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E F Flores,F A Osorio,E L Zanella,S Kit,M Kit Fifteen bovine herpesvirus-1 (BHV-1)-negative calves were vaccinated intramuscularly with 10(7.4) plaque-forming units of a double-deletion BHV-1 mutant (IBRV(NG)dltkdlgIII), and 6 remained as nonvaccinated controls. Thirty days after vaccination, the animals were challenged by nasal instillation o... ( view more )f 10(8.2) CCID50 of a virulent BHV-1 strain (Cooper). The vaccinated calves were protected against wildtype virus challenge as demonstrated by clinical evaluation. Most of the vaccinates developed only a mild rhinitis (lasting an average of 6.5 days) with almost no systemic symptoms, whereas the controls developed a serious illness characterized by rhinitis (mean = 11.5 days), conjunctivitis, hyperthermia, apathy, loss of appetite, and dyspnea. The vaccinates also shed significantly less virus and for a shorter period of time (mean = 5.5 days) than the controls (mean = 9 days). Thirty days after vaccination, the vaccinates were negative in an anti-gIII specific blocking enzyme-linked immunosorbent assay (ELISA), despite the fact that most of them had developed neutralizing antibodies (serum neutralization titers ranging from 1:2 to 1:16). Seroconversion to gIII was detected as early as 7 days postinfection (dpi). Fourteen days after the challenge, all the animals exposed to wildtype BHV-1 had developed anti-gIII antibodies and were positive in this differential serologic test. Six controls plus 8 vaccinates kept in isolation were still positive to gIII when tested at 75 dpi. The use of the IBRV(NG)dltkdlgIII strain in conjunction with an anti-gIII specific blocking ELISA kit represents a powerful tool for BHV-1 control/eradication programs. ( view less ) S Kit,H Otsuka,M Kit Recombinant DNA techniques were used to insert foreign genes into bovine herpesvirus-1 [infectious bovine rhinotracheitis virus (IBRV)] vectors which were attenuated by deletion and/or insertion mutations in the IBRV thymidine kinase (tk) gene. In one recombinant, the regulatory and coding sequence... ( view more )s of the late pseudorabies virus (PRV) glycoprotein gIII gene, were inserted into the early IBRV tk gene. This recombinant efficiently expressed the PRV gIII gene indicating that immediate early IBRV proteins were competent to transactivate the late PRV gIII gene. IBRV vector viruses were also prepared in which the coding sequences of the early PRV tk gene, the late PRV gIII gene, and the E. coli beta-galactosidase gene were ligated to the late IBRV gIII promoter. Genotypes and phenotypes of the recombinant viruses were verified by restriction endonuclease and molecular hybridization experiments, thymidine plaque autoradiography, beta-gal plaque assays, and by immunoprecipitation experiments on extracts from 3H-mannose-labelled cells. The recombinant IBRV expressing beta-gal from the IBRV gIII promoter has been useful as an intermediate in the construction of IBRV vectors harboring foreign DNA sequences. The infectivity of the IBRV recombinant that expressed PRV gIII from the IBRV gIII promoter, was neutralized by polyclonal PRV antisera and by monoclonal antibodies to PRV gIII. The PRV gIII glycoprotein synthesized by the preceding recombinant has been used to coat microtiter test plate wells in a PRV gIII differential diagnostic test kit. ( view less ) S Kit,Y Awaya,H Otsuka,M Kit A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV)-infected pigs from those immunized with a glycoprotein g92(gIII) deletion mutant, PRV(dlg92dltk). The blocking ELISA utilizes 96-well microtiter test plates coated with a cloned PRV ... ( view more )g92(gIII) antigen, a mouse monoclonal antibody against gIII antigen (moMCAgIII): horseradish peroxidase (HRPO) conjugate, and undiluted test sera. Analyses can be completed in less than 3 hours with results printed out by an automated plate reader. Analyses on over 300 pig sera from PRV-free farms, on sera from other species, and on control sera containing antibodies to microorganisms other than PRV showed that the ratio of the optical density at 405 nm for the test sample to the optical density at 405 nm for the negative control (S/N value) was greater than 0.7 for all sera. No false positives were identified. Likewise, the S/N values were greater than 0.7 for over 400 sera obtained from pigs vaccinated twice with more than 1,000 times the standard PRV (dlg92dltk) dose or 1-4 times with the standard dose (2 x 10(5) TCID50/pig). Following challenge exposure to virulent PRV, the S/N values of the vaccinates were 0.1, showing that g92(gIII) antibodies in the sera of experimentally challenged pigs strongly blocked the binding of the moMCAgIII:HRPO conjugate to the antigen-coated wells. Sera of 233 pigs from PRV-infected herds with virus neutralization (VN) titers of 1:4 or greater were tested. All except 2 of these sera had S/N values less than 0.7 and more than 175 had S/N values less than 0.1. Sixteen sera from fetal pigs with VN titers of 1:4 or greater had S/N values of 0.24 or