Nyilasi, Andrea

English

Nyilasi Andrea

University of Szeged

V o l u m e 5 3 ( 1 ) : 5 5 - 7 6 , 2 0 0 9 A c t a B i o l o g i c a S z e g e d i e n s i s http://www.sci.u-szeged.hu/ABS junc t ions . T h e tight junc t ions are composed of t r ansmembrane proteins (occludins. c laudins. junc t ional adhesion molecules) connected to junct ional p laque proteins (i.e. Z O - l ) . The t r ansmembrane proteins of the adherens junct ions are the cadher ins linked through catenins (a lpha, beta, g a m m a ) to the cytoskeleton. T h e blood-brain barrier is involved in a large variety of pathological processes. Little is known about the e f fec ts of nicotine exposure on B B B funct ion . We investigated the changes affect ing the light and adherens junct ion proteins by cigaret te smoke components , especially nicot ine and polyaromat ic hydrocarbons (PAHs). 24h t reatment of cerebral endothelial cel ls with relatively high concentrat ion nicotine led to a decrease in occ ludin . cadher in and Z O - l express ion. Similar but less pronounced ef fec ts were observed af ter 24 h treatment with phenanthrene . Resul ts of the immunof luorescen i analysis conf i rmed western blot data. We also per fo rmed two dimensional electrophoresis in order to explore the cel lular proteins responsive to nicotine and PAHs in brain endothelial cells. We observed different responses of the cells to both nicotine and phenanthrene treatment resulting in altered expression of shock induced proteins, metabol ic enzymes , signaling molecules . Th is conf i rms the cerebral endothe l ium as being a target to cigarette smoke components (Hutamekal in et al. 2008). From clinical point of view, because of the relative impermeabil i ty of the barrier many drugs are unable to reach the C N S in therapeuti-cally relevant concentra t ion, m a k i n g the BBB one of the ma jo r impediments in the treatment of C N S disorders. A number of strategies have been developed to c i rcumvent this problem. One of the successful ly used methods to deliver drugs - especial ly ant i tumorai agents - to the C N S is the osmot ic open ing of the B B B using manni tol . Th is causes a rapid opening (within minutes) of the B B B which is reversible. We investigated the e f f ec t of manni to l t reatment on brain endothel ia l cells and found that manni to l induced a rapid, concentrat ion dependen t , and reversible tyrosine phosphoryla t ion of a broad range of proteins between 50 and 190 kDa. One of the targets of tyrosine phosphoryla t ion turned out to be the adherens junc t ion protein beta-catenin and this phosphorylat ion was Src-kinase dependent (Farkas et al. 2005) . Beside beta-catenin and Src kinase , w e a imed to find new signaling pa thways activated by hypertonici ty in cerebral endothelial cel ls and identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmot ic mannitol . Besides activation. Axl was a lso cleaved in response to osmot ic stress. Specif ic knockdown of Axl increased the rate of apoptos is in hyperosmot ic mannitol-t reated cel ls : therefore , w e a s s u m e that activation of Axl may be a protective mechan i sm against hyper tonic i ty- induced apoptosis . Our results ident i fy Axl as an important e lement of osmot ic s tress- induced signalling. (Wilhelm et al. 2008) . Farkas A. Szatmári E. Orbók A. Wilhelm I. Wejksza K. Nagyoszi P, Hutamekalin P. Bauer H. Bauer HC. Traweger A. Krizbai IA (2005) Hyperosmotic mannitol induces Src kinase-depcndent phosphorylation of beta-catenin in cerebral endothelial cells. J Neurosci Res 80(6):855-86l. Hutamekalin P. Farkas AE. Orbók A. Wilhelm I. Nagyoszi P. Veszelka S. Deli MA. Búzás K. Hunyadi-Gulyás E. Medzihradszky KF. Meksuriyen D. Krizbai IA (2008) Effect of nicotine and polyaromtic hydrocarbons on cerebral endothelial cells. Cell Biol Int 32(2): 198-209. Wilhelm I. Nagyoszi P. Farkas AE. Couraud PO. Romero IA. Weksler B. Fazakas C. Dung NT. Bottka S. Bauer H. Bauer HC. Krizbai IA (2008) Hyperosmotic stress induces Axl activation and cleavage in cerebral endothelial cells. J Neurochem I07(l):l 16-126. Supervisor: István Krizbai Email: nagyoszi@brc.hu Investigation of the maturation of NiFe hydrogenases in Thiocapsa roseopersicina Andrea Nyilasi Depar tment of