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  • Author or Editor: I. Fodor x
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Two indirect ELISAs for the detection of antibodies against glycoprotein E (gE) of Aujeszky's disease virus (ADV) in sera have been developed. The rec-gE-ELISA is based on theE. coli-expressed recombinant protein containing the N-terminal sequences of gE (aa 1-125) fused with the glutathione S-transferase fromSchistosoma japonicum. The affi-gE-ELISA is based on native gE, which was purified from virions by affinity chromatography. The tests were optimised and compared with each other, as well as with the recently developed blocking gE-ELISA (Morenkov et al., 1997b), with respect to specificity and sensitivity. The rec-gE-ELISA was less sensitive in detecting ADV-infected animals than the affi-gE-ELISA (sensitivity 80% and 97%, respectively), which is probably due to the lack of conformation-dependent immunodominant epitopes on the recombinant protein expressed inE. coli. The specificity of the rec-gE-ELISA and affi-gE-ELISA was rather moderate (90% and 94%, respectively) because it was necessary to set such cut-off values in the tests that provided a maximum level of sensitivity, which obviously increased the incidence of false positive reactions. Though the indirect ELISAs detect antibodies against many epitopes of gE, the blocking gE-ELISA, which detects antibodies against only one immunodominant epitope of gE, showed a better test performance (specificity 99% and sensitivity 98%). This is most probably due to rather high dilutions of the sera used in the indirect gE-ELISAs (1:30) as compared to the serum dilution in the blocking gE-ELISA (1:2). We conclude that the indirect gE-ELISAs are sufficiently specific and sensitive to distinguish ADV-infected swine from those vaccinated with gE-negative vaccine and can be useful, in particularly affi-gE-ELISA, as additional tests for the detection of antibodies to gE.

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Authors: Nadja Fodor, S. K. Dube, I. Fodor, E. Horváth, Edith Nagy, V. N. Vakharia and Altancsimeg Rencendorsh

Direct DNA inoculations were used to determine the efficacy of gene immunisation of chickens to elicit protective immune responses against infectious bursal disease virus (IBDV). Thevp2 gene of IBDV strains GP40 and D78, and thevp2-vp4-vp3 encoding segment of strain D78 were cloned in an expression vector which consisted of human cytomegalovirus (HCMV) immediate early enhancer and promoter, adenovirus tripartite leader sequences and SV40 polyadenylation signal. For purification of vaccine-quality plasmid DNA fromE. coli, an effective method was developed. Chickens were vaccinated by inoculation of DNA by two routes (intramuscular and intraperitoneal). Two weeks later, chickens were boosted with DNA, and at 2 weeks post-boost, they were challenged with virulent IBDV strain. Low to undetectable levels of IBDV-specific antibodies and no protection were observed with DNA encoding VP2. However, plasmids encoding VP2-VP4-VP3 induced IBDV-specific antibodies and protection in the chickens. DNA immunisation opens a new approach to the development of gene vaccines for chickens against infectious diseases.

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In vitro antimicrobial sensitivity of 12 Hungarian isolates and the type strain ATCC 33144 of Actinobaculum suis to different antimicrobial compounds was determined both by the agar dilution and by the disc diffusion method. By agar dilution, MIC50 values in the range of 0.05-3.125µg/ml were determined for penicillin, ampicillin, ceftiofur, doxycycline, tylosin, pleuromutilins, chloramphenicol, florfenicol, enrofloxacin and lincomycin. The MIC50 value of oxytetracycline and spectinomycin was 6.25 and 12.5µg/ml, respectively. For ofloxacin, flumequine, neomycin, streptomycin, gentamicin, nalidixic acid, nitrofurantoin and sulphamethoxazole + trimethoprim MIC50 values were in the range of 25-100µg/ml. With the disc diffusion method, all strains were sensitive to penicillin, cephalosporins examined, chloramphenicol and florfenicol, tetracyclines examined, pleuromutilins, lincomycin and tylosin. Variable sensitivity was observed for fluoroquinolones (flumequine, enrofloxacin, ofloxacin), most of the strains were susceptible to marbofloxacin. Almost all strains were resistant to aminoglycosides but most of them were sensitive to spectinomycin. A strong correlation was determined for disc diffusion and MIC results (Spearman's rho 0.789, p<0001). MIC values of the type strain and MIC50 values of other tested strains did not differ significantly. Few strains showed a partially distinct resistance pattern for erythromycin, lincomycin and ampicillin in both methods.

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Previously, we sequenced the HSV-1 Ul39-Ul40 homologue genes of Aujeszky's disease virus (ADV), also designated as pseudorabies virus (Kaliman et al., 1994ab). Now we report the nucleotide sequence of the adjacent DNA that encodes Ul38, the 5'-region (750 bp) of Ul37, and the promoter regions between these divergently arranged two genes. The ADV Ul38 gene encodes a protein of 368 amino acids. Amino acid sequence comparison of ADV Ul38 with that of other herpesviruses revealed significantstructural homology. In a transcription study using RNase protection assay and Northern blot hybridizationwe found that the Ul38 gene had one initiation site, but the Ul37 gene was initiated at two transcription sites with two potential initiator AUGs, one of which was dominant. Comparison of ADV Ul37, Ul38 and ribonucleotide reductase gene expression showed that these genes belong to the same temporal class with early kinetics. Data of structural and transcriptional studies suggest that regulation of the expression of these two ADV genes could differ from that of the HSV-1 virus.