less, but 2 sera with VN titers of 1:4 when tested 5 years prior to the PRV g92(gIII) blocking ELISA test gave false negative S/N values. Twenty-four of 29 pig sera from PRV-infected herds with VN titers less than 1:4 were positive for g92(gIII) antibodies, illustrating the sensitivity of the PRV g92(gIII) blocking ELISA test. Analyses on 7 sera with VN titers of 1:4-1:64 showed that titers obtained with the PRV g92(gIII) blocking ELISA test were from 2- to 16-fold greater than the VN titers. The accuracy and sensitivity of the PRV g92(gIII) blocking ELISA test was further demonstrated by analyses of 40 unknown sera supplied in the National Veterinary Services Laboratories 1988 PRV check test kit. ( view less ) G Ka Kit Leung,W Chun Kit Lee,J M NichollsA 53-year-old woman presented in 1979 with a posterior fossa meningeal haemangiopericytoma (HPC) for which she underwent surgical resection and post-operative radiotherapy. Repeated tumour recurrences occurred 18 years afterwards which were treated with resections and stereotactic radiotherapy. Sur... ( view more )gery for tumour recurrence in 2005 revealed features of rhabdomyosarcomatous transformation. To our knowledge, this is the first reported case of rhabdomyosarcomatous transformation within a HPC which was likely to be radiation-induced, and was associated with relentless disease progression more than 20 years after the initial presentation. ( view less ) Patrick Ying Kit Yue,Nai Ki Mak,Yuen Kit Cheng,Kar Wah Leung,Tzi Bun Ng,David Tai Ping Fan,Hin Wing Yeung,Ricky Ngok Shun Wong In Chinese medicine, ginseng (Panax ginseng C.A. Meyer) has long been used as a general tonic or an adaptogen to promote longevity and enhance bodily functions. It has also been claimed to be effective in combating stress, fatigue, oxidants, cancer and diabetes mellitus. Most of the pharmacological... ( view more ) actions of ginseng are attributed to one type of its constituents, namely the ginsenosides. In this review, we focus on the recent advances in the study of ginsenosides on angiogenesis which is related to many pathological conditions including tumor progression and cardiovascular dysfunctions. Angiogenesis in the human body is regulated by two sets of counteracting factors, angiogenic stimulators and inhibitors. The 'Yin and Yang' action of ginseng on angiomodulation was paralleled by the experimental data showing angiogenesis was indeed related to the compositional ratio between ginsenosides Rg1 and Rb1. Rg1 was later found to stimulate angiogenesis through augmenting the production of nitric oxide (NO) and vascular endothelial growth factor (VEGF). Mechanistic studies revealed that such responses were mediated through the PI3K-->Akt pathway. By means of DNA microarray, a group of genes related to cell adhesion, migration and cytoskeleton were found to be up-regulated in endothelial cells. These gene products may interact in a hierarchical cascade pattern to modulate cell architectural dynamics which is concomitant to the observed phenomena in angiogenesis. By contrast, the anti-tumor and anti-angiogenic effects of ginsenosides (e.g. Rg3 and Rh2) have been demonstrated in various models of tumor and endothelial cells, indicating that ginsenosides with opposing activities are present in ginseng. Ginsenosides and Panax ginseng extracts have been shown to exert protective effects on vascular dysfunctions, such as hypertension, atherosclerotic disorders and ischemic injury. Recent work has demonstrates the target molecules of ginsenosides to be a group of nuclear steroid hormone receptors. These lines of evidence support that the interaction between ginsenosides and various nuclear steroid hormone receptors may explain the diverse pharmacological activities of ginseng. These findings may also lead to development of more efficacious ginseng-derived therapeutics for angiogenesis-related diseases. ( view less ) Yick Fu Wong,Tak Hong Cheung,Keith Wing Kit Lo,So Fan Yim,Loucia Kit Yin Chan,Olivier Buhard,Alex Duval,Tony Kwok Hung Chung,Richard Hamelin The aim of this study was to find the optimal set of microsatellite markers for diagnosis of the microsatellite instability (MSI) phenotype in endometrial cancers. We compared the sensitivity, specificity and ease of use of a reference panel of five markers originally recommended by the National Ca... ( view more )ncer Institute (NCI) for colorectal cancer and a panel of five quasi-monomorphic mononucleotide repeat markers (pentaplex PCR system). We used these panels for establishing the MSI status of a series of 80 sporadic endometrial adenocarcinomas by comparing the allelic profiles of the markers between tumor and matching germline DNA. Both panels detected the same subset of 21 out of 80 (26%) endometrial MSI carcinomas. However, in the MSI cases, the mean instability of the five mononucleotide repeats was 96.1% as compared with a mean instability of 69.8% for the three dinucleotide repeats of the NCI panel, indicating a superiority of mononucleotide repeats over dinucleotide repeats in detecting MSI. The fact that