1-mediated inflammation. The distinct functionality of PRKDC as an individual enzyme, its remarkable sensitivity to inhibition, and its phosphorylation by p38MAPK offer promising therapeutic opportunities to mitigate inflammation while sparing DNA repair processes. These findings expand our understanding of PRKDC biology and open new avenues for targeted anti-inflammatory interventions.

Induction of Systemic Resistance in Hibiscus sabdariffa Linn. to Control Root Rot and Wilt Diseases Using Biotic and Abiotic Inducers.

The possibility of inducing systemic resistance in roselle against root rot and wilt diseases was investigated using biotic and abiotic inducers. The biotic inducers included three biocontrol agents (i.e., Bacillus subtilis, Gliocladium catenulatum, and Trichoderma asperellum) and two biofertilizers (i.e., microbein and mycorrhizeen), while the abiotic inducers included three chemical materials (i.e., ascorbic acid, potassium silicate, and salicylic acid). In addition, preliminary in vitro studies were conducted to evaluate the inhibitory activity of the tested inducers on the growth of pathogenic fungi. The results show that G. catenulatum was the most efficient biocontrol agent. It reduced the linear growth of Fusarium solani, F. oxysporum, and Macrophomina phaseolina by 76.1, 73.4, and 73.2%, respectively, followed by B. subtilis by 71.4, 69, and 68.3%, respectively. Similarly, potassium silicate was the most effective chemical inducer followed by salicylic acid, each at 2000 ppm. They reduced the linear growth of F. solani by 62.3 and 55.7%; M. phaseolina by 60.7 and 53.1%; and F. oxysporum by 60.3 and 53%, respectively. In the greenhouse, all inducers applied as a seed treatment and/or foliar spray strongly limited the development of root rot and wilt diseases. In this regard, G. catenulatum, at 1 × 109 CFU mL-1, achieved the highest values of disease control, followed by B. subtilis; while T. asperellum, at 1 × 105 CFU mL-1, recorded the lowest values. In addition, the plants treated with potassium silicate followed by salicylic acid, each at 4 g/L, recorded the highest disease control compared to ascorbic acid at 1 g/L, which had the lowest values. The mixture of mycorrhizeen + microbein (at 10 g/kg seeds) was the most effective compared to either of them alone. All treatments, applied alone or in combination in the field, significantly reduced the incidence of diseases. The most effective treatments were a mixture of G. catenulatum (Gc) + Bacillus subtilis (Bs) + Trichoderma asperellum (Ta); a mixture of ascorbic acid (AA) + potassium silicate (PS) + and salicylic (SA); G. catenulatum; potassium silicate; and a mixture of mycorrhizeen + microbein. Rhizolix T had the highest disease-reducing efficacy. In response to the treatments, significant improvements in growth and yield, changes in biochemicals, and increased activities of defense enzymes were achieved. This research points to the activity of some biotic and abiotic inducers that can play a vital role in managing the root rot and wilt of roselle through the induction of systemic plant resistance.

Biological Control of Celery Powdery Mildew Disease Caused by Erysiphe heraclei DC In Vitro and In Vivo Conditions.

The present study aimed to investigate the potentiality of certain biocontrol agents, namely Bacillus subtilis, B. pumilus, B. megaterium, Pseudomonas fluorescens, Serratia marcescens, Trichoderma album, T. harzianum and T. viride, as well as the synthetic fungicide difenoconazole to control celery powdery mildew caused by Erysiphe heraclei DC, in vitro (against conidia germination and germ tube length of E. heraclei) and in vivo (against disease severity and AUDPC). In vitro, it was found that the antifungal activity of the tested biocontrol agents significantly reduced the germination percentage of the conidia and germ tube length of the pathogen. The reduction in conidia germination ranged between 88.2% and 59.6% as a result of the treatment with B. subtilis and T. album, respectively compared with 97.1% by the synthetic fungicide difenoconazole. Moreover, the fungicide achieved the highest reduction in germ tube length (92.5%) followed by B. megaterium (82.0%), while T. album was the least effective (62.8%). Spraying celery plants with the tested biocontrol agents in the greenhouse significantly reduced powdery mildew severity, as well as the area under the disease progress curve (AUDPC), after 7, 14, 21 and 28 days of application. In this regard, B. subtilis was the most efficient followed by B. pumilus, S. marcescens and B. megaterium, with 80.1, 74.4, 73.2 and 70.5% reductions in disease severity, respectively. In AUDPC, reductions of those microorganisms were 285.3, 380.9, 396.7 and 431.8, respectively, compared to 1539.1 in the control treatment. On the other hand, the fungicide difenoconazole achieved maximum efficacy in reducing disease severity (84.7%) and lowest AUDPC (219.3) compared to the other treatments. In the field, all the applied biocontrol agents showed high efficiency in suppressing powdery mildew on celery plants, with a significant improvement in growth and yield characteristics. In addition, they caused an increase in the concentration of leaf pigments, and the activities of defense-related enzymes such as peroxidase (PO) and polyphenol oxidase (PPO) and total phenol content (TPC). In conclusion, the results showed the possibility of using tested biocontrol agents as eco-friendly alternatives to protect celery plants against powdery mildew.

