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Immunohistopathological Study of Listeria Monocytogenes Infection in Pregnant Mice

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Immunohistopathological Study of Listeria Monocytogenes Infection in Pregnant Mice Acta Microbiologica et Immunologica Hungarica, 52 (Suppl.), pp. 1–184 (2005) IMMUNOHISTOPATHOLOGICAL STUDY OF LISTERIA MONOCYTOGENES INFECTION IN PREGNANT MICE MAJA ABRAM, DARINKA VUČKOVIĆ, MARINA BUBONJA, MILJENKO DORIĆ Department of Microbiology, Medical Faculty, University of Rijeka, Braće Branchetta 20, 51000 Rijeka, Croatia Listeria monocytogenes is a gram-positive, facultative intracellular pathogen that can cause severe food-borne infections in humans and animals. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Using a murine model of pregnancy-associated listeriosis we have done a careful analysis of the host immune response to L. monocytogenes infection. We studied the kinetics of bacterial clearance, histopathological changes in maternal and fetal tissues, as well as cy- tokine/chemokine pattern during the course of L. monocytogenes infection. Pregnant BALB/c mice challenged intravenously (i.v.) showed failure of maternal anti-listerial im- mune response at the systemic (significantly reduced serum IFN-g levels) and local level (reduced expression of proinflammatory cytokines and chemokines in the liver tissue), leading to devastating necrotizing hemorrhagic hepatitis. The insufficient maternal immune response also facilitated listerial multiplication in the placenta and, ultimately, in the fetus. Placental infection was characterized by large, hemorrhagic necrosis and numerous bacterial clusters. However, the inflammatory reaction in the placenta was confined to single granulocytes, while T-cells and other cellular elements necessary for effective antilisterial defence were absent. During the course of infection, TNF-a, and occasion- ally, IFN-g were transcribed in placental tissue. Increased placental levels of these anti-listerial cyto- kines were not sufficient to control bacterial growth, but may eventually contribute to spontaneous fetal loss and poor pregnancy outcome. PCR BASED STRATEGIES FOR SUBGROUP-SPECIFIC CLONING OF MAP KINASE GENES FROM FILAMENTOUS FUNGI 1 1 1,2 ATTILA L. ÁDÁM , GÁBOR KOHUT , LÁSZLÓ HORNOK 1Group of Mycology HAS, Department of Agricultural Biotechnology and Microbiology, Szent István University, Páter Károly u. 1, H-2103 Gödöllő, Hungary; 2Agricultural Biotechnology Center, Szent-Györgyi A. u. 4, H-2100 Gödöllő, Hungary MAPKs of fungi are divided into three groups: YSAPKs (yeast and fungal stress activated protein kinases), YERK1 and YERK2 (yeast and fungal extracellular regulated kinases). YERK1 and YERK2 MAPKs play an important role in the formation of pathogenesis related morphological structures, whereas members of the YSAPK subfamily are involved in stress adaptation processes. MAPKs are generally cloned by using PCR primers based on the protein kinase subdomains (I-XI) conserved in all MAPKs. The present work aimed at the development of a subgroup-specific approach, suitable for the selective cloning of YSAPK and other fungal MAPKs. Based on multiple sequence alignment of more than 40 MAPK genes from 28 fungal species, we could identify 12 new hallmark sequences in the catalytic core, specific to the YSAPK MAPK sub- family. Only five motifs, I-b, IV-b, V-b, X-b1 and X-b2 showed specific patterns of amino acid resi- dues for YSAPK MAPKs. Two of these motifs, I-b (SA[RK]DQLT) and IV-b (F[IL]SPLED[IV]) are absent in any other eukaryotic proteins. One of the YSAPK motifs, VII-a (IL[VI]NENCDL) coin- cided with a loop spanning 7β-8β sheet present in human p38α (AVNEDCEL) and ERK2 1217-8950/$ 20.00 © 2005 Akadémiai Kiadó, Budapest 2 1ST CENTRAL EUROPEAN FORUM FOR MICROBIOLOGY (LNTTCDLK, consensus in bold face) MAPKs. They may share a common function in interaction with L-X-L docking motifs of transcription factors. Degenerate primers were designed for the above motifs and tested in a nested PCR assay. We could identify 526-991 bp long YSAPK MAPK sequence tags from eight filamentous species. All new se- quence tags from these species, including Fusarium culmorum, Fusarium proliferatum and Tricho- derma harzianum (NCBI GenBank accession numbers are DQ065608, DQ071424 and DQ071423, respectively) contained a TGY dual phosphorylation motif characteristic of YSAPK MAPKs. The right and left side flanking regions of the MAPK sequence tag cloned from F. proliferatum by using the YSAPK specific primers were then isolated by single oligonucleotide nested (SON) PCR (Antal et al., Curr. Genet., 46, 240, 2004). This strategy utilizes two or more subsequent PCRs using only one sequence-specific nested primer for each run. Under low-stringency conditions, the same primer creates new specific annealing site(s) in