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51. Targeted mutational profiling of childhood and adult acute lymphoblastic leukemia patients

Author

Perić, Jelena, Karan-Đurašević, Teodora, Kostić, Tatjana, Marjanović, Irena, Lazić, J., Virijević, M., Krstovski, N., Dokmanović, Lidija, Tomin, D., Vidović, A., Suvajdžić-Vuković, Nada, Janić, D., Pavlović, Sonja, Tošić, Nataša, Perić, Jelena, Karan-Đurašević, Teodora, Kostić, Tatjana, Marjanović, Irena, Lazić, J., Virijević, M., Krstovski, N., Dokmanović, Lidija, Tomin, D., Vidović, A., Suvajdžić-Vuković, Nada, Janić, D., Pavlović, Sonja, and Tošić, Nataša

Published
2017

52. Udruženost ekspresije Bax gena i Bcl2/Bax odnosa sa kliničkim i molekularnim prognostičkim markerima u hroničnoj limfocitnoj leukemiji

Author

Vučićević, Ksenija, Jakovljević, Vladimir, Čolović, Nataša, Tošić, Nataša, Kostić, Tatjana, Glumac, Irena, Pavlović, Sonja, Karan-Đurašević, Teodora, Čolović, Milica, Vučićević, Ksenija, Jakovljević, Vladimir, Čolović, Nataša, Tošić, Nataša, Kostić, Tatjana, Glumac, Irena, Pavlović, Sonja, Karan-Đurašević, Teodora, and Čolović, Milica

Abstract

Uvod: Rezistencija na apoptozu koja karakteriše maligne B limfocite in vivo u hroničnoj limfocitnoj leukemiji (HLL) delimično je uzrokovana unutrašnjim poremecajima apoptotske mašinerije u ovim celijama. Ti poremecaji su rezultat genetičkih promena i aberantne ekspresije regulatora procesa apoptoze, među kojima ključnu ulogu imaju članovi Bcl2 familije. Cilj: Cilj ove studije je bio da se ispita udruženost nivoa ekspresije proapoptotskog Bax gena, kao i Bcl2/Bax odnosa, sa kliničkim karakteristikama bolesnika sa HLL kao i molekularnim prognostičkim markerima, i to mutacionim statusom rearanžiranih gena za teške lance imunoglobulina (IGHV) i ekspresijom gena za lipoproteinsku lipazu (LPL). Metode: Analizirana je ekspresija Bax iRNK i Bcl2/Bax iRNK odnos u mononuklearnim celijama periferne krvi 58 bolesnika sa HLL i 10 zdravih kontrola metodom reverzne transkripcije i lančane reakcije polimeraze u realnom vremenu (qRT-PCR). Rezultati: Detektovana je povišena ekspresija Bax gena u HLL uzorcima u odnosu na kontrolne uzorke (p=0,003), kao i povišen Bcl2/Bax odnos (p= lt 0,001). Kada je u pitanju udruženost sa prognostičkim markerima, Bcl2/Bax odnos je ispoljio negativnu korelaciju sa vremenom udvostručavanja broja limfocita (r=-0,307; p=0,0451), dok je visoka ekspresija Bax bila povezana sa LPL-pozitivnim statusom (p=0,035). I ekspresija Bax gena i Bcl2/Bax odnos su bili viši kod bolesnika sa nemutiranim u odnosu na bolesnike sa mutiranim IGHV genima, ali nije dostignuta statistička značajnost. Zaključak: Rezultati ove studije ukazuju na mogucu ulogu poremecene ekspresije Bcl2 i Bax gena, koja dovodi do visokog Bcl2/Bax odnosa u leukemijskim celijama, u patogenezi i kliničkom toku HLL., Background: In chronic lymphocytic leukemia (CLL), in vivo apoptotic resistance of malignant B lymphocytes results, in part, from the intrinsic defects of their apoptotic machinery. These include genetic alterations and aberrant expression of many apoptosis regulators, among which the Bcl2 family members play a central role. Aim: The aim of this study was to investigate the association of pro-apoptotic Bax gene expression and Bcl2/Bax ratio with the clinical features of CLL patients as well as with molecular prognostic markers, namely the mutational status of rearranged immunoglobulin heavy variable (IGHV) genes and lipoprotein lipase (LPL) gene expression. Methods: We analyzed the expression of Bax mRNA and Bcl2/Bax mRNA ratio in the peripheral blood mononuclear cells of 58 unselected CLL patients and 10 healthy controls by the quantitative reverse-transcriptase polymerase chain reaction. Results: We detected significant Bax gene overexpression in CLL samples compared to non-leukemic samples (p=0.003), as well as an elevated Bcl2/Bax ratio (p= lt 0.001). Regarding the association with prognostic markers, the Bcl2/Bax ratio showed a negative correlation to lymphocyte doubling time (r=-0.307; p=0.0451), while high-level Bax expression was associated with LPL-positive status (p=0.035). Both the expression of Bax and Bcl2/Bax ratio were higher in patients with unmutated vs. mutated IGHV rearrangements, but this difference did not reach statistical significance. Conclusions: Our results suggest that dysregulated expression of Bcl2 and Bax, which leads to a high Bcl2/Bax ratio in leukemic cells, contributes to the pathogenesis and clinical course of CLL.