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Abstract  

In order to determine the radiochemical impurities in pertechnetate solution as well as that of unbound99mTc in its colloid and complex compounds, in indium chloride solution and related compounds, paper chromatography on Whatman No. 1, thin-layer chromatography on silica gel plates, and paper electrophoresis were applied. A simple method for the determination of radionuclidic purity was developed.

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In order to improve the isolation rate of Rhodococcus equi from animals and soil, the efficacy of four previously described selective media (CAZ-NB, M3T, NANAT and TINSDALE) and that of four other media (NC, PNP, TCP and TVP) composed by us was compared and evaluated. Two selective plating media proved to be the best for the isolation of R. equi from contaminated samples. One of them was CAZ-NB containing ceftazidime, novobiocin and cycloheximide, while the other was the newly composed TCP containing trimethoprim, cefoperazone, polymyxin B, cycloheximide and potassium tellurite as selective components. These two media allowed the growth of at least 62-72% of R. equi present in the artificially contaminated samples, and the inhibition of unwanted contaminant bacteria and fungi was satisfactory with both media. TCP medium proved to be superior to CAZ-NB since the colony morphology of R. equi was much more characteristic (shiny, smooth, black colonies 3-5 mm in diameter) on it, and it inhibited the unwanted contaminant bacterial and fungal flora more effectively, especially in the case of faecal and soil samples. Therefore, TCP is recommended as a new, highly selective plating medium for the isolation of R. equi from contaminated samples.

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Authors: I. Bata-Vidács, A. Lugasi, J. Farkas, O. Németh and P. Fodor
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Authors: E Burgettiné Böszörményi, S Németh, A Fodor, K Bélafiné Bakó, D Vozik, Z Csima and I Barcs

Introduction

The prevalence of invasive fungal diseases shows an increasing trend. Due to the frequent but unprofessional usage of antifungal medications, the fungi show decreasing susceptibility towards these agents and this trend may lead to the emergence of resistant pathogens. There is a great need to develop antifungal medications with new mechanisms. One of these options is to apply proteins with natural antifungal effects. The objective was to measure the antifungal efficacy of Xenorhabdus budapestensis in vitro on clinical Candida species (Candida albicans, Candida lusitaniae, Candida krusei, Candida kefyr, Candida tropicalis, and Candida glabrata). Materials and methods: We defined the sensitivity of the Candida species towards antibiotics. We conducted agar diffusion tests with the cleaned biopreparation of X. budapestensis (100%) and its dilutions (80%, 60%, 40%, and 20%). Zones of inhibition were measured after 24, 48, and 96 hr.

Results

Most of the tested Candida species have shown sensitivity to the biopreparation and its 40% dilution. The area of the zones of inhibition did not decrease after several days. The most sensitive species was C. lusitaniae and the least sensitive was C. krusei.

Conclusion

We assume that the proteins produced by X. budapestensis have antifungal effect, as the area of the zones of inhibition did not change.

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Authors: J Fodor, A Gomba-Tóth, T Oláh, E Zádor, Zs Cs Tóth, I Ioannis, B Molnár, I Kovács and L Csernoch

Atherosclerosis is a disease caused by a build-up of fatty plaques and cholesterol in the arteries. The lumen of the vessels is obliterated resulting in restricted blood supply to tissues. In ischemic conditions, the cytosolic Ca2+ level of skeletal muscle may increase, indicating the alteration of Ca2+ removal mechanisms. Ca2+ is transported from cytosol into the sarcoplasmic reticulum by Ca2+ ATPase (SERCA), with its 1a isoform expressed in adult, while its 1b isoform in neonatal and regenerating fast-twitch skeletal muscle. To investigate the role of these isoforms in ischemic skeletal muscle, biopsies from musculus biceps femoris of patients who underwent amputation due to atherosclerosis were examined. Samples were removed from the visibly healthy and hypoxia-affected tissue. Significantly increased SERCA1a expression was detected under the ischemic conditions (246 ± 69%; p < 0.05) compared with the healthy tissue. Furthermore, the ratio of SERCA1a-positive fibers was slightly increased (46 ± 4% in healthy tissue and 60 ± 5% in ischemic tissue; p > 0.05), whereas SERCA2a did not change. In addition, in primary cultures derived from hypoxia-affected tissue, the diameter and fusion index of myotubes were significantly increased (30 ± 1.6 µm vs. 41 ± 2.4 µm and 31 ± 4% vs. 45 ± 3%; p < 0.05). We propose that the increased SERCA1a expression indicates the existence and location of compensating mechanisms in ischemic muscle.

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Authors: Anna Tóth, Katalin Fodor, P. Blazsó, I. Cserpán, Tünde Praznovszky, V. Tubak, A. Udvardy, Gy. Hadlaczky and R. Katona

Direct reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPS) was achieved recently by overexpression of four transcription factors encoded by retroviral vectors. Most of the virus vectors, however, may cause insertional mutagenesis in the host genome and may also induce tumor formation. Therefore, it is very important to discover novel and safer, non-viral reprogramming methods. Here we describe the reprogramming of somatic cells into iPS cells by a novel protein-based technique. Engineered Oct4, Sox2 and Klf4 transcription factors carrying an N-terminal Flag-tag and a C-terminal polyarginine tail were synthesized by a recently described mammalian artificial chromosome expression system (ACEs). This system is suitable for the high-level production of recombinant proteins in mammalian tissue culture cells. Recombinant proteins produced in this system contain all the post-translational modifications essential for the stability and the authentic function of the proteins. The engineered Oct4, Sox2 and Klf4 proteins efficiently induced the reprogramming of mouse embryonic fibroblasts by means of protein transduction. This novel method allows for the generation of iPS cells, which may be suitable for therapeutic applications in the future.

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