Dynamic associations of transcription factors with the rat liver nuclear matrix are functionally related to differential alpha-2-macroglobulin gene expression

Participation of the nuclear matrix in regulation of alpha-2-macroglobulin (α2M) gene transcription during rat liver development and the acute-phase (AP) response are examined. DNA affinity chromatography of fetal and adult liver internal nuclear matrix proteins under basal and AP conditions with the α2M gene promoter (-852/+12) and immunoblot analysis revealed diverse patterns of association of transcription factors with the nuclear matrix. HNF-6, C/EBPα, and STAT5b were involved in basal and C/EBPβ, STAT1, and STAT3 in AP-stimulated α2M expression. These findings support the assumption that transcription factor-nuclear matrix interactions serve to channel gene regulatory proteins to DNA sequences.Cilj rada je ispitivanje učešća jedarnog matriksa u regulaciji transkripcije gena za alfa-2-makroglobulin tokom razvića jetre pacova i akutno faznog odgovora (AFO). Nakon DNK afinitetne hromatografije proteina unutrašnje mreže jedarnog matriksa fetalne i adultne jetre, u bazalnim i AFO uslovima, sa promotorskim elementom gena za α2M (-852/+12) i imunoblot analize, identifikovane su dinamičke asocijacije transkripcionih faktora uključenih u regulaciju ekspresije gena za α2M sa jedarnim matriksom. HNF-6, C/EBPα, STAT5b su uključeni u regulaciju bazalne ekspresije gena za α2M, dok C/EBPβ, STAT1, STAT3 posreduju u regulaciji ekspresije ovog gena tokom AFO. Opisane interakcije doprinose razumevanju predloženih mehanizama kojima se transkripcioni faktori usmeravaju ka ciljnim regulatornim elementima DNK.Projekat ministarstva br. 143002

GABA-Producing Natural Dairy Isolate From Artisanal Zlatar Cheese Attenuates Gut Inflammation and Strengthens Gut Epithelial Barrier in vitro

Probiotic bacteria are recognized for their health-promoting properties, including maintenance of gut epithelial integrity and host immune system homeostasis. Taking into account the beneficial health-promoting effects of GABA, the presence of the gadB gene, encoding glutamate decarboxylase that converts L-glutamate to GABA, was analyzed in Lactic Acid Bacteria (LAB) natural isolates from Zlatar cheese. The results revealed that 52% of tested Lactobacillus spp. and 8% of Lactococcus spp. isolates harbor the gadB gene. Qualitative and quantitative analysis of GABA production performed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) revealed the highest GABA production by Lactobacillus brevis BGZLS10-17. Since high GABA-producing LAB natural isolates are the most valuable source of naturally produced GABA, the probiotic properties of BGZLS10-17 were characterized. This study demonstrated high adhesion of BGZLS10-17 strain to Caco2 cells and the ability to decrease the adhesion of Escherichia coli ATCC25922 and Salmonella enterica C29039. Treatment of differentiated Caco-2 cells monolayer with BGZLS10-17 supernatant containing GABA alleviated inflammation (production of IL-8) caused by IL-1 beta and significantly stimulated the expression of tight junction proteins (zonulin, occludin, and claudin 4), as well as the expression of TGF-b cytokine leading to the conclusion that immunosuppression and strengthening the tight junctions can have significant role in the maintenance of intestinal epithelial barrier integrity. Taken together the results obtained in this study support the idea that using of GABA producing BGZLS10-17 probiotic strain could be a good strategy to modulate immunological response in various inflammatory diseases, and at the same time, it could be a good candidate for adjunct starter culture for production of GABA-enriched dairy foods and beverages offering new perspectives in designing the novel functional foods

Transcription factor p53 exhibits increased binding to the A2-macroglobulin gene promoter and decreased glycosylation in fetal and adult rat liver during the acute-phase response