Published
2016

53. Assessment of BAALC and MN1 gene expression level could contribute to improved prognostic stratification of the aml-nk patients

Author

Marjanović, Irena, Karan-Đurašević, Teodora, Kostić, Jelena, Kostić, Tatjana, Virijević, M., Djunić, I., Suvajdžić-Vuković, Nada, Tomin, D., Pavlović, Sonja, Tošić, Nataša, Marjanović, Irena, Karan-Đurašević, Teodora, Kostić, Jelena, Kostić, Tatjana, Virijević, M., Djunić, I., Suvajdžić-Vuković, Nada, Tomin, D., Pavlović, Sonja, and Tošić, Nataša

Published
2016

54. Ekspresija Bcl2 gena kod pacijenata sa hroničnom limfocitnom leukemijom

Author

Vučićević, Ksenija, Jakovljević, Vladimir, Sretenović, Jasmina, Tošić, Nataša, Kostić, Tatjana, Glumac, Irena, Čolović, Milica, Čolović, Nataša, Pavlović, Sonja, Karan-Đurašević, Teodora, Vučićević, Ksenija, Jakovljević, Vladimir, Sretenović, Jasmina, Tošić, Nataša, Kostić, Tatjana, Glumac, Irena, Čolović, Milica, Čolović, Nataša, Pavlović, Sonja, and Karan-Đurašević, Teodora

Abstract

Hronična limfocitna leukemija (HLL) se manifestuje kao klonska ekspanzija zrelih B limfocita, čija se akumulacija pripisuje prvenstveno poremećajima procesa apoptoze. U HLL su uočene genetičke promene i aberantna ekspresija različitih članova Bcl2 genske familije, koji imaju ključnu ulogu u regulaciji unutrašnjeg, mitohondrijskog puta aktivacije apoptoze. U ovom radu je analizirana ekspresija anti-apoptotskog Bcl2 gena u grupi od 58 pacijenata obolelih od HLL. Metodom kvantitativnog RT-PCRa detektovana je povišena ekspresija Bcl2 mRNA u HLL uzorcima u odnosu na kontrolne uzorke (p= lt 0.001). 'Receiver operating characteristic' (ROC) analiza je pokazala da nivo ekspresije Bcl2 ima visoku moć diskriminacije između pacijenata i zdravih kontrola (A=0.98, 95% CI=0.95-1.009, p lt 0.0001)., Chronic lymphocytic leukaemia (CLL) manifests as clonal expansion of mature B lymphocytes, whose accumulation is primarily attributed to the dysregulation of apoptosis. Aberrant expression, as well as genetic alterations within various Bcl2 family members and central regulators of the intrinsic, mitochondriamediated apoptotic pathway all hasve been observed in CLL. Here, we report the expression analysis of the anti-apoptotic Bcl2 gene in a cohort of 58 CLL patients. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) analysis revealed a significant overexpression of Bcl2 mRNA in CLL samples compared to control samples (p= lt 0.001). Receiver operating characteristic (ROC) analysis showed that the level of Bcl2 expression exerts a high discriminatory power between patients and healthy subjects (A=0.98, 95% CI=0.95-1.009, p lt 0.0001).

Published
2015

55. Prognostic impact of wt1 gene expression quantification during minimal residual disease monitoring of acute promyelocytic leukemia

Author

Mitrović, M., Tošić, Nataša, Suvajdžić, Nada, Djunić, I., Vidović, A., Virijević, M., Čolović, Nataša, Glumac, Irena, Kostić, Tatjana, Pavlović, Sonja, Elezović, I., Tomin, D., Mitrović, M., Tošić, Nataša, Suvajdžić, Nada, Djunić, I., Vidović, A., Virijević, M., Čolović, Nataša, Glumac, Irena, Kostić, Tatjana, Pavlović, Sonja, Elezović, I., and Tomin, D.

Published
2015

56. JAK2V617F mutacija kod bolesnika sa B ćelijskom hroničnom limfocitnom leukemijom i prefibrotičkom primarnom mijelofibrozom

Author

Ristić, Slobodan, Radojković, Milica, Kostić, Tatjana, Spasovski, Vesna, Pavlović, Sonja, Čemerikić-Martinović, Vesna, Ristić, Slobodan, Radojković, Milica, Kostić, Tatjana, Spasovski, Vesna, Pavlović, Sonja, and Čemerikić-Martinović, Vesna

Abstract

Uvod Sekundarni maligniteti, naročito solidni tumori, česti su kod bolesnika s hroničnom limfocitnom leukemijom (HLL), ali retko se sreće udruženost mijeloproliferativnih neoplazmi i HLL. Prikaz bolesnika Prikazujemo muškarca starog 67 godina sa B ćelijskom HLL kod koga se nakon devet godina razvila primarna mijelofibroza (PMF). Bolesnik je lečen alkilišućim agensima i analozima purina, što može biti predisponirajući faktor za razvoj mijeloproliferativnog oboljenja. JAK2V617F mutacija nije otkrivena prilikom postavljanja dijagnoze HLL, ali je utvrđena posle devet godina, kada se razvila PMF, što ukazuje na to da su B ćelijska HLL i PMF neoplazme koje potiču od različitih ćelijskih klonova. Zaključak Patogenetski mehanizmi udruženosti mijeloproliferativne i limfoproliferativne neoplazme kod bolesnika nisu razjašnjeni. Potrebna su dalja istraživanja radi utvrđivanja da li ove maligne bolesti potiču od dva različita ćelijska klona ili nastaju od iste pluripotentne matične ćelije hematopoeze., Introduction Secondary malignancies, particularly solid tumors, are common in patients with chronic lymphocytic leukemia (CLL), but association of myeloproliferative neoplasms and chronic lymphocytic leukemia in the same patient is very rare. Case Outline We report of a 67-year-old man with B-cell chronic lymphoid leukemia (B-CLL) who developed primary myelofibrosis (PMF) nine years after initial diagnosis. Patient received alkylation agents and purine analogue, which can be a predisposing factor for the development of myeloproliferative neoplasms. JAK2V617F mutation was not present initially at the time of CLL diagnosis, but was found after nine years when PMF occurred, which indicates that B-CLL and PMF represent two separate clonal origin neoplasms. Conclusion Pathogenic mechanisms for the development of myeloproliferative and lymphoproliferative neoplasms in the same patient are unknown. Further research is needed to determine whether these malignancies originate from two different cell clones or arise from the same pluripotent hematopoietic stem cell.