Negative regulation of Wingless signaling by D-axin, a Drosophila homolog of axin.

Wnt/Wingless directs many cell fates during development. Wnt/Wingless signaling increases the amount of beta-catenin/Armadillo, which in turn activates gene transcription. Here the Drosophila protein D-Axin was shown to interact with Armadillo and D-APC. Mutation of d-axin resulted in the accumulation of cytoplasmic Armadillo and one of the Wingless target gene products, Distal-less. Ectopic expression of d-axin inhibited Wingless signaling. Hence, D-Axin negatively regulates Wingless signaling by down-regulating the level of Armadillo. These results establish the importance of the Axin family of proteins in Wnt/Wingless signaling in Drosophila.

Toward gene therapy of uterine fibroids: targeting modified adenovirus to human leiomyoma cells.

BACKGROUND: To circumvent the paucity of the primary adenovirus (Ad5) receptor and the non-specific Ad5 tropism in the context of uterine leiomyoma cells, Ad5 modification strategies would be beneficial. METHODS: We screened several modified adenoviruses to identify the most efficient and selective virus toward human leiomyoma cells to be used as candidate for delivering therapeutic genes. We propagated: wild-type Ad5-luc, fiber-modified viruses: ad5 RGD-luc, Ad5-Sigma-luc, Ad5/3-luc and Ad5-CAV2-luc, as well as transcriptional targeted viruses: ad5 survivin-luc, Ad5-heparanase-luc, Ad5-MSLN-CRAD-luc and Ad5-SLPI-luc, on 293 cells and purified them by double CsCL density centrifugation. Then we transfected primary cultures of human leiomyoma cells derived from fibroids of four different patients, telomerase-immortalized human leiomyoma cell line (huLM), telomerase-immortalized normal human myometrial cell line (HM9) and immortalized normal human liver cells (THLE3) with the viruses at 5, 10 and 50 plaque-forming units (PFU)/cell. After 48 h, luciferase activities were measured and normalized to the total cellular protein content. RESULTS: Ad5-RGD-luc and Ad5-CAV2-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc at 5, 10 and 50 pfu/cell showed significantly higher expression levels of luciferase activity in both primary and immortalized human leiomyoma cells when compared with Ad5-Luc. Additionally, these modified viruses demonstrated selectivity toward leiomyoma cells, compared with myometrial cells and exhibited lower liver cell transduction, compared with Ad5-luc, at the same dose levels. CONCLUSIONS: Ad5-CAV2-luc, Ad5-RGD-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc are promising delivery vehicles in the context of leiomyoma gene therapy.

Flagellin Encoded in Gene-Based Vector Vaccines Is a Route-Dependent Immune Adjuvant.

Flagellin has been tested as a protein-based vaccine adjuvant, with the majority of studies focused on antibody responses. Here, we evaluated the adjuvant activity of flagellin for both cellular and humoral immune responses in BALB/c mice in the setting of gene-based immunization, and have made several novel observations. DNA vaccines and adenovirus (Ad) vectors were engineered to encode mycobacterial protein Ag85B, with or without flagellin of Salmonella typhimurium (FliC). DNA-encoded flagellin given IM enhanced splenic CD4+ and CD8+ T cell responses to co-expressed vaccine antigen, including memory responses. Boosting either IM or intranasally with Ad vectors expressing Ag85B without flagellin led to durable enhancement of Ag85B-specific antibody and CD4+ and CD8+ T cell responses in both spleen and pulmonary tissues, correlating with significantly improved protection against challenge with pathogenic aerosolized M. tuberculosis. However, inclusion of flagellin in both DNA prime and Ad booster vaccines induced localized pulmonary inflammation and transient weight loss, with route-dependent effects on vaccine-induced T cell immunity. The latter included marked reductions in levels of mucosal CD4+ and CD8+ T cell responses following IM DNA/IN Ad mucosal prime-boosting, although antibody responses were not diminished. These findings indicate that flagellin has differential and route-dependent adjuvant activity when included as a component of systemic or mucosally-delivered gene-based prime-boost immunization. Clear adjuvant activity for both T and B cell responses was observed when flagellin was included in the DNA priming vaccine, but side effects occurred when given in an Ad boosting vector, particularly via the pulmonary route.