Biotechnology, University of Szeged, Szeged, Hungary O u r model organism, Thiocapsa roseopersicina BBS is an anaerobic, phototrophic purple sulfur bacterium. There are at least two membrane-bound ( H y n S L and H u p S L ) and one soluble ( H o x E F U Y H ) [NiFe] hydrogenases in the cells. A typical [NiFe] hydrogenase is composed of a large and a small subuni t . The large subunit harbors a specific NiFe catalytic metal locenter associated with C O and C N ligands (Volbeda et al. 1995.) The maturat ion of these complex e n z y m e s require numerous accessory proteins. Most of these auxil iary genes were found using t ransposon mutagenes is , one of them was the hupK gene . (Mar6ti et al. 2003.) The product of this gene, the HupK protein is present only in o rgan i sms conta in ing at least o n e membrane -bound [NiFe] hydrogenase enzyme. T h e role of HupK is not known yet. In order to investigate the role of this protein. MiupK mutant strains were created, then the hy-d rogenase act ivi t ies of the wild type and the mutant s t rains were compared . The results clearly showed that HupK protein is important for the format ion of the funct ional ly active membrane -bound hydrogenases . but not for the biosynthesis of the soluble enzyme. (Maroti et al. 2003. ) More detai led informat ion can be obtained f rom biochemical experiments. Special expression vector was used to produce active, tagged HupK protein in h o m o l o g o u s host . The tagged HupK protein was purified under mild condi t ions to retain all protein-protein interactions and the copur i f ied proteins were analyzed by m a s s spectrometry . From cells grown under standard condi t ions, only one protein partner, namely the G r o E L chaperon in cou ld be fished out. T h e specif ic role of GroEL in the hydrogenase maturat ion is not likely, therefore al-ternat ive growth condi t ions were used to find the specif ic partners. Nickel starvation of the cells is supposed to result in the accumulat ion of the in termedia tes of the post translat ional process. Therefore , the tagged HupK protein was purified f rom such cells, however only one co-pur i fy ing band was observed: the PuhA protein, which is the H subunit of the photosynthet ic reaction centre. 70 V o l u m e 5 3 ( 1 ) : 5 5 - 7 6 , 2 0 0 9 A c t a B i o l o g i c a S z e g e d i e n s i s http://www.sci.u-szeged.hu/ABS Metal content determinat ion of the purified HupK protein f rom homologous host was per formed. T h e H y p K p r o t e i n - w a s s ty jwn. tq contain nickel a tom in 1:2 molar ratio, and no Fe a tom was detected in the sample . In order to determine, which amino acids assist in binding the nickel a tom, the sequences of the HupK and the large subunits of the hydrogenases 'Wére compared /Cbnse rv r t l regions Cdulcf be recognized at the N- and C-termini . while the middle pan of the proteins was variable. The al ignment uncovered two conserved cysteine residues as candidates fo r coordinat ion of the metal. One of them is in the R-X-F-X-X-C motif at the amino terminus , otHér'órie is'ftftíife1 D-P-C-X-X-F motif at the carboxyl terminus. In order to examine the putative role of these residues. siteTdirected mutagenes i s were per-fo rmed and the effects of the mutat ions were monitored via the hydrogenase activties of the membrane-associa ted hydrogenases . A mutant carrying alanine instead of Cys378 had only 6 5 % activity of the wild-type strain. However , the replacement of the Cys54 by alanine led to a considerable reduct ion in the hydrogenase activity (to 2 5 % of the wild type level). Ni content investigation of the C y s 5 4 A l a mutant HupK protein revealed that it conta ins the s a m e amoun t of Ni a tom like the wi ld- type protein. T h e R-X-C-X-X-C and the D-P-C-X-X-C sequences in the large subunits of NiFe hydrogenases have been shown to be essential fo r their activity and the cyste ine residues have been proposed to form a coordinat ion sphere sur rounding the NiFe cen te r (Przybyla et al. 1992, Volbeda et al . ,1995.) . In the HupK protein, phenylalanines subtitute the first and the last cysteins. In order to confe r the mot i f s of the hydrogenas large subunit on H u p K , the two phenylalanines were replaced by cysteines. T h e ef fec t of these