Published
2015

57. HLA genotyping in pediatric celiac disease patients

Author

Stanković, Biljana, Radlović, Nedeljko, Leković, Zoran, Ristić, Dragana, Radlović, Vladimir, Nikević, Gordana, Kotur, Nikola, Vucicević, Ksenija, Kostić, Tatjana, Pavlović, Sonja, Zukić, Branka, Stanković, Biljana, Radlović, Nedeljko, Leković, Zoran, Ristić, Dragana, Radlović, Vladimir, Nikević, Gordana, Kotur, Nikola, Vucicević, Ksenija, Kostić, Tatjana, Pavlović, Sonja, and Zukić, Branka

Abstract

Celiac disease (CD) is a chronic inflammatory disease in the small intestine triggered by gluten uptake that occurs in genetically susceptible individuals. HLA-DQ2 protein encoded by HLA-DQA1*05 and DQB1*02 alleles is found in 9o-95% of CD patients. All of the remaining patients carry HLA-DQ8 protein encoded by HLA-DQA1*03 and DQB1*03:02 alleles. Specific HLA-DQ genotypes define different risk for CD incidence. Presence of susceptible HLA-DQ genotypes does not predict certain disease development, but their absence makes CD very unlikely, close to 100%. Here we presented for the first time the distribution of HLA-DQ genotypes in the group of pediatric celiac patients from the University Children's Hospital, Belgrade, Serbia and estimated risk for CD development that these genotypes confer. Seventy three celiac disease patients and 62 healthy individuals underwent genotyping for DQA1, DQB1 alleles and DRB1 allele. 94.5% of patients carried alleles that encode DQ2 protein variant and 2.7% carried alleles that encode DQ8 protein variant. Two patients carried single DQB1 *02 allele. No patients were negative for all the alleles predisposing to CD. The highest HLA-DQ genotype risk for CD development was found in group of patients homozygous for DQ2.5 haplotype, followed by the group of heterozygous carriers of DQ2.5 haplotype in combination with DQB1*02 allele within the other haplotype. The lowest risk was observed in carriers of a single copy of DQB1*02 or DQA1*05 allele or other non-predisposing alleles. HLA genotyping, more informative than serological testing commonly used, proved to be a useful diagnostic tool for excluding CD development.

Published
2014

58. Adverse prognostic significance of wilms tumor gene 1 overexpression in acute promyelocytic leukemia

Author

Mitrović, M., Tošić, Nataša, Suvajdžić, Nada, Djunić, I., Vidović, A., Virijević, M., Čolović, Nataša, Kostić, Tatjana, Glumac, Irena, Pavlović, Sonja, Elezović, I., Tomin, D., Mitrović, M., Tošić, Nataša, Suvajdžić, Nada, Djunić, I., Vidović, A., Virijević, M., Čolović, Nataša, Kostić, Tatjana, Glumac, Irena, Pavlović, Sonja, Elezović, I., and Tomin, D.

Published
2014

59. HLA genotyping in pediatric celiac disease patients

Author

Stanković, Biljana, primary, Radlović, Nedeljko, additional, Leković, Zoran, additional, Ristić, Dragana, additional, Radlović, Vladimir, additional, Nikčević, Gordana, additional, Kotur, Nikola, additional, Vučićević, Ksenija, additional, Kostić, Tatjana, additional, Pavlović, Sonja, additional, and Zukic, Branka, additional

Published
2014
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60. Adverse prognostic significance of FLT3 mutations in acute promyelocytic leukemia

Author

Mitrović, M., Suvajdžić, Nada, Tošić, Nataša, Bogdanović, A., Djunić, I., Sretenović, A., Vidović, A., Virijević, M., Čolović, Nataša, Kostić, Tatjana, Karan-Đurašević, Teodora, Glumac, Irena, Spasovski, Vesna, Pavlović, Sonja, Elezović, I., Tomin, D., Mitrović, M., Suvajdžić, Nada, Tošić, Nataša, Bogdanović, A., Djunić, I., Sretenović, A., Vidović, A., Virijević, M., Čolović, Nataša, Kostić, Tatjana, Karan-Đurašević, Teodora, Glumac, Irena, Spasovski, Vesna, Pavlović, Sonja, Elezović, I., and Tomin, D.

Published
2013

61. Molecular basis of genetic rearrangements induced by IS5 elements in polA1 mutator strain of E.coli K12

Author

Kostić, Tatjana, Knežević-Vukčević, Jelena, Simić, Draga, and Topisirović, Ljubiša

Subjects
delecije, PCR, IS5, mutageneza, polA1, deletions, polA1- recombination, mutagenesis, rekombinacija
Abstract