Gene-based neonatal immune priming potentiates a mucosal adenoviral vaccine encoding mycobacterial Ag85B.

Tuberculosis remains a major public health hazard worldwide, with neonates and young infants potentially more susceptible to infection than adults. BCG, the only vaccine currently available, provides some protection against tuberculous meningitis in children but variable efficacy in adults, and is not safe to use in immune compromised individuals. A safe and effective vaccine that could be given early in life, and that could also potentiate subsequent booster immunization, would represent a significant advance. To test this proposition, we have generated gene-based vaccine vectors expressing Ag85B from Mycobacterium tuberculosis (Mtb) and designed experiments to test their immunogenicity and protective efficacy particularly when given in heterologous prime-boost combination, with the initial DNA vaccine component given soon after birth. Intradermal delivery of DNA vaccines elicited Th1-based immune responses against Ag85B in neonatal mice but did not protect them from subsequent aerosol challenge with virulent Mtb H37Rv. Recombinant adenovirus vectors encoding Ag85B, given via the intranasal route at six weeks of age, generated moderate immune responses and were poorly protective. However, neonatal DNA priming following by mucosal boosting with recombinant adenovirus generated strong immune responses, as evidenced by strong Ag85B-specific CD4+ and CD8+ T cell responses, both in the lung-associated lymph nodes and the spleen, by the quality of these responding cells (assessed by their capacity to secrete multiple antimicrobial factors), and by improved protection, as indicated by reduced bacterial burden in the lungs following pulmonary TB challenge. These results suggest that neonatal immunization with gene-based vaccines may create a favorable immunological environment that potentiates the pulmonary mucosal boosting effects of a subsequent heterologous vector vaccine encoding the same antigen. Our data indicate that immunization early in life with mycobacterial antigens in an appropriate vaccine setting can prime for protective immunity against Mtb.

Orthovanadate increased the frequency of aneuploid mouse sperm without micronucleus induction in mouse bone marrow erythrocytes at the same dose level.

The objective of the current study was to investigate the ability of orthovanadate to induce aneuploidy in mouse sperm and micronuclei in mouse bone marrow cells at the same dose levels. The BrdU-incorporation assay was performed to test if the chemical treatment altered the duration of the meiotic divisions. It was found that orthovanadate (25mg/kg bw) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated by intraperitoneal (i.p.) injection with 5, 15 or 25mg/kg bw orthovanadate and sperm were sampled from the Caudae epididymes 22 days later. Fluorescence in situ hybridization (FISH) was performed with DNA-probes for chromosomes 8, X or Y. Significant increases in the frequencies of total hyperhaploid sperm (p<0.01) were found with 15 and 25mg/kg bw orthovanadate, indicating induced non-disjunction during male meiosis. The dose-response was described best by a linear equation. Orthovanadate did not significantly increase the frequencies of diploid sperm at any of the three doses tested, indicating that no complete meiotic arrest occurred. Orthovanadate was investigated also by the micronucleus test at i.p. doses of 1, 5, 15 or 25mg/kg bw, followed by bone marrow sampling 24h after treatment. None of the orthovanadate doses caused a significant increase in the rates of micronuclei (MN). Since the results show that orthovanadate induced non-disjunction during male meiosis without an accompanying induction of MN in bone marrow erythrocytes under the present experimental conditions and doses, it is concluded that male germ cells (meiosis) are more sensitive to the aneugenic effects of orthovanadate than somatic cells (mitosis). However, induction of micronuclei was reported in the literature with orthovanadate, vanadylsulfate and ammonium metavanadate, which contradicts the notion that vanadium compounds might be unique germ cell aneugens.

Stem cell intervention ameliorates epigallocatechin-3-gallate/lipopolysaccharide-induced hepatotoxicity in mice.