bordering regions of the known sequence tag. Using this ap- proach, the entire copy of a YSAPK MAPK gene, named Fpmk3 was cloned from F. proliferatum. The catalytic core of the deduced FpMK3 protein showed 99.5 and 87.3 % identity with the corre- sponding regions of Osm1 and HOG1 YSAPK MAPKs from Magnaporthe grisea and Saccharomy- ces cerevisiae, respectively. Acknowledgement: Supported by grants from OTKA (T 46529 and T 43221). EFFECTS OF COMBINED TREATMENTS OF MAP AND IRRADIATION ON ALFALFA SPROUTS 1 1 1 2 RÉKA ÁGOSTON , CSILLA MOHÁCSI-FARKAS , GABRIELLA KISKÓ , ISTVÁN DALMADI 1Department of Microbiology and Biotechnology, Corvinus University of Budapest; Somlói út 14-16, H-1118 Budapest, Hungary; 2Department of Refrigeration and Livestock Products Technology, Corvinus University of Budapest; Ménesi út 43-45, H-1118 Budapest, Hungary Sprouts represent a specific issue because the sprouting procedure (conducted under high humidity at higher/elevated temperatures) is extremely favourable for the growth of bacterial pathogens. The be- haviour of vegetable-associated pathogens after irradiation and MAP treatment will help to determine the applicability of these preservation treatments to these products as an antimicrobial intervention. Examination of the effect of irradiation on the survival of foodborne pathogens, such as Listeria monocytogenes and Bacillus cereus strains, inoculated on sprouts to improve the microbiological safety of these products has been carried out in the frame of an IAEA co-ordinated international pro- ject (11619/R0). Two different gas mixtures were applied in MAP: 2% O2, 4% CO2, 96% N2 (1) and 3-5% O2, 10-15% CO2 balanced with N2 (2). Besides the effect of MAP, we examined the effect of the combination of low dose irradiation (1 and 2 kGy) and MAP on raw and inoculated alfalfa sprouts. Samples were stored at 5°C for 10 days. Microbiological and gas composition analysis were periodically carried out in triplicates. For the determination of radiation survivals, parallel with selective plating, Thin Agar Layer (TAL) method was performed to promote the recovery of the sublethally demaged cells. Be- cause the traditional detection methods are labour- and time consuming, experiments were carried out to adapt a new, rapid impedimetric method for potential detection of Listeria monocytogenes on treated sprouts. Non-inoculated samples were used for sensory testing; after the combined treatment and at the end of the storage, panels of judges rated the samples on the basis of hedonic scores on colour, odour, taste and texture. Furthermore, another aim was to examine a possible relationship be- tween the electronic nose results and irradiation doses. The oxigen concentration was reduced to zero during storage at the 2nd (1) and 6th (2) days, respec- tively. Under the applied atmospheres, irradiation reduced the initial levels of the examined patho- Acta Microbiologica et Immunologica Hungarica 52, 2005 ABSTRACTS 3 gens and that of the total microflora. During storage, L. monocytogenes, and mainly lactic acid bacte- ria from the natural microbiota regrew both on the irradiated and on the control samples. There were no differences between the selective plating and the TAL methods. The developed impedimetric method can be used to detect and enumerate L. monocytogenes present in numbers higher than 3 log CFU/g within 24 hours. The sensory analysis showed no significant differences between treated samples. Results of electronic nose investigations indicated a significant difference between control (samples packed in air) and treated samples (MAP and MAP+ irradiation) at the day of the irradiation treatment. Combination of low dose gamma-irradiation with modified atmosphere packaging and refrigerated storage can improve the microbiological safety and shelflife of alfalfa sprouts. Further investigations are necessary to develop the composition of head-space in MAP to be able to prevent regrowth of surviving pathogens during storage. CONSPIRACY THEORY ON MAPK PATHWAY ELEMENTS AS INHIBITORS OF HIV-1 VPR PROTEIN JUDIT ANTAL, MIKLÓS PESTI Department of General and Environmental Microbiology, University of Pécs, Ifjúság ú. 6, H-7624 Pécs, Hungary HIV-1 is the most common type of the HIV viruses, and, similarly to SIV and other lentiviruses, a 96 -amino acid, 15 kDa virion associated protein Vpr is encoded by the viral genome. HIV-1 Vpr has been shown before to be responsible for G2/M cell cycle arrest which helps the viral replication cycle and enhances virus-associated pathology. Induction of Vpr expression causes a number of cellular disfunctions including disruption in nuclear envelope structure, trans-activation of the viral promoter, and induction of apoptosis both in human and fission yeast cells. Some of these functions are thought to be caused
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