U našem prethodnom radu na polA1 mutantima pokazali smo da polA1 kreira mutacije isključivo tipa minus fs i delecija. U mutacionom spektru histidinskih auksotrofa bila je prisutna i jedna specifična kategorija delecija, velike delecije, koje su prolazile preko celog his operona. Veličina delecija je ukazivala da one nastaju u procesu rekombinacije izmedju homologih sekvenci, a poznato je da u polA mutantima dolazi do povećane frekvence rekombinacije. Ovaj rad predstavlja završni deo naše originalne studije polA1 indukovane mutageneze praćene na histidinskom operonu hromozoma E.coli. Analizirani su molekularni mehanizmi nastajanja velikih delecija, ili proces rekombinacije koji promoviše ove genomske rearanžmane. Pokazali smo da velike delecije nastaju kao rezultat rekombinacije izmedju dve IS5 sekvence koje se nalaze sa obe strane his operona. Sve četiri nezavisno izolovane delecije su bile jednake i iznosile su 35 kb. Analizom sekvence rekombinovanih IS5 elemenata pokazali smo da rekombinacija uvek prolazi kroz levi terminus IS5 i da se odigrava po tipu “site specific” rekombinacije. Levi terminus IS5 elementa ima karakteristično zakrivljenje DNK uslovljeno njenom primarnom strukturom i sadrži mesto za vezivanje IHF proteina. Rezultati ovog rada zajedno sa podacima iz literature ukazuju na moguću ulogu IHF proteina u nastajanju velikih delecija i u procesu rekombinacije. Primenom PCR tehnologije pokazli smo da je frekvenca nastajanja velikih delecija povećana kod polA1 i dam mutanata i da velike delecije nastaju na recA nezavisan način. PolA1 ne pokazuje nikakvu specifičnost jer se rekombinacija dešava na isti način i kod divljeg soja, ali je frekvenca rekombinacije dva puta povećana u polA1 mutantima u odnosu na divlji soj. In our previous work on polA1 mutants we have shown that polA1 creates exclusively minus fs and deletions types. In mutational spectra of histidine auxotrophs, one specific category of deletions, large deletions, that cover all his operon, was present. The size of deletions pointed out that they appear in a process of recombination between homology sequences; it is known that polA1 mutants show an increased frequency of recombination. This work presents final part of our original study of polA1-induced mutagenesis followed on histidine operon of chromosome E.coli. Molecular mechanisms of appearances of large deletions, or process of recombination promoting this chromosomal rearrangement, was analysed. We showed that large deletions appear as a result of recombination between two IS5 sequences located on both sides of his operon. Four independently isolated deletions were identical, 35 kb long. Sequence analysis of recombinant IS5 elements was show that recombination always goes throught the left terminus of IS5, as a “site specific” recombination. Left terminus of IS5 contains a characteristic sequence-directed bent DNA, including a binding site for IHF protein. The data from literature, including the present results, point to a possible role of IHF protein in appearance of large deletions and process of recombination. Applying a PCR technology, we show that the frequency of large deletions is increased in polA1 and dam mutants, and that large deletions appear on recA independent way. PolA1 is not sequence specific because the process of recombination between two IS5 is identical in wild type and polA mutants, but the frequency of recombination is two times higher in regard to wild type.

Published
2001

62. Mutational Status and Gene Repertoire of IGHV-IGHD-IGHJ Rearrangements in Serbian Patients With Chronic Lymphocytic Leukemia

Author

Karan-Đurašević, Teodora, Palibrk, Vuk, Kostić, Tatjana, Spasovski, Vesna, Nikčević, Gordana, Srzentić Dražilov, Sanja, Colović, Milica, Čolović, Nataša, Vidović, Ana, Antić, Darko, Mihaljević, Biljana, Pavlović, Sonja, Tošić, Nataša, Karan-Đurašević, Teodora, Palibrk, Vuk, Kostić, Tatjana, Spasovski, Vesna, Nikčević, Gordana, Srzentić Dražilov, Sanja, Colović, Milica, Čolović, Nataša, Vidović, Ana, Antić, Darko, Mihaljević, Biljana, Pavlović, Sonja, and Tošić, Nataša

Abstract

The mutational status and configuration of immunoglobulin heavy variable (IGHV) gene rearrangements was analyzed in 85 Serbian patients with chronic lymphocytic leukemia (CLL). We found that 55.3% of cases belonged to mutated and 44.7% to unmutated CLL, progressive disease predominating in the unmutated subset. IGHV gene use resembled that obtained for Mediterranean countries, except for underrepresentation of the IGHV4 subgroup in our cohort. Background: Chronic lymphocytic leukemia (CLL) results from the clonal expansion of mature B lymphocytes and is characterized by extreme clinical heterogeneity. One of the most reliable prognostic markers in chronic lymphocytic leukemia (CLL) is the mutational status of immunoglobulin heavy variable (IGHV) genes, which defines 2 subsets, mutated CLL (M-CLL) and unmutated CLL (U-CLL), with different clinical courses. Biased IGHV gene use between M-CLL and U-CLL clones, as well as population differences in the IGHV gene repertoire have been reported. Patients and Methods: In this study, mutational status and configuration of IGHV-IGHD-IGHJ rearrangements in 85 Serbian patients were analyzed using reverse transcriptase-polymerase chain reaction (RT-PCR) and sequencing methodology. Results: We found that 55.3% of cases belonged to M-CLL and 44.7% belonged to U-CLL, with progressive disease predominating in the unmutated subset. Most frequently expressed was the IGHV3 subgroup (55.7%), followed by IGHV1 (27.3%), IGHV4 (12.5%), IGHV5 (2.3%), IGHV2 (1.1%), and IGHV6 (1.1%). The distribution of IGHD subgroups was as follows: IGHD3, 39.1%; IGHD2, 21.8%; IGHD6, 12.6%; IGHD1, 10.3%; IGHD4, 8%; IGHD5, 6.9%; and IGHD7, 1.1%. The most frequent IGHJ gene was IGHJ4 (48.9%), followed by IGHJ6 (28.4%), IGHJ3 (11.4%), and IGHJ5 (11.4%). In 15.3% of cases, heavy complementarity-determining region 3 (VH CDR3) amino acid sequences could be assigned to previously defined stereotyped clusters. Conclusions: Our study showed a strong correlation betwee

Published
2012

63. Expression of bcl2l12 gene in chronic lymphocytic leukemia: association with clinical and molecular prognostic markers

Author

Karan-Đurašević, Teodora, Palibrk, V., Tošić, Nataša, Kostić, Tatjana, Spasovski, Vesna, Nikčević, Gordana, Srzentić Dražilov, Sanja, Glumac, Irena, Colović, M., Čolović, Nataša, Antić, Darko, Mihaljević, B., Scorilas, A., Kontos, C., Pavlović, Sonja, Karan-Đurašević, Teodora, Palibrk, V., Tošić, Nataša, Kostić, Tatjana, Spasovski, Vesna, Nikčević, Gordana, Srzentić Dražilov, Sanja, Glumac, Irena, Colović, M., Čolović, Nataša, Antić, Darko, Mihaljević, B., Scorilas, A., Kontos, C., and Pavlović, Sonja

Published
2012

64. Mehanizmi delovanja atrazina na steroidogenu aktivnost Leydig-ovih ćelija peripubertalnih pacova

Author

Kovačević, Radmila, Kuhajda, Ksenija, Kostić, Tatjana, Cvijić, Gordana, Pogrmić, Kristina, Kovačević, Radmila, Kuhajda, Ksenija, Kostić, Tatjana, Cvijić, Gordana, and Pogrmić, Kristina