Stem cells are identified as a novel cell therapy for regenerative medicine because of their ability to differentiate into many functional cell types. We have shown earlier a new model of hepatotoxicity in mice by administering (1500 mg/kg) epigallocatechin-3-gallate (EGCG) intragastric (IG) for 5 days after a single intraperitoneal dose (6 mg/kg) of lipopolysaccharide (LPS). In this study, we aimed to study the effect of intrahepatic (IH) injection of mouse embryonic stem cells (MESCs) on the hepatotoxicity induced by EGCG/LPS in mice. Mice were administered EGCG/LPS and rested for 3 days. MESCs were obtained from American Type Culture Collection and cultured in vitro for 4 days. Stem cells were injected IH. Seven days later, a single dose of LPS (6 mg/kg) followed by daily doses of IG administration of EGCG were re-administered for 5 days. At the end of the experiment, blood samples were collected for analysis of biochemical parameters associated with liver. Results showed that the group of mice that were administered MESCs prior to EGCG/LPS showed lower levels of alanine amino transferase, alkaline phosphatase, and bilirubin, higher albumin/globulin ratio, and less remarkable histopathological lesions. Also, that group of mice showed less expression of oxidative stress biomarkers (oxidized low-density lipoprotein Ox.LDL and chemokine CXCL16), less expression of nuclear protein receptors (retinoic acid receptor and retinoid X receptor), and less expression of inflammatory biomarkers (tumor necrosis factor α and transforming growth factor ß1) compared with other groups of mice that were not given MESCs. In conclusion, MESCs can ameliorate EGCG/LPS-induced hepatotoxicity in mice.

Role of polymorphic CYP2E1 and CYP2D6 genes in NNK-induced chromosome aberrations in cultured human lymphocytes.

Polymorphisms in genes of xenobiotic-metabolizing enzymes are largely responsible for interindividual differences in ability to activate and detoxify genotoxic agents and therefore may influence individual susceptibility to environmental cancer. The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), requires metabolic activation by cytochrome P450 (CYP) enzymes to generate DNA-reactive intermediates that induce mutations and cancer. In the current study, we investigated the role of the polymorphic CYP2E1 and CYP2D6 genes in the genotoxicity of NNK using the tandem-probe fluorescence in-situ hybridization (FISH) chromosome aberration assay as a marker. Our results, using whole blood cultures from 39 volunteers, indicated that NNK (0.12, 0.24 or 0.72 mM) induced a concentration-dependent increase in the frequency of chromosome aberration. The potential role of CYP2E1 and CYP2D6 in NNK-induced genetic damage in cultured human lymphocytes was characterized using specific CYP inhibitors. Treatment of blood cultures with 25 microM diethyldithiocarbamate (DDC), a specific CYP2E1 inhibitor, or 0.5 microM quinidine, a specific CYP2D6 inhibitor, simultaneously with NNK, significantly decreased NNK-induced chromosome aberration. We also studied the role of CYP2E1 and CYP2D6 allelic variants on NNK-induced chromosome aberration. Our results indicate that NNK induced a significantly higher level of chromosome aberration in cells with the CYP2E1 WT/*5B genotype compared to cells with the CYP2E1 WT/WT. In contrast, no difference in NNK-induced chromosome aberration was observed between cells with the CYP2D6 extensive metabolizers compared to cells with the CYP2D6 poor metabolizer genotypes. These results underscore the important role of polymorphic metabolizing genes in influencing the genotoxic responses to environmental mutagens and provide support to the reported findings linking CYP2E1 polymorphism to smoking-related lung cancer.

Role of polymorphic GSTM1 and GSTT1 genotypes on NNK-induced genotoxicity.

Polymorphisms in chemical metabolizing genes are known to influence individual susceptibility to environmental cancer. We investigated the role of GSTM1 and GSTT1 polymorphisms in modifying the genotoxicity of a tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) using the sister chromatid exchange (SCE), and the tandem-probe fluorescence in situ hybridization chromosome aberration (CA) assays. NNK (0.24, 0.72 or 1.44 mM) induced a significant concentration-dependent increase in the mean number of SCE regardless of genotypes. In comparing the effects between genotypes, significant increase was observed in GSTM1 null cells compared with GSTM1 positive cells only at the low concentration of NNK (0.24 mM). No significant difference was observed between cells with the null and positive GSTT1 genotypes. Using the CA assay, treatment with NNK (0.12, 0.24 or 0.72 mM) induced a significant concentration-dependent increase in the frequency of CA. In addition, cells with the null GSTM1 genotype had significantly increased CA compared with cells with GSTM1 positive genotype at the three concentrations of NNK. Regarding GSTT1 polymorphism, no significant effect was observed between the null and the positive genotypes. Treatment of the cells with 1 mM glutathione monoethyl ester (GSHME) significantly reduced NNK-induced CA in all cells regardless of their genotypes. The effect was clearly more evident in cells with the GSTM1 positive genotype. Therefore, GSHME is protective against NNK-induced CA with more dominant effect in cells with the GSTM1 positive genotype. Our study indicates that GSTM1 may influence NNK-induced genotoxicity and subsequent tobacco-related health effects.