the two panels of markers detect the same set of MSI tumors is due to the presence of two mononucleotide repeats within the NCI panel. As demonstrated previously in gastric and colon MSI cases, the pentaplex PCR reaction using mononucleotide repeats is thus an easier and more sensitive method than the NCI panel, for the screening of MSI status in endometrial tumors. ( view less ) Yan Kit Fong,Sam Oon Hui Peh,Siew Hong Ho,Foo Cheong Ng,Pearllyn Leng Choo Quek,Kok Kit Ng BACKGROUND: Lower pole spatial anatomy is an important determinant of success after extracorporeal shockwave lithotripsy. In the present study, we determine whether there is a significant relationship between lower pole ratio (infundibular length : infundibular width) on preoperative intravenous ur... ( view more )ograms and stone fragment clearances after shockwave lithotripsy. METHODS: A total of 42 patients with isolated lower pole stones were retrospectively reviewed. Anatomical factors, such as infundibular length, width and infundibulopelvic angle were measured and the lower pole ratio was calculated on pretreatment intravenous urogram. Stone fragment clearance was assessed at three months with a plain abdominal X-ray. RESULTS: The overall three-month stone-free rate was 62%. Mean stone size +/- SD was 10 +/- 4.8 mm, mean infundibular length was 21.7 +/- 6.9 mm, mean infundibular width was 6.1 +/- 2.3 mm, mean infundibulopelvic angle was 62.1 +/- 30.1 degrees and mean lower pole ratio was 4.3 +/- 2.8. Stone-free status after shockwave lithotripsy was significantly related to infundibular length and width as well as to lower pole ratio, but not to infundibulo-pelvic angle. Infundibular length less than 30 mm, width greater than 5 mm and lower pole ratio less than 3.5 were noted to have an improved three-month stone-free rate (P = 0.049, 0.01 and <0.01, respectively). CONCLUSION: Caliceal anatomy is an important consideration for lower pole stone clearance after shockwave lithotripsy. The present study suggests that a lower pole ratio of less than 3.5, which considers both infundibular length and width, is a promising predictor for stone-free status. ( view less ) Yick Fu Wong,Zachariah E Selvanayagam,Nien Wei,Joseph Porter,Ragini Vittal,Rong Hu,Yong Lin,Jason Liao,Joe Weichung Shih,Tak Hong Cheung,Keith Wing Kit Lo,So Fan Yim,Shing Kai Yip,Danny Tse Ngong,Nelson Siu,Loucia Kit Ying Chan,Chun Sing Chan,Tony Kong,Elena Kutlina,Randall D McKinnon,David T Denhardt,Khew-Voon Chin,Tony Kwok Hung Chung PURPOSE: The incidence and mortality rates of cervical cancer are declining in the United States; however, worldwide, cervical cancer is still one of the leading causes of death in women, second only to breast cancer. This disparity is at least partially explained by the absence of or comparatively... ( view more ) ineffective screening programs in the developing world. Recent advances in expression genomics have enabled the use of DNA microarray to profile gene expression of various cancers. These expression profiles may be suitable for molecular classification and prediction of disease outcome and treatment response. We envision that expression genomics applied in cervical cancer may provide a more rational approach to the classification and treatment of the disease. EXPERIMENTAL DESIGN: In this report, we examined the expression profiles of cervical cancer compared with normal cervical tissues in DNA microarrays that contained approximately 11,000 features that correspond to either human transcripts with known function or anonymous expressed sequence tags. RESULTS: Our results showed that normal cervical tissues were completely segregated from the cancer samples using about 40 genes whose expressions were significantly different between these specimens. In addition, clinical stage IB and stage IIB tumors could also be classified based on their signature expression patterns. Most importantly, some of the tumor samples were further stratified into two major groups based on their response to radiotherapy, and we were able to predict the response of these patients to radiotherapy from their expression profiles. CONCLUSIONS: Gene expression profiling by DNA microarray may be used for further molecular classification of disease stages and prediction of treatment response in cervical cancer. ( view less ) J A Galeota,E F Flores,S Kit,M Kit,F A Osorio Using quantitative polymerase chain reaction (PCR) we have studied the latency established by wildtype (WT) bovine herpesvirus-1 (BHV-1) after challenge of cattle that had been vaccinated with a double deletion (gC-/tk-) mutant BHV-1 vaccine. Fourteen animals were vaccinated intramuscularly with 2 ... ( view more )ml containing 10(7.4) CCID50 (cell culture infectious dose 50%) of IBRV (NG) dltkdlgC and challenged, along with six unvaccinated control animals, 30 days later with 10(8.2) CCID50 of WT BHV-1 (Cooper). The ability of this vaccine to prevent acute clinical BHV-1 infection after this challenge has been previously reported. Sixty days after challenge, eight of the vaccinates and the six control