Abstract

Rezultati prikazani u ovom radu opisuju efekte in vivo primene atrazina (2-hloro-4- etilamino-6-izopropilamino-s-triazin) na ex vivo steroidogenezu u Leydig-ovim ćelijama peripubertalnih pacova (tretiranih sa 50 mg/kg i 200 mg/kg telesne mase od 23. do 50. dana starosti). Dobijeni rezultati jasno ukazuju da 28-dnevna in vivo primena atrazina snažno inhibira testikularnu steroidogenezu, smanjujući ekspresiju gena za steroidogene enzime i druge regulatorne proteine uključene u kontrolu testikularne steroidogeneze u Leydig-ovim ćelijama peripubertalnih pacova. Rezultati in vivo primene atrazina pokazuju da atrazin snažno inhibira ekspresiju gena za luteinizirajući hormon receptor (LHR), skevendžer receptor-B1 (SR-B1), steroidogeni faktor-1 (SF-1), steroidogeni akutni regulatorni protein (StAR), translokator protein (TSPO), fosfodiesterazu 4B, 3β−hidroksisteroid dehidrogenazu (ΗSD), CYP17A1 i 17βHSD. Rezultati u okviru ovih istraživanja pokazuju da primena atrazina tokom prepubertalnog perioda razvoja mužjaka pacova dovodi do dozno-zavisnog smanjenja nivoa cAMP i snažne inhibicije androgeneze u prisustvu hCG. Obzirom na blokadu ekspresije LHR, prvog elementa u aktivaciji cAMP-signalnog puta, moglo bi se predpostaviti da je to uzrok blokade androgeneze kod atrazinom-tretiranih životinja. Takođe, rezultati ukazuju na inhibiciju supstrat-stimulisane produkcije androgena paralelno sa redukcijom ekspresije steroidogenih enzima CYP17A1 i 17βHSD. U drugom delu ove doktorske disertacije, ispitivan je efekat direktne in vitro primene različitih doza atrazina (1 nM, 1 μM, 20 μM, 50 μM) na ekspresiju i aktivnost steroidogenih enzima u kulturi prečišćenih Leydig-ovih ćelija testisa peripubertalnih pacova, pri čemu je zabeleženo stimulatorno dejstvo pomenutog herbicida. Naime, zabeleženo je povećanje bazalne i hCG-stimulisane produkcije testosterona praćeno povećanim nivoom cAMP u medijumu tretiranih ćelija. Pri ispitivanju ekspresije gena za steroidogene enzime i regulatorne protei, In the present study, we investigated the effects of oral dosing of atrazine (2-chloro-4- ethylamino-6-isopropylamino-s-triazine) to peripubertal male rats (50 mg/kg and 200 mg/kg body weight daily from postnatal day 23 to 50) on ex vivo Leydig cell steroidogenesis. Leydig cells from treated rats were characterised by significant decline in mRNA transcripts of several genes responsible for steroidogenesis: luteinizing hormone receptor (LHR), scavenger receptor-B1, steroidogenic acute regulatory protein (StAR), translocator protein, steroidogenic factor-1 (SF-1), phosphodiesterase 4B, 3β−hydroxysteroid dehydrogenase (ΗSD), CYP17A1 and 17βHSD. In the presence of human chorion gonadotropin, the dose-dependent decrease in extra cellular cAMP level and accordingly strong inhibition of androgenesis were obtained. The transcription of LHR gene in Leydig cells of atrazine-treated rats was down-regulated in a dose-dependent manner, which could be the reason for reduction in cAMP level and expression of cAMPdependent genes. The results also indicated inhibition of substrate-stimulated androgen production in parallel with reduced expression of steroidogenih enzymes CYP17A1 and 17βHSD. In the second part of this study we examined direct 24 h in vitro effect of different doses of atrazine (1 nM, 1 μM, 20 μM, 50 μM) on expression and activity of steroidogenic enzymes in purified Leydig cells obtained from peripubertal rats. Obtained results indicated that 24 h-incubation of peripubertal Leydig cells in the presence of atrazine increased steroidogenic capacity of that cells. Increased basal and hCGstimulated testosterone production were accompanied by increasing levels of cAMP in the medium of treated cells. Also, in comparison to controls, gene expression revealed increased expression of SF-1, StAR, CYP17A1 and 17β-HSD. When Leydig cells were challenged with progesterone and Δ4–androstenedione, testosterone production was increased in atrazine chalenged Leydig cells. To address t

Published
2010

65. Analysis of FLT3 mrna expression level in adult aml

Author

Tošić, Nataša, Karan-Đurašević, Teodora, Spasovski, Vesna, Kostić, Tatjana, Čolović, Nataša, Colović, M., Pavlović, Sonja, Tošić, Nataša, Karan-Đurašević, Teodora, Spasovski, Vesna, Kostić, Tatjana, Čolović, Nataša, Colović, M., and Pavlović, Sonja

Published
2010

66. Uloga sistema FasR/FasL u patogenezi mijeloproliferativnih neoplazija

Author

Spasovski, Vesna, Tošić, Nataša, Kostić, Tatjana, Zukić, Branka, Stojiljković, Maja, Čolović, Milica, Pavlović, Sonja, Spasovski, Vesna, Tošić, Nataša, Kostić, Tatjana, Zukić, Branka, Stojiljković, Maja, Čolović, Milica, and Pavlović, Sonja