Two mammalian cell systems for propagation of the hepatitis B virus genome in extrachromosomal and chromosomally integrated states: production of the surface and e antigens.

A recombinant plasmid consisting of (i) the entire genome of hepatitis B virus (HBV) DNA, (ii) the replication origin of SV40 virus, and (iii) a deletion derivative of pBR322 was introduced either into COS cells of monkey origin which constitutively express SV40 large T antigen, or into thymidine kinase(TK)-deficient mouse L cells together with the TK DNA of Herpes simplex virus. In the COS cell system, the transfecting recombinant DNA replicates via SV40 origin and is maintained in an autonomously replicating state. The cells carrying these extrachromosomal elements express the hepatitis B surface antigen gene at moderate rate, and release the products into the culture medium. However, neither core antigen nor e antigen expression was detected in this system. In the L cell system, the transformed L cells carry the recombinant DNA in a chromosomally integrated state. Such cells express the surface antigen gene at high rate, and release the products into the culture medium. This system also excretes the e antigen into the culture medium. The core antigen was not detected.

Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells.

Recombinant baculoviruses that produce a putative non-structural protein 1 (NS1) of hepatitis C virus (HCV), predicted to be the second envelope glycoprotein, were constructed. The recombinant NS1 protein (re-NS1) produced in infected insect cells was localized on the cell surface and was apparently glycosylated, because it was susceptible to treatment with both tunicamycin and N-glycanase. Furthermore, re-NS1 was effectively secreted into the culture supernatant when the putative NS1 signal peptide (SP) was replaced by the SP of rabies virus G protein, and the C-terminal hydrophobic region was eliminated. The secreted re-NS1 was tagged with six His residues at the C terminus and purified simply by native Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) affinity column chromatography. An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of HC using purified re-NS1. Anti-NS1 antibody (Ab) was detected in 55 of 60 patients (92%) with chronic HC liver diseases. Thus, this ELISA for Ab directed against HCV re-NS1 produced in insect cells is useful for the detection of chronic HC patients.

Expression of herpes simplex virus glycoprotein B gene in yeast.

The DNA sequence coding for herpes simplex virus type 1 glycoprotein B was placed under control of the acid phosphatase promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of replicating in both yeast and Escherichia coli. Yeast transformed by the plasmid synthesized immunologically active glycoprotein B polypeptide.

A multiplex-PCR/RFLP procedure for simultaneous CYP2E1, mEH and GSTM1 genotyping.

Inter-individual variation in metabolism of environmental toxicants, which is attributed to genetic polymorphism, may be a major risk factor in determining who will develop adverse health effects. This priority research area is the focus of many laboratories, and new techniques need to be developed to enhance the efficiency in generating data. We have developed and validated a new multiplex-polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) procedure for simultaneous genotyping of cytochrome P450 II E1 (CYP2E1), microsomal epoxide hydrolase (mEH), and glutathione S-transferase mu (GSTM1). Enzymes from these three polymorphic genes are involved with the phase I and II metabolism of a variety of environmental toxicants. Therefore, simultaneous characterization of these genes will not only reduce costs but will increase the efficiency of data collection, thereby contributing to health risk assessment efforts.

The antioxidant properties of zinc and metallothionein.

Support for the hypothesis that metallothionein isoforms participate in intracellular defense against reactive oxygen and nitrogen species is derived from observations that substances causing oxidative stress, such as ethanol and iron, and agents involved in inflammatory processes, such as interleukin-1 and tumour necrosis factor alpha, induce the synthesis of metallothionein. Moreover, animals deficient in metallothionein isoforms exhibit greater susceptibility to oxidative stress; metallothionein genes are transcriptionally activated in cells and tissues during oxidative stress; and over expression of metallothionein reduces the sensitivity of cells and tissues to free radical-induced injury. In this study, we have shown that the i.c.v. administration of ZnSO4 increases the synthesis of metallothionein I mRNA and metallothionein II mRNA. In addition, the i.c.v. administration of ZnSO4 enhances the concentration of zinc and in direct proportion the synthesis of metallothionein mRNAs. Agents known to generate free radicals and to cause oxidative stress such as 6-hydroxydopamine, iron, hydrogen peroxide, and various alcohols lead to induction of metallothionein in the hippocampal neurons in primary culture and in Chang liver cells in culture. In view of the fact that zinc and 6-hydroxydopamine induce the level of brain metallothionein and its mRNAs and zinc and metallothionein concentrations vary in different regions of the brain, it is postulated that metallothionein may play a major role in nullifying the iron-mediated generation of free radicals and in protecting against oxidative stress in the brain.

Neurotrophins and their receptors in nerve injury and repair.

Cytokines are a heterogenous group of polypeptide mediators that have been associated with activation of numerous functions, including the immune system and inflammatory responses. The cytokine families include, but are not limited to, interleukins (IL-I alpha, IL-I beta, ILIra and IL-2-IL-15), chemokines (IL-8/ NAP-I, NAP-2, MIP-I alpha and beta, MCAF/MCP-1, MGSA and RANTES), tumor necrosis factors (TNF-alpha and TNF-beta), interferons (INF-alpha, beta and gamma), colony stimulating factors (G-CSF, M-CSF, GM-CSF, IL-3 and some of the other ILs), growth factors (EGF, FGF, PDGF, TGF alpha, TGF beta and ECGF), neuropoietins (LIF, CNTF, OM and IL-6), and neurotrophins (BDNF, NGF, NT-3-NT-6 and GDNF). The neurotrophins represent a family of survival and differentiation factors that exert profound effects in the central and peripheral nervous system (PNS). The neurotrophins are currently under investigation as therapeutic agents for the treatment of neurodegenerative disorders and nerve injury either individually or in combination with other trophic factors such as ciliary neurotrophic factor (CNTF) or fibroblast growth factor (FGF). Responsiveness of neurons to a given neurotrophin is governed by the expression of two classes of cell surface receptor. For nerve growth factor (NGF), these are p75NTR (p75) and p140trk (referred to as trk or trkA), which binds both BDNF and neurotrophin (NT)-4/5, and trkC receptor, which binds only NT-3. After binding ligand, the neurotrophin-receptor complex is internalized and retrogradely transported in the axon to the soma. Both receptors undergo ligand-induced dimerization, which activates multiple signal transduction pathways. These include the ras-dependent pathway utilized by trk to mediate neurotrophin effects such as survival and differentiation. Indeed, cellular diversity in the nervous system evolves from the concerted processes of cell proliferation, differentiation, migration, survival, and synapse formation. Neural adhesion and extracellular matrix molecules have been shown to play crucial roles in axonal migration, guidance, and growth cone targeting. Proinflammatory cytokines, released by activated macrophages and monocytes during infection, can act on neural targets that control thermogenesis, behavior, and mood. In addition to induction of fever, cytokines induce other biological functions associated with the acute phase response, including hypophagia and sleep. Cytokine production has been detected within the central nervous system as a result of brain injury, following stab wound to the brain, during viral and bacterial infections (AIDS and meningitis), and in neurodegenerative processes (multiple sclerosis and Alzheimer's disease). Novel cytokine therapies, such as anticytokine antibodies or specific receptor antagonists acting on the cytokine network may provide an optimistic feature for treatment of multiple sclerosis and other diseases in which cytokines have been implicated.

Comparison of the spinal anti-nociceptive effects of ES-242-1 and MK-801, two different NMDA antagonists, in rats.

The purpose of this study was to determine whether ES-242-1, a novel N-methyl-D-aspartate (NMDA) receptor antagonist of microbial origin, has anti-nociception at the spinal level and to evaluate how its anti-nociceptive effect differs from that of MK-801, a non-competitive NMDA receptor antagonist. Agents were injected intrathecally (0.1, 1.0 and 10 microg) through a previously implanted PE tube in rats. Formalin (2%, 100 microl) was injected subcutaneously into the left hindpaw 15 min after each antagonist administration. Licking time as a nociceptive behavior was measured in three stages after formalin-injection, such as early phase (0-9 min), late first phase (10-29 min) and late second phase (30-60 min). In the early phase, the largest dose of ES-242-1 significantly decreased total licking time, although MK-801 did not show any significant reduction. With the treatment of 1.0 and 10 microg MK-801, total licking time in both late first and second phases was significantly suppressed, although the smallest dose (0.1 microg) of ES-242-1 showed a significant reduction in the late second phase. These results indicate that ES-242-1 is highly effective against tonic pain, such as inflammatory pain.