The binding affinity of p53 for the MG promoter was assessed by DNA-affinity chromatography with the extended α2-macroglobulin (MG) gene promoter (-852/+12) and immunoblot analysis. During the increased MG gene transcription observed in the fetus and the acute-phase (AP) response in both the fetus and the adult, p53 exhibited increased binding to the MG promoter. This increase was accompanied by decreased O-linked N-acetyl glucosamine glycosylation of p53. We suggest that the enzymatic removal of sugar moieties in vivo serves to activate the MG gene promoter binding potential of p53 and its participation in upregulated MG gene transcription.DNK afinitetna hromatografija, sa promotorskim regionom gena za alfa2-makroglobulin (MG) (-852/+12), i imunoblot analiza su pokazale povećan afinitet vezivanja p53 za promotorski region gena za MG u kontrolnoj jetri fetusa, kao i ulogup53 u transkripcionoj regulaciji gena za MG i u fetalnoj i adultnoj jetri tokom akutno-faznog odgovora (AFO). Povećanje vezivanja p53 za promotorski region gena za MG je praćeno smanjenjem stepena glikozilacije p53. Rezultati sugerišu da u in vivo uslovima uklanjanje šećernih ostataka sa p53 omogućava njegovo vezivanje za promotorski region MG gena, kao i ulogu u transkripcionoj aktivaciji ispitivanog gena tokom AFO.Projekat ministarstva br. 143002

The radioprotective efficacy of the rat acute-phase protein alpha2-macroglobulin on bone marrow cells

The rat acute phase protein α2-macroglobulin (α2M) plays an important role in the restoration of disrupted homeostasis by inhibiting different types of non-specific proteases and facilitating the transport of cytokines, growth factors and hormones. Previously, we observed that administration of α2M to experimental animals prior to the infliction of life- threatening trauma in the form of scalding or total-body irradiation, significantly improved their survival rates. The aim of the present work was to evaluate the radioprotective effect on blood cells of α2M that, when administered 30 min before irradiation with 6.7 Gy (LD50/30), provides 100% survival of experimental animals where in unprotected irradiated rats the said dose results in 50% lethality. We observed that rats pretreated with α2M, after an initial decline, exhibited complete recovery of the leukocyte count due to the preservation of bone marrow cells, observed as a stable mitotic index. In untreated irradiated rats the decrease of the mitotic index reflected the significant destruction of bone marrow cells that resulted in a protracted decline in the leukocyte count. We conclude that the radioprotection provided by α2M was in part mediated through cytoprotection of new blood cells produced in the bone marrow.Pacovski alfa2-makroglobulin (α2M) ima važnu ulogu u uspostavljanju narušene homeostaze inhibicijom različitih tipova nespecifičnih proteaza i olakšavajući transport citokina, hormona rasta i hormona. Naša ranija istraživanja su pokazala da administracija α2M eksperimentalnim životinjama u traumama tipa opekotine ili ozračivanja celog organizma značajno povećava njihovu stopu preživljavanja. Cilj ove studije je bio izučavanje radioprotektivne uloge α2M na ćelije kosne srži. α2M je apliciran 30 minuta pre ozračivanja pacova dozom od 6.7 Gy (LD50/30) X-zraka i omogućio je 100% preživljavanje pacova za razliku od ozračenih pacova bez tretmana kod kojih je smrtnost bila 50%. Rezultati su pokazali da tretiranje životinja sa α2M, nakon inicijalnog pada, omogućavaju potpuni oporavak broja leukocita kao posledica očuvanja ćelija kosne srži, što se uočava preko stabilnog mitotskog indeksa. Kod ozračenih pacova bez tretmana signifikantno smanjenje mitotskog indeksa, kao posledica narušavanja ćelija kosne srži, rezultuje i u prolongiranom padu broja leukocita. Na osnovu ovih rezultata može se zaključiti da se radioprotektivna uloga α2M delom odvija putem citoprotekcije novih krvnih ćelija u kosnoj srži.Projekat ministarstva br. 143002

GABA potentiate the immunoregulatory effects of Lactobacillus brevis BGZLS10-17 via ATG5-dependent autophagy in vitro

The characterization of mechanisms involved in the positive effects of probiotic bacteria in various pathophysiological conditions is a prerogative for their safe and efficient application in biomedicine. We have investigated the immunological effects of live bacteria-free supernatant collected from GABA-producing Lactobacillus brevis BGZLS10-17 on Concanavalin A-stimulated mesenteric lymph node cells (MLNC), an in vitro model of activated immune cells. We have shown that GABA containing and GABA-free supernatant of Lactobacillus brevis BGZLS10-17 have strong immunoregulatory effects on MLNC. Further, GABA produced by this strain exhibit additional inhibitory effects on proliferation, IFN-gamma and IL-17 production by MLNC, and the expression of MHCII and CD80 on antigen presenting cells. At the other hand, GABA-containing supernatants displayed the strongest stimulatory effects on the expression of immunoregulatory molecules, such as Foxp3(+), IL-10, TGF-beta, CTLA4 and SIRP-alpha. By looking for the mechanisms of actions, we found that supernatants produced by BGZLS10-17 induce autophagy in different MLNC, such as CD4(+) and CD8(+) T lymphocytes, NK and NKT cells, as well as antigen presenting cells. Further, we showed that the stimulation of Foxp3(+), IL-10 and TGF-beta expression by BGZLS10-17 produced GABA is completely mediated by the induction of ATG5 dependent autophagy, and that other molecules in the supernatants display GABA-, ATG5-, Foxp3(+)-, IL-10- and TGF-beta- independent, immunoregulatory effects