animals were euthanitized and the trigeminal ganglia (TG) examined for the amount of WT BHV-1 DNA by an internal standard quantitative PCR. The quantitative protocol that we used is based on co-amplification of BHV-1 gC specific sequences (present in WT BHV-1 but absent in the vaccine strain) and sequences from the bovine growth hormone (BGH) gene, which is used as an internal standard. The TG of the eight vaccinates contained BHV-1 WT DNA, but in a statistically significantly lower amount than the unvaccinated controls. These results are significant from the standpoint that, to our knowledge, this is the first report of a systematic quantitative approach to the study of the effect of BHV-1 vaccines on latency. This technique could be used to measure and compare the efficiency of various BHV-1 vaccines in preventing or diminishing latency, which is a significant factor for the perpetuation of BHV-1 in cattle populations. ( view less ) B Lawhorn,S McConnell,M Kit,S Kit To test the hypothesis that newborn pigs with pseudorabies virus (PRV) colostral antibodies might be actively immunized with a PRV glycoprotein gIII-deleted vaccine (Omnimark-PRV), 23 piglets were obtained from four sows that had been immunized 4 weeks and 2 weeks before farrowing with this vaccine... ( view more ). Thirteen piglets were immunized with Omnimark-PRV when they were less than 3 days old and ten piglets served as non-vaccinated controls. Piglets were weaned at 28 days of age and challenged with virulent PRV (Shope) when they were 49 days old, at which time the vaccinated and control pigs were seronegative for PRV virus neutralizing (VN) and gIII antibodies, and all control pigs and ten vaccinees were seronegative for PRV antibodies by the latex agglutination test (LAT). Two vaccinees were LAT(+) and one was LAT(+/-). Central nervous system signs and/or respiratory disease signs were observed in six of ten control pigs with the death of one control, while two of 13 vaccinees showed only very mild and transient clinical disease signs and there were no fatalities. Non-vaccinees lost weight until postchallenge day (PCD) 6 and did not regain prechallenge weight until PCD 8. All vaccinees gained weight after challenge and at PCD 11 had mean weight gains nearly twice that of the controls. On PCD 11, the geometric mean titre for VN antibodies of non-vaccinees was 9.3, while that of vaccinees was 49.0, indicating that the vaccinated group had been immunologically primed. ( view less ) S Kit,M Kit,S McConnell,B Lawhorn Maternal antibodies interfere with active immunization of swine by gI-deleted pseudorabies virus [(PRV); Aujeszky's disease virus] vaccines. To test the hypothesis that modified-live (MLV) vaccines retaining the PRV gI and with deletions in the PRV glycoprotein gIII and thymidine kinase (TK) genes ... ( view more )might be efficacious in circumventing colostral antibody interference, the OMNI-MARK-PRV (gI+ gIII- TK-) vaccine was administered intramuscularly to 13 newborn pigs with colostral antibodies, while 10 pigs from the same litters served as nonvaccinated controls. At 49 days of age, when PRV virus neutralization (VN) antibodies were negative and all nonvaccinated pigs as well as 10 vaccinates were latex agglutination test (LAT)-negative, the pigs were challenged intranasally with the virulent PRV(SHOPE) strain. In support of the hypothesis, it was found that several central nervous system and respiratory disease signs developed in 6 of 10 nonvaccinates, with one fatality, while 2 of 13 vaccinates showed only very mild and transient disease signs. Nonvaccinates lost weight until post challenge day (PCD) 6, did not regain prechallenge weight until PCD 8, and at PCD 11 had gained only 4.9 pounds/pig. Vaccinates gained weight after challenge and at PCD 11 showed a 9.4 pounds/pig weight gain. On PCD 11, the geometric mean titer (GMT) for VN antibodies of the nonvaccinates was 9.3, while the GMT of the vaccinates for VN antibodies was 49.0, showing that vaccinated pigs had been immunologically primed. ( view less ) S Kit,S McConnell,M Kit,B Lawhorn Maternal antibodies interfere with the immunization of swine by modified live-virus pseudorabies virus (PRV) vaccines. To test the hypothesis that a PRV vaccine attenuated by deletions in the thymidine kinase (TK) and gIII genes might reduce interference by maternal antibodies, pigs with moderate t... ( view more )o low levels of colostral PRV antibodies were immunized with the TK- gIII-OMNIMARK-PRV vaccine. Vaccinates and non-vaccinates were challenged intranasally with virulent PRV at 7 weeks of age. In support of the hypothesis, it was found that central nervous system (CNS) and/or respiratory disease developed in six out of 10 controls with a fatal outcome in one, while two out of 13 vaccinates showed only very mild and transient CNS or respiratory disease signs with no fatalities. All vaccinates gained weight while non-vaccinates initally lost weight. At post-challenge day (PCD) 11, vaccinates showed 4.5 lb/pig greater weight gain than non-vaccinates. Virus neutralization (VN) analyses before and after challenge showed that vaccinates had been primed immunologically. In another experiment, newborn pigs from a pseudorabies disease-quarantined herd with high VN antibody titres were vaccinated, respectively, with the gIII-TK-OMNIMARK-PRV vaccine, a TK-gI-gX-vaccine, or no vaccine and challenged with virulent PRV at 14 weeks of age when VN titres were < 1:2. By PCD 9, the TK-gIII-group had outgained the TK-gI-gX- and the control groups, respectively, by 6.0 and 3.2 lb per pig. ( view less ) S Kit,H Otsuka,M Kit A sensitive and specific blocking enzyme-linked immunosorbent assay (ELISA) was developed to distinguish infectious bovine rhinotracheitis virus (IBRV)-infected animals from those immunized with a glycoprotein gIII deletion mutant, IBRV(NG)dltkdlgIII. For this ELISA, undiluted test sera are used to... ( view more ) block the binding of an anti-IBRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate to gIII antigen. TMB substrate is used for color development. Negative S/N values (defined as the absorbance at 650 nm of test sera/absorbance at 650 nm of negative control sera) of > 0.80 were obtained with immune sera from gnotobiotic cattle immunized with several bovine viruses, with bovine antisera to bovine herpesvirus-2, and vesicular stomatitis virus, with porcine antisera to pseudorabies virus and parvovirus, and with normal sera from heterologous species. Negative S/N values were also obtained with sera from rabbits twice vaccinated with IBRV(NG)dltkdlgIII. However, the S/N values became positive (S/N < 0.8) 10 to 17 days after the rabbits were challenge exposed to virulent IBRV(Cooper). Most of 116 sera (84%) from feedlot cattle with virus neutralization (VN) titers of < 1:2 or < 1:4 had negative S/N values > 0.8, but 18 sera with negative VN titers had positive S/N values, consistent with observations indicating that an IBRV outbreak was occurring in one of the feedlot herds. Thirty nine sera (98%) from feedlot cattle with VN titers of 1:2 to 1:128 had positive S/N values (< 0.8). One serum with a VN titer of 1:2 had a borderline (+/-) S/N value of 0.81. After immunization with a commercial gIII-positive IBRV vaccine, 115/116 sera with VN titers of 1:2 to 1:256 had positive S/N values (< 0.8). One serum with a VN titer of 1:2 had a negative S/N value of 0.83. Serum from one vaccinated animal that failed to seroconvert after vaccination (VN < 1:4) showed a strongly positive ELISA S/N of 0.48. ( view less ) K Pity?ski,A Skawina,J Gorczyca,M Kitli?ski,Z Kitli?skiIn 50 human fetuses of the crown-rump length ranging from 88 mm to 185 mm (12 to 20 weeks), the variability of the arterial vascularization of the vermiform appendix was investigated using the injection method. 3 vascularization types were distinguished on the basis of the size and number of the su... ( view more )pplying arteries. The obtained results were compared with those reported by other authors who studied vermiform appendix vascularization in adults. In most cases the vermiform appendix is supplied by single ramus arising from ileocolic artery. ( view less ) M Kit,S Kit,S P Little,R D Di Marchi,C Gale A recombinant infectious bovine rhinotracheitis virus (IBRV) vector has been constructed to express bovine growth hormone signal sequence plus a foot-and-mouth disease virus [FMDV (O1K)] capsid protein (VP1) epitope as the N-terminal sequence of an IBRV glycoprotein gIII fusion protein on the surfa... ( view more )ce of virus infected cells and on the surface of virus particles. Sequences encoding the first 38 amino acids of IBRV gIII were deleted from the recombinant to avoid redundant glycoprotein signal sequences, but IBRV gIII epitopes detected by anti-gIII monoclonal antibodies were retained. Phenotypes were confirmed by in situ immunostaining of virus plaques with anti-FMDV peptide sera, by immunogold staining of permeabilized- and non-permeabilized infected cells, and by virus neutralization experiments with anti-FMDV peptide sera. Vaccination with the IBRV-FMDV recombinant induced protective levels of anti-FMDV antibodies in calves and protected them from challenge with virulent IBRV. ( view less ) M Kit,S Kit A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV) (Aujeszky's disease virus) -infected pigs from those immunized with a glycoprotein g92 (gIII) deletion mutant, PRV (dlg92dltk) [OMNIMARK-PRV]. This blocking ELISA test utilizes an an... ( view more )ti-PRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate, TMB for color development and a cloned PRVg92 (gIII) antigen to coat wells of microtiter test plates. Undiluted sera are used to block the binding of the mAbgIII-HRPO conjugate to the antigen. The gIII blocking ELISA is specific and has a sensitivity comparable to screening ELISA and latex agglutination tests. PRV-negative sera and sera from pigs vaccinated once, twice, or four times with the gIII-negative vaccine all showed negative S/N values of greater than 0.70 (S/N defined as the optical density at 630 nm of test sera/optical density at 630 nm of negative control sera). Sera from PRV-infected herds, sera from pigs experimentally infected with virulent PRV, and sera from pigs vaccinated with modified-live or inactivated gIII+ vaccines were positive for gIII antibodies (S/N less than 0.7). Sera from pigs experimentally infected with 200 PFU virulent PRV seroconverted to gIII+ antibodies 7-10 days postinfection. Sera from pigs vaccinated with gpX- and gI- vaccines seroconverted to gIII+ antibodies 7-8 days after vaccination. The gIII antibodies persisted after gIII+ vaccinated for at least 376 days postvaccination. Sera from pigs protected by vaccination with PRV (dlg92dltk) and then challenge exposed to virulent PRV at 21 days postvaccination showed gIII+ antibodies by 14 days postchallenge. The specificity and sensitivity of the gIII blocking ELISA assay was further demonstrated on the United States Department of Agriculture-National Veterinary Services Laboratory (USDA-NVSL) sera from the 1988 PRV check set and the 1989 gIII PRV check set by comparing the gIII blocking ELISA assay with virus neutralization, screening/verification ELISA and latex agglutination assays. ( view less ) S Kit,M Kit,R D DiMarchi,S P Little,C Gale Modified-live, attenuated infectious bovine rhinotracheitis (IBR) hybrid virus vaccines have been constructed by inserting in the major IBRV glycoprotein gIII gene chemically synthesized deoxyribonucleotide sequences encoding the bovine growth hormone signal sequence and monomeric or dimeric forms ... ( view more )of the foot and mouth disease virus (FMDV) VP 1 epitope sequences. The foreign DNA sequences were inserted at the N-terminal end of the IBRV gIII coding sequence and were driven by the IBRV gIII promoter. The sequences encoding the first 38 and the first 21 amino acids of the IBRV gIII were deleted from the hybrid viruses containing inserts of the monomeric and dimeric FMDV epitope sequences, respectively, to avoid redundant signal sequences. Plaque immunoassay experiments with guinea pig and bovine anti-FMDV peptide antisera, and with anti-IBRV gIII monoclonal antibodies demonstrated that IBRV-FMDV fusion proteins were expressed in virus-infected MDBK cells. Immunoelectron microscopy analyses demonstrated that the IBRV-FMDV fusion proteins were expressed as repeated structures on the surface of virus particles. Experiments showed that the recombinant IBRV-FMDV viruses protected cattle from IBRV (Cooper) challenge and induced anti-FMDV peptide antibodies, thereby demonstrating that the FMDV epitopes were expressed in vivo. ( view less ) S Kit,M Sheppard,H Ichimura,M Kit A modified-live pseudorabies virus (PRV) vaccine, designated PRV(dlg92/d1tk), with deletions in the thymidine kinase (tk) and glycoprotein-gIII (g92) genes, was derived from the PRV (Bucharest [BUK]-d13) vaccine strain. The vaccine virus also contained a deletion in glycoprotein gI. Despite 3 delet... ( view more )ions, PRV(dlg92/d1tk) replicated to high titers in cell culture from 30 C to 39.1 C. Enzyme assays and autoradiography revealed that PRV(dlg92/d1tk) did not induce a functional tk activity in infected tk- RAB(BU) cells (rabbit skin). Rabbit skin cells were infected with PRV(dlg92/d1tk), with vaccine strains derived from BUK or Bartha K strains of PRV or with the virulent Illinois (ILL), Indiana-Funkhauser (IND-F), and Aujeszky (Auj) strains of PRV and were labeled with [3H]mannose from 4 or 5 to 24 hours after infection to investigate whether these viruses induced the synthesis of glycoprotein gIII. Nonionic detergent extracts were prepared and immunoprecipitated with antisera from pigs vaccinated with tk(-)-PRV(BUK-d13) or tk+-Bartha K, pigs vaccinated with tk+-PRV(BUK) strains and then challenge exposed to tk+-PRV(IND-F), naturally infected domestic or feral pigs, and pigs vaccinated with tk-)-PRV(dlg92/d1tk). Mouse monoclonal antibodies against PRV glycoproteins gIII, gp50, and gII were also studied. After immunoprecipitation, labeled PRV-specific proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and autoradiography. The PRV glycoprotein-gII complex, but not glycoprotein gIII, was synthesized in PRV(dlg92/d1tk)-infected cells. Glycoprotein gII and gIII were made in cells infected with PRV vaccine strains BUK, Bartha K, and BUK-d13 and with virulent PRV strains ILL, IND-F, and Auj. Cells infected with PRV(dlg92/d1tk) and with PRV strains ILL, IND-F, Auj, Bartha K, BUK, and BUK-d13, excreted into the cell culture medium a highly sulfated glycoprotein gX of about 90 kilodaltons. Antibodies to glycoprotein gIII were not detected in the sera of pigs inoculated with PRV(dlg92/d1tk), but were found in all other swine sera. ( view less ) S Kit,M Kit,S McConnellTo test the safety and efficacy of a thymidine kinase-negative (TK-), temperature-resistant (TR) mutant of bovine herpes virus-1 (BHV-1) in pregnant cows, seronegative cows, 2-5 months pregnant, were vaccinated intramuscularly (i.m.) or intravaginally (i.vag.) with this candidate vaccine virus. I.m... ( view more ). vaccinated cows did not shed virus i.vag. or intranasally (i.n.), but i.vag. vaccinated cows replicated virus i.vag. for 8-9 days postvaccination (p.v.) Some of the cows were challenge exposed i.n. at 46 days p.v. with virulent TK+ BHV-1(Cooper). Vaccinated cows showed no clinical disease signs p.v. or postchallenge and responded anamnestically postchallenge. All cows delivered live calves. Pre-colostrum sera of the calves were negative for BHV-1 antibodies. ( view less ) S Kit,M Kit,H Ichimura,R Crandell,S McConnell The bovine herpesvirus type 4 (BHV-4) group has a slow replication cycle, a narrow host range, and cytopathogenic effects characteristic of cytomegaloviruses (CMV), but a Group B genome structure similar to that of lymphotropic Herpesvirus saimiri (HVS). Reference BHV-4 strain DN599 and BHV-4 strai... ( view more )ns N124 and FHV-2 induced in the cytosol fraction of thymidine kinase-negative (TK-) rabbit skin (RAB-BU) cell mutants a novel TK activity. The BHV-4-induced thymidine kinase (TK) differed from the principal cytosol TK of mock-infected cells in PAGE mobility (Rm) under non-denaturing conditions and in the capacity to efficiently substitute CTP for ATP as a phosphate donor. The BHV-4 thymidine phosphorylating activity could also be distinguished from many common herpesvirus-induced TKs because it lacked iododeoxycytidine phosphorylating activity. Iododeoxyuridine, trifluorothymidine and bromovinyldeoxyuridine inhibited [3H]thymidine (0.01 mM) phosphorylation by the BHV-4 enzyme in a dose-dependent manner, but arabinosylthymine and 2'-fluoro-5-methyl-arabinosyluracil (FMAU) were poor inhibitors of [3H]thymidine phosphorylation, and acyclovir and (dihydroxy-2-propoxymethyl)guanine (DHPG) did not inhibit at all at 60 and 40 times the concentrations of [3H]thymidine, respectively. ( view less ) S Kit,M Kit,E C Pirtle A thymidine kinase (TK)-negative (TK-) deletion mutant of the Bucharest (BUK) strain of pseudorabies virus (PRV) was isolated. The mutant, designated as PRV (BUK d13), did not revert to TK-positive (TK+), even when propagated in medium that selected for TK+ viruses. The mutant also replicated equal... ( view more )ly well at 39.1 C and 34.5 C, and was easily distinguished from other PRV strains by molecular hybridization experiments, restriction nuclease fingerprints, and plaque autoradiography or other assays for the TK phenotype. The PRV (BUK d13) had greatly reduced virulence for mice and rabbits, compared with parental TK+ strains, PRV (BUK-5) and PRV (BUK-5A-R1), and provided mice with solid protection against the TK+ BUK and Aujeszky strains of PRV. Experiments were done in 5- to 6-week-old pigs to assess the safety and efficacy of PRV (BUK d13) in the natural host. In one experiment, pigs were vaccinated IM with 7.5 X 10(8) plaque-forming units of TK- PRV (BUK d13), and were then challenge exposed intranasally (IN) with 4.3 X 10(8) TCID50 of virulent PRV [Indiana-Funkhauser (IND-F)]. Vaccinated pigs did not have clinical signs of illness after vaccination or after challenge exposure. One nonvaccinated control pig died on postchallenge day 4; a 2nd nonvaccinated control pig became moribund, but eventually recovered. Pigs developed virus-neutralizing antibodies after vaccination, and had a secondary immunologic response after challenge exposure; however, PRV was not isolated from the tonsils or trigeminal ganglia of vaccinated pigs at postchallenge exposure day 11.(ABSTRACT TRUNCATED AT 250 WORDS) ( view less ) S Kit,M Kit,H Qavi,D Trkula,H Otsuka To analyze the boundaries of the functional coding region of the HSV-2(333) thymidine kinase gene (TK gene), deletion mutants of hybrid plasmid pMAR401 H2G, which contains the 17.5 kbp BglII-G fragment of HSV-2 DNA, were prepared and tested for capacity to transform LM(TK-) cells to the thymidine k... ( view more )inase-positive phenotype. These studies showed that hybrid plasmids containing 2.2-2.4 kbp subfragments of HSV-2 BglII-G DNA transformed LM(TK-) cells to the thymidine kinase-positive phenotype and suggested that the region critical for transformation might be less than 2 kbp. That the activity expressed in the transformants was HSV-2 thymidine kinase was shown by experiments with type-specific enzyme-inhibiting rabbit antisera and by disc-polyacrylamide gel electrophoresis analyses. DNA fragments of the HSV-2 TK gene were subcloned in phage M13mp9 and M13mp8. A sequence of 1656 bp containing the entire coding region of the TK gene and the flanking sequences was determined by the dideoxynucleotide chain termination method. Comparisons with the HSV-1(Cl 101) TK gene revealed that PstI, PvuII, and EcoRI cleavage sites had homologous locations as did promoter, translational start and stop, and polyadenylation signals. Extensive homology was observed in the nucleotide sequence preceding the ATG translational start signal and in portions of the coding region of the genes. Comparisons of the predicted amino acid sequences of the HSV-1 and HSV-2 thymidine kinase polypeptides revealed that both were enriched in alanine, arginine, glycine, leucine, and proline residues and that clear, but interrupted homology existed within several regions of the polypeptide chains. Stretches of 15-30 amino acid residues were identical in conserved regions. The possibility is suggested that domains containing some of the conserved amino acid sequences might have a role in substrate binding and as major antigenic determinants. ( view less ) S Kit,H Otsuka,H Qavi,M Kit The recombinant plasmid pAGO contains the Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and consists of a 2-kb PvuII fragment of HSV-1 DNA inserted into the PvuII site of pBR322. A deletion mutant of pAGO, designated pMH110, has been isolated which removes the normal HSV-1 TK gene ... ( view more )promoter but places the promoter of the pBR322 tetracycline-resistance (tetr) gene only about 400 bp from the translational start codon of the HSV-1 TK polypeptide. In contrast to pAGO, which transforms mouse LM(TK-) cells to TK+ but is only weakly expressed in TK- bacteria, pMH110 not only efficiently transforms LM(TK-) cells to TK+ but also enables TK- Escherichia coli K-12 cells to form colonies on selective plates containing 5-fluorodeoxyuridine (FdUrd) plus thymidine (dThd) and to exhibit fully restored ability to incorporate [3H]dThd into DNA. The levels of TK activity expressed by bacteria harboring pMH110 were about as high as those expressed by bacteria harboring plasmid pTK3, which contains the wild-type E. coli TK gene. The TK activity expressed in bacteria harboring pMH110 was partially purified and shown to be HSV-1-specific by serological and disc PAGE analyses and by experiments demonstrating that this enzyme phosphorylated [125I]deoxycytidine. ( view less ) S Kit,G Kit Disc polyacrylamide gel electrophoresis (disc PAGE) analyses have revealed that mouse, human, and monkey cytosal deoxycytidylate (dCMP) deaminases differ in electrophoretic mobility, so that mixtures of mouse and human, mouse and monkey and human and monkey enzymes can be separated. To learn whethe... ( view more )r the genes for dCMP deaminase and thymidine (dT) kinase are genetically linked, disc PAGE analyses of cytosol fractions from human-mouse and monkey-mouse somatic cell hybrids were carried out. The interspecific somatic cell hybrids were derived from the fusion of cytosol dT kinase deficient mouse cells with cytosol dT kinase-positive human and monkey cells: they contained mostly mouse chromosomes and a few primate chromosomes, including the determinant for primate cytosol dT kinase. The disc PAGE analyses demonstrated that the human-mouse and monkey-mouse somatic cell hybrids contained a dCMP deaminase activity with an electrophoretic mobility characteristic of mouse dCMP deaminase. Enzymes with electrophoretic mobilities characteristic of human and monkey dCMP deaminases were not demonstrable. these findings suggest that primate cytosol dT kinase and dCMP deaminase are coded on different chromosomes, or that the formation in hybird cells of an active primate dCMP deaminase is suppressed. Chick-mouse somatic cell hybrids containing chick but not mouse cytosol dT kinase were also analyzed, but it was not possible to establish whether the enzyme was of murine or avian origin because of the similarity in electrophoretic mobility between the chick and mouse enzymes. Human and mouse cells contained low levels of mitochondrial dCMP deaminase activity. In contrast to dT kinase isozymes, however, mitochondrial and cytosol dCMP deaminases were electrophoretically indistinguishable. ( view less ) Chun Hay Ko,Karen Li,Pak Cheung Ng,Kwok Pui Fung,Raymond Pui-On Wong,Kit Man Chui,Goldie Jia-Shi Gu,Edmund Yung,Tai Fai Fok Glucose-6-phosphate dehydrogenase (G6PD)-deficient subjects are susceptible to chemical-induced oxidative haemolysis. Little is known concerning the haemolytic properties of Chinese herbal medicine on G6PD-deficient subjects. Our objective was to investigate the pro-oxidative effect of 18 commonly ... ( view more )used Chinese herbal medicine (CHM) on human G6PD-deficient red blood cells. G6PD-deficient (n=10) and normal (n=10) whole blood samples were incubated with water extracts of CHM. The resulting levels of reduced glutathione (GSH) and methaemoglobin (MetHb) were determined by biochemical assays. Rhizoma Coptidis significantly reduced GSH level by 48.9+/-5.4% (at 1mg/mL) in the G6PD-deficient erythrocytes (P<0.001) compared with the respective control group without challenge. Similar dose-dependent responses were observed at higher concentrations of Cortex Moutan, Radix Rehmanniae, Radix Bupleuri, Rhizoma Polygoni Cuspidati and Flos Chimonanthi (P<0.01, 5-10mg/mL). In addition, the levels of MetHb were elevated significantly when challenged with Rhizoma Coptidis (2.8 fold at 5mg/mL) and Cortex Moutan (3.4 fold at 10mg/mL). This is the first report on the pro-oxidative action of CHM on G6PD-deficient blood samples in vitro as demonstrated by the decrease of GSH and increase of MetHb. G6PD-deficient subjects should restrain from excessive consumption of these pro-oxidative herbs. ( view less )
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