mutat ions on the b iosynthes is of the membrane -bound hydrogenase is be ing investigated. Maróti G. Fodor BD. Rákhely G. Kovács AT. Arvani S. Kovács KL (2003) Accessory proteins functioning selectively and pleiotropically in the biosynthesis of (NiFe] hydrogenases in Thiocapsa roseopersicina. Eur. J Biochem 270( I0):2218-227. Przybyla AE. Robbins J. Menon N. Peck HD Jr. (1992) Structure-function relationships among the nickel-containing hydrogenases. FEMS Microbiol Rev 8(2): 109-135 Volbeda A. Charon MH. Piras C, Hatchikian EC, Frey M. Fontecilla-Camps JC 11995) Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas. Nature 373(6515):580-587. Supervisors: Gábor Rákhely. Kornél Kovács E-mail: nyilasi@brc.hu Anthropological Analysis of the Medieval Cemetery of 'Szeged-Vár' Krisztina Prazsák-Hajnal Department of Anthropology, University of Szeged, Szeged, Hungary The environs of Szeged was a populated area f rom the primitive age; archeological finds prove there were Roman inhabi tants at the t ime of Roman Empire , and later habitation dur ing the great migrations of Huns. Gep ids and Avars. This area was important because this is where Maros runs into the Tisza River, the Maros River be ing an excellent and cheap transport possibility of salt f rom Transylvania . The first mention of the Medieval town is f rom 1246. As a result of the Turkish occupat ion of Hungary limited data is available, making the archeological excavation of Szeged-Vár very important. The Medieval cemetery of Szeged-Vár was used f rom the Hungarian conquest to 1543. and f rom 1686 until 1713. The excavat ions have been going on since 1999. and by now approximate ly 700 graves have been excavated, along with some objects and crypts . In this study, w e have researched 425 graves excavated between 1999 and 2004. The basis of the anthropological analysis was the determinat ion of sex and age of death , inclusion metric data, and paleopathological and taxonomica l analysis. To determine these data, w e have used c o m m o n anthropological methods . Paleopathological and taxonomica l examinat ions have been carried out us ing m a c r o m o r p h o -logical methods, though in certain cases radiographical analysis was also applied. Af te r the determinat ion of sex and age, we could establish that in this populat ion the sex ratio was 50%-50%; the percentage of infants ( INF1 . INF2, J U V ) was 4 9 % , and elderly (SEN) 7%. By means of the measu remen t s of humerus , radius, ulna, tibia and f emur w e determined the height of people. T h e average of the height of adult males was 170 c m . adult females 160 c m : the highest was 181 c m both a m o n g males and females , the m i n i m u m height was 157.5 c m a m o n g males and 147.7 cm a m o n g females . In accordance with general medieval health, many of the skeletons showed di f ferent f o r m s of paleopathological lesions: periostitis, os teomyeli t is , arthritis; minor developmental anomalies : sacralisation. lumbarisat ion, spina bifida, dislocation of the hip: t raumas: f rac-tures of humerus , radius, ulna, r ibs or clavicle. There also were infect ious bony lesions due to T B and syphilis. We found some metabol ic dis turbances of bone: os teoporosis ; and circulatory and hematologic disorders as well: cribra orbitalia and cribra crani i . The taxonomical analysis could yield s o m e very interesting informat ion because there is no such data available for this town. T h e r e are some finds in the area of a ' K u n ' population ( for example in Kiskundorozsma) . and we suppose there was a Mongol id populat ion in Szeged af ter the Hungarian conques t . For taxonomical analysis w e have to research anatomical variations like sutura metopica , os Wormiana . torus palatínus, torus mandibu lae and maxil lae, fossa canina and several dental variat ions. Th is presentat ion is fo r the preliminary results only. Supervisors: Antónia Marcsik, György Pálfi E-mail: aurorakr@citromail.hu 71 

Investigation of the maturation of NiFe hydrogenases in Thiocapsa roseopersicina

http://acta.bibl.u-szeged.hu/23030/1/biologica_053_numb_001_070-071.pdf

Investigation of the maturation of NiFe hydrogenases in Thiocapsa roseopersicina

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