Abstract

Mijeloproliferativne neoplazije (MPN) su hematološki maligniteti koji se karakterišu nekontrolisanom ćelijskom proliferacijom i poremećajem u procesu apoptoze. Sistem FasR/FasL je uključen u kontrolu apoptoze u različitim tipovima ćelija. U ovom radu je izučavana uloga sistema FasR/FasL u patogenezi mijeloproliferativnih neoplazija. Upoređena je ekspresija FasR i FasL između pacijenata sa MPN (24) i zdravih kontrola korišćenjem metode 'real-time' PCR. Detektovana je povećana ekspresija FasR kod pacijenata sa MPN. Nije utvrđena razlika u ekspresiji FasL. Mutacija B617F u JAK2 genu, karakteristična za MPN, je nađena kod 13 od 24 pacijenta. Pokazano je da ekspresija FasR i FasL nije povezana sa prisustvom B617F JAK2 mutacije., Myeloproliferative neoplasms (MPN) are hematological malignancies characterized by uncontrolled cell proliferation and impaired apoptosis. The FasR/FasL system is involved in the control of apoptosis in different cell types. Here we have investigated the role of FasR/FasL in the pathogenesis of MPNs. We compared FasR/FasL expression between MPN patients (24) and healthy individuals using the real-time PCR assay. We found an increase of FasR expression in MPN patients. No difference was detected in FasL expression. Mutation V617F in the JAK2 gene, a hallmark of MPN, was detected in 13/24 patients. We found that neither FasR nor FasL expression were related to the presence of JAK2 V617F mutation.

Published
2010

67. Mutacija JAK2-V617F kod bolesnika s mijeloproliferativnim neoplazijama - veza sa mutacijom FLT3-ITD

Author

Spasovski, Vesna, Tošić, Nataša, Kostić, Tatjana, Pavlović, Sonja, Čolović, Milica, Spasovski, Vesna, Tošić, Nataša, Kostić, Tatjana, Pavlović, Sonja, and Čolović, Milica

Abstract

Uvod. Stečena somatska mutacija V617F u genu za Janus kinazu 2 (JAK2) uzrok je nekontrolisane proliferacije ćelija kod bolesnika s mijeloproliferativnim neoplazijama. Poznato je da do proliferacije ćelija mijeloidne loze dolazi i pod uticajem mutacija u drugim genima, kao što su mutacije u genu za tirozin-kinazni receptor FLT3, koji je najčešći mutirani gen u akutnim mijeloidnim leukemijama. Od posebnog je značaja to što mutirani protein FLT3 koristi isti signalni put kao protein JAK2. To je signalni put preko proteina STAT5, čija je aktivacija važna za samoobnavljanje matičnih ćelija hematopoeze. Cilj rada. Cilj istraživanja je bio da se otkrije mutacija V617F u genu za JAK2 kod bolesnika s mijeloproliferativnim neoplazijama. Ispitano je i istovremeno prisustvo mutacije FLT3-ITD kod ovih bolesnika radi rasvetljavanja hipoteze o sličnoj ulozi ova dva molekularnogenetička markera u hematološkim malignitetima. Metode rada. Metodom alel-specifičnog PCR (engl. polymerase chain reaction) analiziran je 61 bolesnik sa potvrđenom dijagnozom mijeloproliferativne neoplazije na mutaciju V617F u genu za JAK2 ili sumnjom na ovu dijagnozu. Kod bolesnika sa mutacijom u genu JAK2 je zatim ispitano postojanje mutacije FLT3-ITD metodom PCR. Rezultati. Kod 18 ispitanika je otkrivena mutacija V617F u genu za JAK2. Među njima je kod osam bolesnika dijagnostikovana policitemija vera, a kod deset esencijalna trombocitemija. Ni kod jednog ispitanika s mutacijom V617F u genu za JAK2 nije otkrivena mutacija FLT3-ITD. Zaključak. Rezultati ovog istraživanja podržavaju hipotezu da je za malignu transformaciju matične ćelije hematopoeze dovoljna jedna mutacija koja izaziva poremećaj proliferacije ćelije., Introduction. An acquired somatic mutation V617F in Janus kinase 2 gene (JAK2) is the cause of uncontrolled proliferation in patients with myeloproliferative neoplasms. It is known that uncontrolled myeloid cell proliferation is also provoked by alteration in other genes, e.g. mutations in receptor tyrosine kinase FLT3 gene. FLT3 represents the most frequently mutated gene in acute myeloid leukaemia. Interestingly, mutated FLT3- ITD (internal tandem duplication) protein is a member of the same signalling pathway as JAK2 protein, the STAT5 signalling pathway. STAT5 activation is recognized as important for selfrenewal of haematopoetic stem cells. Objective. The aim of this study was the detection of JAK2- V617F mutation in patients with myeloproliferative neoplasms. Additionally, we investigated the presence of FLT3-ITD mutation in JAK2-V617F-positive patients in order to shed the light on the hypothesis of a similar role of these two molecular markers in haematological malignancies. Methods. Using allele-specific PCR, 61 patients with known or suspected diagnosis of myeloproliferative neoplasms were tested for the presence of JAK2-V617F mutation. Samples that were positive for JAK2 mutation were subsequently tested for the presence of FLT3-ITD mutation by PCR. Results. Eighteen of 61 analysed patients were positive for JAK2-V617F mutation. Among them, 8/18 samples were diagnosed as polycythaemia vera, and 10/18 as essential thrombocythaemia. None of JAK2-V617F-positive patient was positive for FLT3-ITD mutation. Conclusion. This study suggests that one activating mutation is sufficient for aberrant cell proliferation leading to malignant transformation of haematopoetic stem cell.

Published
2010

70. Application of targeted next generation sequencing for the mutational profiling of patients with acute lymphoblastic leukemia

Author

Janic Dragana, Peric Jelena, Karan-Djurasevic Teodora, Kostic Tatjana, Marjanovic Irena, Stanic Bojana, Pejanovic Nadja, Dokmanovic Lidija, Lazic Jelena, Krstovski Nada, Virijevic Marijana, Tomin Dragica, Vidovic Ana, Suvajdzic-Vukovic Nada, Pavlovic Sonja, and Tosic Natasa

Subjects
acute lymphoblastic leukemia, next generation sequencing, somatic mutations, Biochemistry, QD415-436
Abstract

Background: Acute lymphoblastic leukemia (ALL) is the most common cancer in children, whereas it is less common in adults. Identification of cytogenetic aberrations and a small number of molecular abnormalities are still the most important risk and therapy stratification methods in clinical practice today. Next generation sequencing (NGS) technology provides a large amount of data contributing to elucidation of mutational landscape of childhood (cALL) and adult ALL (aALL). Methods: We analyzed DNA samples from 34 cALL and aALL patients, using NGS targeted sequencing TruSeq Amplicon - Cancer Panel (TSACP) which targets mutational hotspots in 48 cancer related genes. Results: We identified a total of 330 variants in the coding regions, out of which only 95 were potentially protein-changing. Observed in individual patients, detected mutations predominantly disrupted Ras/RTK pathway (STK11, KIT, MET, NRAS, KRAS, PTEN). Additionally, we identified 5 patients with the same mutation in HNF1A gene, disrupting both Wnt and Notch signaling pathway. In two patients we detected variants in NOTCH1 gene. HNF1A and NOTCH1 variants were mutually exclusive, while genes involved in Ras/RTK pathway exhibit a tendency of mutation accumulation. Conclusions: Our results showed that ALL contains low number of mutations, without significant differences between cALL and aALL (median per patient 2 and 3, respectively). Detected mutations affect few key signaling pathways, primarily Ras/RTK cascade. This study contributes to knowledge of ALL mutational landscape, leading to better understanding of molecular basis of this disease.

Published
2020

71. Molekularna osnova genetičkih rearanžmana izazvanih IS5 elementima u polA1 mutatorskom soju Escherichia coli K12

Author

Knežević-Vukčević, Jelena, Simić, Draga, Topisirović, Ljubiša, Kostić, Tatjana, Knežević-Vukčević, Jelena, Simić, Draga, Topisirović, Ljubiša, and Kostić, Tatjana

Abstract

U našem prethodnom radu na polA1 mutantima pokazali smo da polA1 kreira mutacije isključivo tipa minus fs i delecija. U mutacionom spektru histidinskih auksotrofa bila je prisutna i jedna specifična kategorija delecija, velike delecije, koje su prolazile preko celog his operona. Veličina delecija je ukazivala da one nastaju u procesu rekombinacije izmedju homologih sekvenci, a poznato je da u polA mutantima dolazi do povećane frekvence rekombinacije. Ovaj rad predstavlja završni deo naše originalne studije polA1 indukovane mutageneze praćene na histidinskom operonu hromozoma E.coli. Analizirani su molekularni mehanizmi nastajanja velikih delecija, ili proces rekombinacije koji promoviše ove genomske rearanžmane. Pokazali smo da velike delecije nastaju kao rezultat rekombinacije izmedju dve IS5 sekvence koje se nalaze sa obe strane his operona. Sve četiri nezavisno izolovane delecije su bile jednake i iznosile su 35 kb. Analizom sekvence rekombinovanih IS5 elemenata pokazali smo da rekombinacija uvek prolazi kroz levi terminus IS5 i da se odigrava po tipu “site specific” rekombinacije. Levi terminus IS5 elementa ima karakteristično zakrivljenje DNK uslovljeno njenom primarnom strukturom i sadrži mesto za vezivanje IHF proteina. Rezultati ovog rada zajedno sa podacima iz literature ukazuju na moguću ulogu IHF proteina u nastajanju velikih delecija i u procesu rekombinacije. Primenom PCR tehnologije pokazli smo da je frekvenca nastajanja velikih delecija povećana kod polA1 i dam mutanata i da velike delecije nastaju na recA nezavisan način. PolA1 ne pokazuje nikakvu specifičnost jer se rekombinacija dešava na isti način i kod divljeg soja, ali je frekvenca rekombinacije dva puta povećana u polA1 mutantima u odnosu na divlji soj., In our previous work on polA1 mutants we have shown that polA1 creates exclusively minus fs and deletions types. In mutational spectra of histidine auxotrophs, one specific category of deletions, large deletions, that cover all his operon, was present. The size of deletions pointed out that they appear in a process of recombination between homology sequences; it is known that polA1 mutants show an increased frequency of recombination. This work presents final part of our original study of polA1-induced mutagenesis followed on histidine operon of chromosome E.coli. Molecular mechanisms of appearances of large deletions, or process of recombination promoting this chromosomal rearrangement, was analysed. We showed that large deletions appear as a result of recombination between two IS5 sequences located on both sides of his operon. Four independently isolated deletions were identical, 35 kb long. Sequence analysis of recombinant IS5 elements was show that recombination always goes throught the left terminus of IS5, as a “site specific” recombination. Left terminus of IS5 contains a characteristic sequence-directed bent DNA, including a binding site for IHF protein. The data from literature, including the present results, point to a possible role of IHF protein in appearance of large deletions and process of recombination. Applying a PCR technology, we show that the frequency of large deletions is increased in polA1 and dam mutants, and that large deletions appear on recA independent way. PolA1 is not sequence specific because the process of recombination between two IS5 is identical in wild type and polA mutants, but the frequency of recombination is two times higher in regard to wild type.

Published
2001

73. Nucleotide sequence of the Escherichia coli K12 histidine operon revisited

Author

Jovanović, Goran, Kostić, Tatjana, Janković, M., Savić, D.J., Jovanović, Goran, Kostić, Tatjana, Janković, M., and Savić, D.J.

Abstract

We report on a significant difference between the published nucleotide sequence of the Escherichia coli K12 histidine operon and our sequencing results repeatedly obtained from a number of different E. coli K12 strains. The discrepancies include 39 base-pair changes and one addition located predominantly in the proximal portion of the operon. Our data also suggest that neutral and near-neutral mutations do not accumulate to a significant extent in the histidine operon of E. coli strains harbouring strong mutator alleles.

Published
1994

75. Cellular role of DNA polymerase I

Author

Savić, D.J., Janković, M., Kostić, Tatjana, Savić, D.J., Janković, M., and Kostić, Tatjana

Abstract

Escherichia coli possesses three well‐established DNA polymerases, I, II and III. DNA polymerase I (Pol 1) is the main repair polymerase in E. coli and also has a minor but important role in chromosomal replication. A major advantage of Pol I as an experimental system is its simplicity: unlike other replication enzymes, it is active as a single subunit. To a large extent, mutagenesis appears to be the result of (dis) functions of the DNA replication machinery. It is the purpose of this review to provide an integrated view of this relationship with particular emphasis on the role of Pol I in mutagenic events. Copyright

Published
1990

76. DNA sequence analysis of spontaneous histidine mutations in a polA1 strain of Escherichia coli K12 suggests a specific role of the GTGG sequence

Author

Janković, M., Kostić, Tatjana, Savić, D.J., Janković, M., Kostić, Tatjana, and Savić, D.J.

Abstract

Spontaneously arising histidine mutations in an Escherichia coli K12 strain deficient for DNA polymerase I were analysed at the DNA sequence level. We screened approximately 150000 colonies and isolated 106 histidine auxotrophs. Of these, 98 were unstable hisC mutations; 12 representative mutants analysed were shown to have arisen by the excision of a single quadruplet repeat in the sequence 5′-GCTGGCTGGCTGGCTG-3′. Of the eight mutations at other sites, three hisA deletions and one hisD deletion occurred as a consequence of misalignment of tandemly repeated pentamers (hisD) or decamers (hisA). A single hisA point mutation was found to be a missense mutation. Two extended deletions, covering the his operon were not analysed. We could not identify the hisC deletion by sequencing. We conclude that polA1 is a strong imitator that induces mutations mostly of the minus frameshift and deletion type by a Streisinger-type of mispairing in repetitive DNA sequences. Finally, the possible role of a 5′-GTGG-3′ sequence and its inverted or direct complements, which are found in the vicinity of all the deletions and frameshifts, is discussed.

Published
1990

82. EFFECTS OF ACUTE AND REPEATED IMMOBILIZATION STRESS ON OXYGEN CONSUMPTION OF THE ISOLATED INTERSTITIAL RATS' TESTES CELLS.

Author

Kojić, Zvezdana, Videnović-Ivanov, Jelica, and Kostić, Tatjana

Subjects
*ANIMAL immobilization, *OXYGEN consumption, *OXIDATIVE stress, *RESPIRATION, *LABORATORY rats, *OXYGEN electrodes
Abstract

The aim of this study was to investigate the effects of acute and repeated immobilization stress on oxygen consumption of the isolated interstitial rats' testes cells (ISC). The ISC testes cells were isolated acording to Anakwe et al. The oxygen consumption by ISC testes was measured polarographically in vitro with a Clark-type oxygen electrode (YSI-5331, Yellow Springs Instrument), which was done in two phases of respiration: in phase V4 (without ADP) and in phase V3 (with ADP). Repeated immobilization stress (2 hours daily for 10 consecutive days) induced a fall in oxygen consumption in both phases of ISC rat's testes respiration (-10% V4, -4% V3), but this inhibition of respiration was not statistically significant (p>0.05). Acute immobilization stress (2 h) induced decrease in oxygen consumption in both phases of ISC rats' testes respiration (-49% V4, -31% V3) which was statistically significant (p<0.01). Our data suggest that acute and repeated immobilization stress reduce oxygen consumption of ISC testes cells. However, the mechanisams by which immobilization stress induces mitochondrial dysfunction, as well as mechanisms which develop an adaptive response to repeated immobilization remain unclear, so that further investigations of this mechanisms are required. [ABSTRACT FROM AUTHOR]

Published
2009
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83. Uloga glukokortikoida u cirkadijalnoj sinhronizaciji endokrinefunkcije Leydig-ovih ćelija

Author

Kostić, Tatjana, Andrić, Silvana, Kaišarević, (Zorić), Sonja, Kojić, Zvezdana, and Đorđević, Ana

Subjects
Glukokortikoidi, testosteron, cirkadijalni ritam, stres, Leydigove ćelije, Glucocorticoids, testosterone,circadian rhythm, stress, Leydig cells
Abstract

Dva sistema, cirkadijalni časovnik i mehanizam odgovora na stres, ključni su za prilagođavanje i održavanje dinamičke ravnoteže organizma. Uticaj stresa, posredovan glukokortikoidima, u cirkadijalnoj sinhronizaciji endokrinih funkcija Leydig-ovih ćelija odraslih pacova praćen je kroz različite in vivo i ex vivo eksperimentalne modele. Eksperimentalni modeli obuhvatali su primenu stresa imobilizacije (IMO), u različitim cirkadijalnim vremenima, i različitom broju ponavljanja, kao i tretman primene sintetičkih glukokortikoida. Preciznije, ove studije su dizajnirane da ispitaju (1) da li je efekat glukokortikoida na obrazac ekspresije gena časovnika, gena povezanih sa steroidogenezom, kao i drugih gena od značaja za endokrinu funkciju Leydig-ovih ćelija, vremenski zavisan, kao i (2) da li je zavisan od tipa stresnog događaja (akutni i ponovljeni stres). U analiziranim eksperimentalnim modelima, utvrđeno je da je stres generalno povećao nivo glukokortikoida i smanjio nivo testosterona u krvi. Međutim, zapaženo je da je efekat ponovljenog stresa na nivou testostorena u krvi najmanje bio izražen u tamnoj (aktivnoj) fazi dana. Transkripciona analiza gena je otkrila različitu osetljivost na stresne događaje u zavisnosti od cirkadijalnog vremena: većina gena povezanih sa steroidogenezom (Lhcgr, Nr3c1, Cyp11a1, Cyp17a1, Hsd3b1/2) pokazala je smanjenu ekspresiju u neaktivnoj (svetloj) fazi, dok je u tamnoj fazi dana, njihova ekspresija nepromenjena ili čak stimulisana. Stres je takođe uticao i na ekspresiju gena i proteina časovnika i stimulisao ekspresiju Bmal1/BMAL1, Per1/2/PER1. Tretman pacova primenom sintetičkog glukokortikoida pokazao je sličan odgovor kao i IMO tretman. