HLA-DPB1 and anti-HBs titer kinetics in hepatitis B booster recipients who completed primary hepatitis B vaccination during infancy.

Previously we reported significant associations of the human leukocyte antigen (HLA)-DPB1 05:01 with memory against hepatitis B (HB) vaccination. However, the effects of HLA-DPB1 on antibodies to hepatitis B surface antigen (anti-HBs) kinetics were not explored. We followed up a cohort of 1974 HB booster recipients and quantified their 1-month and 1-year post-booster anti-HBs titers. A total of 681 subjects were randomly selected and typed for HLA-DPB1. We found that male subjects, undetectable pre-booster titers, and 05:01 homozygotes led to significantly lower post-booster anti-HBs titers. The geometric means (95% confidence interval (CI)) of 1-month post-booster anti-HBs titers were 4.68 (2.69-8.12), 23.01 (14.96-35.40) and 50.06 (27.20-92.13) mIU ml(-1) for subjects carrying two, one and no HLA-DPB1 05:01 allele. The corresponding figures for 1-year post-booster anti-HBs titers were 1.26 (0.73-2.18), 4.72 (3.08-7.25) and 7.32 (3.75-13.56) mIU ml(-1). There were significant associations of post-booster anti-HBs titers with the number of HLA-DPB1 risk and protective alleles. Among booster responders, anti-HBs decay rates were significantly reduced in subjects who had detectable pre-booster anti-HBs titers and the HLA-DPB1 05:01 allele. Our results indicated that HLA-DPB1 influences the kinetics of anti-HBs. The long-term memory against hepatitis B surface antigen (HBsAg) and the residual serum titers of anti-HBs after HB vaccination may be influenced by different mechanisms as evidenced by their inverse trend of associations with the 05:01 allele.

Intranasal Treatment of Ferrets with Inert Bacterial Spores Reduces Disease Caused by a Challenging H7N9 Avian Influenza Virus.

Background: Influenza is a respiratory infection that continues to present a major threat to human health, with ~500,000 deaths/year. Continued circulation of epidemic subtypes in humans and animals potentially increases the risk of future pandemics. Vaccination has failed to halt the evolution of this virus and next-generation prophylactic approaches are under development. Naked, "heat inactivated", or inert bacterial spores have been shown to protect against influenza in murine models. Methods: Ferrets were administered intranasal doses of inert bacterial spores (DSM 32444K) every 7 days for 4 weeks. Seven days after the last dose, the animals were challenged with avian H7N9 influenza A virus. Clinical signs of infection and viral shedding were monitored. Results: Clinical symptoms of infection were significantly reduced in animals dosed with DSM 32444K. The temporal kinetics of viral shedding was reduced but not prevented. Conclusion: Taken together, nasal dosing using heat-stable spores could provide a useful approach for influenza prophylaxis in both humans and animals.

Radiocarbon dating of petroleum-impregnated bone from tar pits at Rancho La Brea, California.

A liquid-chromatography method has been developed for the separation of amino acids with the same specific activity in radiocarbon from bones impregnated with isotopically dead petroleum compounds found in the La Brea tar pits. This technique permits the application of radiocarbon dating to such bone assemblages.

Emodin is a novel alkaline nuclease inhibitor that suppresses herpes simplex virus type 1 yields in cell cultures.

BACKGROUND AND PURPOSE: Most antiviral therapies directed against herpes simplex virus (HSV) infections are limited to a small group of nucleoside analogues that target the viral polymerase. Extensive clinical use of these drugs has led to the emergence of resistant viral strains, mainly in immunocompromised patients. This highlights the need for the development of new anti-herpesviral drugs with novel targets. Herein the effects of a plant anthraquinone, emodin, on the HSV-1 alkaline nuclease activity and virus yields were investigated. EXPERIMENTAL APPROACH: HSV-1 alkaline nuclease activity was examined by nuclease activity assay. Inhibition of virus yields was measured by plaque reduction assay and immunohistochemical staining. Interaction between emodin and alkaline nuclease was analysed by docking technology. KEY RESULTS: Emodin specifically inhibited the nuclease activity of HSV-1 UL12 alkaline nuclease in a biochemical assay. Plaque reduction assay revealed that emodin reduced the plaque formation with an EC(50) of 21.5+/-4.4 muM. Immunohistochemical staining using the anti-nucleocapsid protein antibody demonstrated that emodin induced the accumulation of viral nucleocapsids in the nucleus in a dose-dependent manner. Docking analysis further suggested that the inhibitory effect of emodin on the UL12 activity may result from the interaction between emodin and critical catalytic amino acid residues of UL12. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that emodin is a potent anti-HSV agent that inhibits the yields of HSV-1 via the suppression of a novel target, UL12.

Timing and duration of nursing from birth affect neonatal porcine uterine matrix metalloproteinase 9 and tissue inhibitor of metalloproteinase 1.

Nursing for 2 d from birth supports neonatal porcine uterine and cervical development. However, it is not clear how timing or duration of lactocrine signaling from birth (postnatal day = PND 0) affects development of neonatal female reproductive tract tissues. Therefore, studies were conducted to determine effects of age at first nursing and duration of nursing from birth on specific elements of the matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) system in uterine and cervical tissues at PND 2. When nursing was initiated at 0 h or 30 min of age, targeted proteins, including proMMP9 and MMP9, were detected in uterine and cervical tissues on PND 2, as was uterine TIMP1. However, these proteins were undetectable when nursing was delayed for 12 h and when gilts were fed milk replacer for 48 h from birth. Increasing the duration of nursing from 30 min to 12 h from birth increased uterine (P < 0.05) and cervical (P < 0.001) MMP9 levels to those observed in gilts nursed for 48 h. Similarly, uterine TIMP1 levels increased with duration of nursing. Uterine MMP2 levels were detectable but unaffected by age at first nursing or duration of nursing from birth. Uterine MMP2 and MMP9 activities, monitored by zymography, reflected immunoblotting data. Results provide evidence for the utility of MMP9 and TIMP1 as markers of age- and lactocrine-sensitive porcine female reproductive tract development.

Residual analgesic effects of morphine in 55 four-period crossover analgesic studies.

Subjective response data from 55 postoperative pain studies were examined for the residual analgesic effects of morphine. The studies were planned as four-period crossover designs for four treatments. Each patient received 5 and 10 mg of morphine and two doses of a test preparation. Two measures of analgesia were used: Sum of the Pain Intensity Difference (SPID) and Total Pain Relief (TOTPAR). To facilitate analysis, two two-period groups were defined. Morphine data for periods 1 and 2 were designated as group A, and morphine data for periods 3 and 4 were designated as group B. Residual analgesic effects were 0.12 for both SPID and TOTPAR in group A and were 0.65 and 0.17 for SPID and TOTPAR, respectively, in group B. In these 55 studies, there was no evidence of significant residual analgesic effects. Thus the crossover design is an appropriate method for the evaluation of selected parenteral analgesics in the postoperative pain model.

Identification of a pseudorabies virus UL12 (deoxyribonuclease) gene.

We characterized the gene encoding the pseudorabies virus (PrV) homologue of the herpes simplex virus 1 UL12 open reading frame that encodes the alkaline nuclease. The deduced PrV UL12 product was 492 amino acid residues and exhibited three conserved regions among herpesviruses. Northern blot analysis indicated that three transcripts (3.2, 1.6 and 1 kb) were encoded in this region and the UL12 corresponds to the 1.6-kb transcript. Primer extension and UL12-specific cDNA cloning were performed to verify the precise location of the UL12 transcript. These data indicated that the transcription start site of UL12 was located at 47-62 nucleotides upstream of the UL12 translation start site and the polyadenylation cleavage site was located at 15 or 16 nucleotides downstream the typical polyadenylation signal. Furthermore, the 53-kDa UL12 product, which indeed has deoxyribonuclease activity, was evidenced by in vitro expression.

Analysis of pseudorabies virus genes by cDNA sequencing.

Cloning and sequencing of cDNA could provide a complementary approach to functional analysis of the pseudorabies virus (PrV) genome. Using colony hybridization, Southern hybridization, and DNA sequencing, four species of PrV-specific cDNA were identified. Among these four species of PrV-specific cDNA, three unidentified genes, UL26, UL29, and UL31, were mapped and a novel gI-11K bicistronic cDNA was confirmed. Thus, analysis of PrV-specific transcripts provided a way for identifying genes and a foundation to further study the roles of these transcripts in PrV infection.

Identification, expression, and characterization of the pseudorabies virus DNA-binding protein gene and gene product.

The pseudorabies virus (PRV) gene encoding a DNA-binding protein (DBP) was first identified in this study. The DBP gene has an open reading frame of 3531 nucleotides, capable of coding a 1177-amino-acid polypeptide of 125 kDa. The deduced DBP exhibits a conserved zinc-binding motif and a conserved DNA-binding region, suggesting the similar DNA-binding mechanism occurs among alphaherpesviral DBP homologs. To further identify the biochemical properties of PRV DBP, this protein was expressed in Escherichia coli by using a pET expression vector and purified to homogeneity. The PRV DBP binds cooperatively and preferentially to single-stranded DNA with no significant base preference, judged by agarose gel electrophoresis and competitive nitrocellulose filter binding assays. Taken together, these results suggest that PRV DBP may play an important role in PRV DNA replication by binding cooperatively and nonspecifically to single-stranded DNA that is formed during the replication origin unwinding and replication fork movement.

Pseudorabies virus early protein 0 trans-activates the TATA-associated promoter by stimulating the transcription initiation.

Pseudorabies virus (PRV) early protein 0 (EP0) is a transactivator containing a RING finger domain. To assess the transactivation mechanism of PRV EP0, we performed the in vitro transcription by combining HeLa nuclear extract, purified recombinant EP0 and simple promoter constructs, and evaluated the results by primer extension. The data showed that EP0 could significantly activate the TATA-containing synthetic promoters. Moreover, EP0 activated transcription by stabilizing the formation of transcription initiation complex instead of enhancing the elongation rate. To further understand the role of EP0 on assembling the transcription initiation complex, we performed the pull-down assay using affinity precipitation of proteins from HeLa nuclear extracts and bacterially expressed glutathione-S-transferase EP0 RING finger fusion. The data showed that at least six nuclear proteins physically interacted with the EP0 RING finger. Overall, the transactivation of PRV EP0 is accomplished by enhancing the transcription initiation and is associated with at least six nuclear proteins.

Rapid screening of pseudorabies virus-specific cDNAs from a cDNA library.

In order to reduce the time and cost for screening of pseudorabies virus (PRV)-specific cDNAs, a rapid and inexpensive method was developed that involved subtractive hybridization of the plasmid, which contained cDNA fragment, to PRV genomic DNA which was bound to nylon membranes. Ninety percent of DNA background was subtracted successfully by this method and the eluted DNA in the form of plasmid could be used to transform bacteria directly. Applying this technique, 200 colonies were screened from a cDNA library containing 30000 colonies. Furthermore, 17 colonies containing PRV-specific cDNAs, including PRV43, UL42, gII, DNase, EP0, 11K, gX, and RSP40, were identified from the 200 colonies by colony hybridization, Southern hybridization, and DNA sequencing. Thus, the subtractive hybridization can be used to construct and successfully establish the PRV cDNA library from PRV-infected cells.

Analysis of upregulated cellular genes in pseudorabies virus infection: use of mRNA differential display.

Virus infection usually alters the host cell and shuts off the synthesis of cellular macromolecules. In order to screen the upregulated cellular transcripts during pseudorabies virus (PRV) infection, we employed the mRNA differential display technique. The screen is based on positive selection at the mRNA level for genes expressed in normal cells but increased in corresponding PRV-infected cells. Over 14000 species of mRNA, isolated from mock-infected and PRV-infected Madin-Darby bovine kidney cell at 1 h post infection, were screened, and 40 candidate clones were recovered. Southern blot analysis revealed that 17 out of 40 candidate clones, were enhanced in PRV-infected cells. Partial DNA sequences demonstrated that 17 clones were distinct cellular genes, including those encoding the modulators of signal transduction (saposin, 14-3-3, adenylate kinase, adenylyl cyclase, protein kinase C-alpha), those encoding the components of translation (fau, ribosomal proteins S11, L31, L36), other cellular genes (peptidase, cyclin E, rch1, oligo-C-rich single-stranded nucleic acid binding protein, rap, arginyl-tRNA synthetase), and two unknown genes. Thus, this study identifies successfully the transcriptionally regulated cellular genes which are associated with PRV infection. Furthermore, this study provides support for the use of mRNA differential display as a method to rapidly isolate differentially expressed genes in virus infection.

Identification of the copper-zinc superoxide dismutase activity in Mycoplasma hyopneumoniae.

Copper-zinc superoxide dismutase (Cu/ZnSOD), a key enzyme in defense against toxic oxygen-free radicals, is widespread in eukaryotes and several species of gram-negative bacteria. The presence of this enzyme in Mycoplasma hyopneumoniae (M. hyopneumoniae), the primary pathogen of mycoplasmal pneumonia in pigs, was examined since the polyclonal antibody against bovine Cu/ZnSOD was dominantly cross-reactive with the M. hyopneumoniae Cu/ZnSOD from whole cellular proteins. In situ activity staining on SDS-PAGE showed that the molecular mass of M. hyopneumoniae Cu/ZnSOD in reducing form was approximately 17kDa. The presence of Cu and Zn ions at the active site of the enzyme was confirmed on the basis of inhibition by KCN and by H(2)O(2). The activity of M. hyopneumoniae Cu/ZnSOD on both SDS- and native-polyacrylamide gels was completely inhibited by 2mM KCN and the gels showed no iron-containing SOD (FeSOD) or manganese-containing SOD (MnSOD) in the crude extracts. The activity of M. hyopneumoniae Cu/ZnSOD in crude extract was 70units/mg protein and was 55% inhibited by 5mM KCN and 56% inactivated by 40mM H(2)O(2). This enzyme was growth-stage dependent and evidenced markedly higher production during the early log phase. Different expression levels of Cu/ZnSOD activity in field isolates were also detected. Taken together, the presence of Cu/ZnSOD in M. hyopneumoniae was identified for the first time.

Inhibitory effect of anti-pyretic and anti-inflammatory herbs on herpes simplex virus replication.

The increasing clinical use of acyclovir, ganciclovir, and foscarnet against herpes simplex virus (HSV), varicella-zoster virus, and cytomegalovirus has been associated with the emergence of drug-resistant herpesvirus strains. To develop anti-HSV compounds from plants, 31 herbs used as antipyretic and anti-inflammatory agents in Chinese medicine were screened. Five different preparations (cold aqueous, hot aqueous, ethanolic, acid ethanolic, and methanolic) from 31 herbs were analyzed by plaque reduction assay, and 7 extracts. which showed significant antiviral activities, were further elucidated for their antiviral mechanisms. Our results showed that ethanolic extract of Rheum officinale and methanolic extract of Paeonia suffruticosa prevented the process of virus attachment and penetration. Aqueous extract of P. suffruticosa and ethanolic extract of Melia toosendan inhibited virus attachment to cell surface. Aqueous extract of Sophora flavescens and methanolic extract of M. toosendan showed no effect on virus attachment and penetration. These data indicated that these 4 herbs have a potential value as a source of new powerful anti-HSV compounds.

The effectiveness of Tsu-San-Li (St-36) and Tai-Chung (Li-3) acupoints for treatment of acute liver damage in rats.

This study investigated the effects of acupuncture on carbon tetrachloride (CCl4) induced acute liver injury in male rats (n=36). The experimental groups were injected with CCl4 before, during, or after acupuncture therapy. Acupoints similar to the human Tsu-San-Li (St-36) and Tai-Chung (Li-3) were needled bilaterally. Rats treated with CCl4 had higher levels of serum glutamate-oxalate-transaminase (sGOT) and serum glutamate-pyruvate-transaminase (sGPT). Comparing the experimental groups, biochemical and pathological parameters of liver injury were significantly reduced when rats were acupunctured after, not before, CCl4-induced hepatotoxicity. Acupuncture at the Tsu-San-Li and Tai-Chung acupoints cannot prevent acute liver injury but may be effective in treating liver injury induced by carbon tetrachloride in rats.

Comprehensive evaluation of a novel nuclear factor-kappaB inhibitor, quinoclamine, by transcriptomic analysis.

BACKGROUND AND PURPOSE: The transcription factor nuclear factor-kappaB (NF-kappaB) has been linked to the cell growth, apoptosis and cell cycle progression. NF-kappaB blockade induces apoptosis of cancer cells. Therefore, NF-kappaB is suggested as a potential therapeutic target for cancer. Here, we have evaluated the anti-cancer potential of a novel NF-kappaB inhibitor, quinoclamine (2-amino-3-chloro-1,4-naphthoquinone). EXPERIMENTAL APPROACH: In a large-scale screening test, we found that quinoclamine was a novel NF-kappaB inhibitor. The global transcriptional profiling of quinoclamine in HepG2 cells was therefore analysed by transcriptomic tools in this study. KEY RESULTS: Quinoclamine suppressed endogenous NF-kappaB activity in HepG2 cells through the inhibition of IkappaB-alpha phosphorylation and p65 translocation. Quinoclamine also inhibited induced NF-kappaB activities in lung and breast cancer cell lines. Quinoclamine-regulated genes interacted with NF-kappaB or its downstream genes by network analysis. Quinoclamine affected the expression levels of genes involved in cell cycle or apoptosis, suggesting that quinoclamine exhibited anti-cancer potential. Furthermore, quinoclamine down-regulated the expressions of UDP glucuronosyltransferase genes involved in phase II drug metabolism, suggesting that quinoclamine might interfere with drug metabolism by slowing down the excretion of drugs. CONCLUSION AND IMPLICATIONS: This study provides a comprehensive evaluation of quinoclamine by transcriptomic analysis. Our findings suggest that quinoclamine is a novel NF-kappaB inhibitor with anti-cancer potential.

Increased gene expression in human promyeloid leukemia cells exposed to trans,trans-muconaldehyde, a hematotoxic benzene metabolite.

The hematotoxicity of benzene, a human leukemogen, has been postulated to be mediated by reactive metabolites and involve cell damage caused by reactive oxygen species. Because expression of the transcription factors AP-1 and NF-kappaB is sensitive to the redox state in eukaryotic cells, the DNA binding activity of AP-1 and NF-kappaB was examined in HL-60 promyeloid leukemia cells exposed to trans,trans-muconaldehyde, a microsomal hematotoxic metabolite of benzene. There was little AP-1 binding activity in nuclear extracts from control HL-60 cells based on electrophoretic mobility shift assays. Exposure to 0.1 microM MUC for 4 h resulted in significantly increased levels of nuclear protein with high sequence specificity for the consensus AP-1 sequence. In addition, electrophoretic mobility shift assays showed a strong increase in the binding of a factor to the NF-kappaB site. The latter was highest in nuclear extracts from HL-60 cells treated with 1.0 microM muconaldehyde and cultured for 4 h. Exposure of HL-60 cells to muconaldehyde resulted in an increase in c-fos and c-jun mRNA levels. Western blot analysis showed that the protein levels of c-jun increased in HL-60 cells treated with 1 microM muconaldehyde and cultured for 4-6 h and subsequently decreased gradually. Increased AP-1 binding was observed in bone marrow cells from B6C3F1 mice 2 h after administration of 440 mg/kg benzene. We suggest that increased gene expression of NF-kappaB and AP-1 binding activity and up-regulation of c-fos and c-jun may play a role in the mechanism of benzene leukemogenesis.

Polypeptide pattern of human breast epithelial cells following human chorionic gonadotropin (hCG) treatment.

Numerous attempts have made to describe the particular protein pattern of malignant cells by using high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The placental hormone human chorionic gonadotropin (hCG) inhibits tumor initiation and progression in experimental animals and has an inhibitory effect on the proliferation of human breast epithelial cells (HBEC) in vitro. The inhibitory effect on the immortalized HBEC MCF-10F is accompanied by the immunocytochemical expression of inhibin alpha and beta subunits by treated cells. With the purpose of clarifying the molecular mechanisms involved in this effect, the pattern of protein synthesis and mRNA were studied by 2-D PAGE in the immortalized HBEC MCF-10F cells treated in vitro 1001U for 24 h. The effect of hCG treatment on the synthesis of MCF-10F cells was monitored by labeling both control and treated cells with [S35]methionine and separation by 2-D PAGE. At least 11 proteins were preferentially synthesized and five specific polypeptides were decreased in hCG treated cells in comparison with controls. The hCG induced at least four new mRNAs which encoded protein in the molecular mass range of 24-72 kDa. It also increased the expression of at least six mRNAs and reduced the expression of least four mRNAs in comparison with control cells. The hCG-treated cells actively synthesized a 33-kDa polypeptide which was not present in control cells. The nature of this hCG-inducible 33 kDa protein elucidated by immunoprecipating [S35]methionine-labeled proteins with antisera directed against rat inhibin subunit alpha and beta b.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA vaccination induces a long-term antibody response and protective immunity against pseudorabies virus in mice.

In order to investigate the mechanism of long-term immunity and the effect of protective immunity induced by DNA vaccination, we constructed the expression plasmid containing a pseudorabies virus (PRV) gD gene encoding an envelope glycoprotein. Intramuscular vaccination of mice with the plasmid DNA induced a strong antibody response which lasted for one year after final vaccination. An IgM to IgG class switch occurred, indicating helper T-lymphocyte activity. We further analyzed the persistence and expression of gD gene by polymerase chain reaction and reverse transcriptase polymerase chain reaction. The results showed that gD gene was present and expressed in the muscle cell up to one year after final booster injection. Furthermore, mice vaccinated with the plasmid DNA were protected against a subsequent lethal challenge with PRV. Therefore, the DNA vaccination does induce a protective immunity and long-term antibody response against PRV, which could be maintained by persistent expression of gD gene in muscle cells.