Expression of CYP1A in the hepatopancreas of Merluccius merluccius, Trigla lucerna, and Liza ramada (pisces) in the wider vicinity of Bar harbor Montenegro

The expression of CYP1A, a biomarker for the presence of xenobiotic compounds, was examined in three fish species from the wider vicinity of Bar harbor in winter and spring. Induction of CYP1A was observed in winter and increased further in spring. Several PCBs were found in seawater in winter. They decreased below the limit of detection in spring, when the PAH fluorene was detected. It is concluded that the constant presence of CYP1A expression is probably due to pollutants in the environment, whereas increased expression of CYP1A in spring results from exposure of the fish to fluorene.Ekspresija CYP1A, biomarkera na prisustvo ksenobiotika, posmatrana je u tri vrste riba ulovljenih sa šireg lokaliteta barske luke tokom zime i proleća. Indukcija CYP1A koja je uočena u zimskom periodu, dodatno je uvećana u prolećnom. Nekoliko PCB koji su pronađeni u morskoj vodi tokom zime, nije detektovano u proleće kada je otkriven PAH fluoren. Zaključeno je da je stalno prisustvo ekspresije CYP1A najverovatnije posledica prisustva polutanata u životnoj sredini; dodatno uvećanje ekspresije CYP1A u proleće posledica je izlaganja riba fluorenu.nul

Effets motivationnels et sommatifs de l'évaluation par réussite-échec à paliers

Résumé à la fin des ptiesÉgalement disponible en version papierTitre de l'écran-titre (visionné le 3 mars. 2010)Comprend des bibliogr

Dynamic associations of transcription factors with the rat liver nuclear matrix are functionally related to differential alpha-2-macroglobulin gene expression

Participation of the nuclear matrix in regulation of alpha-2-macroglobulin (α2M) gene transcription during rat liver development and the acute-phase (AP) response are examined. DNA affinity chromatography of fetal and adult liver internal nuclear matrix proteins under basal and AP conditions with the α2M gene promoter (-852/+12) and immunoblot analysis revealed diverse patterns of association of transcription factors with the nuclear matrix. HNF-6, C/EBPα, and STAT5b were involved in basal and C/EBPβ, STAT1, and STAT3 in AP-stimulated α2M expression. These findings support the assumption that transcription factor-nuclear matrix interactions serve to channel gene regulatory proteins to DNA sequences.Cilj rada je ispitivanje učešća jedarnog matriksa u regulaciji transkripcije gena za alfa-2-makroglobulin tokom razvića jetre pacova i akutno faznog odgovora (AFO). Nakon DNK afinitetne hromatografije proteina unutrašnje mreže jedarnog matriksa fetalne i adultne jetre, u bazalnim i AFO uslovima, sa promotorskim elementom gena za α2M (-852/+12) i imunoblot analize, identifikovane su dinamičke asocijacije transkripcionih faktora uključenih u regulaciju ekspresije gena za α2M sa jedarnim matriksom. HNF-6, C/EBPα, STAT5b su uključeni u regulaciju bazalne ekspresije gena za α2M, dok C/EBPβ, STAT1, STAT3 posreduju u regulaciji ekspresije ovog gena tokom AFO. Opisane interakcije doprinose razumevanju predloženih mehanizama kojima se transkripcioni faktori usmeravaju ka ciljnim regulatornim elementima DNK.Projekat ministarstva br. 143002

Induction of acute phase reaction and suppression of the immune system in the paraoxon-intoxicated rats

Widespread use of organophosphates, especially in agriculture, led us to study their toxic effects. Our investigations were focused on paraoxon an oxygenated analog of a phosphothionate pesticide. This points to an analogy between the response to organophosphate intoxication and the acute phase reaction to injury. The capacity of paraoxon to elicit the acute phase response was assessed by studying two major processes characteristic of acute inflammation, the expression of acute phase proteins (APP) and the immunosuppressive activity of serum. After an LDso paraoxon administration to rats, the serum APP levels increased with time reaching a maximal level at the 24 h time point. The several-fold increases of AGP, MG, Hp and TST concentrations in the circulation of intoxicated rats, as well as a significant immunosuppressive activity of examined animal serum, pointed to the role of APP, especially AGP and MG, as immune modulators. These processes are analog to those observed during the acute phase response to injury and aimed at reestablishing homeostasis.Široka upotreba organofosfatnih jedinjenja u industriji, a posebno u poljoprivredi, navela nas je da ispitujemo njihove toksične efekte. Istraživanja su bila fokusirana ka paraoksonu, kiseoničnom analogu fosfotionatnog pesticida, sa ciljem da se utvrdi analogija između odgovora organizma na intoksikaciju paraoksonom i akutno fazne reakcije na povredu. Sposobnost paraoksona da indukuje akutno fazni odgovor analizirana je ispitivanjem dva glavna procesa karakteristična za inflamaciju: ekspresije akutno faznih proteina i imunosupresivne aktivnosti seruma. Nakon ubrizgavanja LDso paraoksona u pacove, nivo akutno faznih proteina u serumu životinja se povećava dostižući maksimalni nivo u 24 satu. Višestruko povećanje koncentracije al- kiselog glikoproteina (AGP), a2-makroglobulina (MG), haptoglobina i tiostatina u cirkulaciji tretiranih pacova kao i značajna imunosupresivna aktivnost seruma tretiranih životinja ukazali su na ulogu ovih proteina, naročito AGP i MG, kao finih modulatora imunološkog odgovora. Ovi procesi su analogni procesima tokom akutno faznog odgovora organizma na povredu i odvijaju se u cilju uspostavljanja fiziološke ravnoteže.nul

Ameliorating effects of antioxidative compounds from four plant extracts in experimental models of diabetes

Given that oxidative stress plays a major role in pancreatic β-cell dysfunction and ultimate destruction, as well as in different complications of diabetes, therapy with antioxidants has assumed an important place in the management of diabetes. The relatively limited effects of established antioxidant compounds have stimulated efforts to develop new therapeutic strategies, e.g. to increase the endogenous antioxidant defences through pharmacological modulation of key antioxidant enzymes. Plant extracts are gaining popularity in treating diabetes because many substances synthesized by higher plants and fungi possess antioxidant activities and can prevent or protect tissues against the damaging effects of free radicals. This review summarizes experimental models of diabetes and possible mechanisms that lie behind the antioxidative effects of α-lipoic acid (LA), a powerful antioxidant and compound that stimulates cellular glucose uptake, as well as of plant extracts from sweet chestnut (Castanea sativa), edible mushroom (Lactarius deterrimus) and natural products containing β-glucans in the treatment of diabetes. Their roles in preventing pancreatic β-cell death and in ameliorating the effects of severe diabetic complications are discussed.Terapija antioksidansima zauzima značajno mesto u lečenju dijabetesa s obzirom da oksidativni stres u velikoj meri doprinosi narušavanju funkcije i strukture β-ćelija pankreasa kao i razvoju komplikacija u dijabetesu. Zbog ograničenog dejstva postojećih antioksidativnih jedinjenja traga se za novim terapijskim rešenjima u tretmanu dijabetesa, kao što je povećanje endogene antioksidativne zaštite organizma putem farmakološke modulacije ključnih antioksidativnih enzima. Primena biljnih ekstrakata u lečenju dijabetesa postaje sve popularnija. Mnoge supstance koje se nalaze u sastavu viših biljaka i gljiva poseduju antioksidativna svojstva koja mogu da zaštite tkiva od štetnih uticaja slobodnih radikala. U ovom revijalnom radu opisani su eksperimentalni modeli dijabetesa kao i mogući mehanizmi koji leže u osnovi antioksidativnog dejstva α-liponske kiseline (LA), snažnog antioksidansa i jedinjenja koje stimuliše ćelijsku apsorpciju glukoze, kao i biljnih ekstrakata izolovanih iz slatkog kestena (Castanea sativa), jestivih pečuraka (Lactarius deterrimus) i prirodnih proizvoda koji sadrže β-glukan u lečenju dijabetesa. Opisani su njihova uloga u sprečavanju smrti β-ćelija pankreasa kao i blagotvorno dejstvo na komplikacije u dijabetesu.Projekat ministarstva br. 17302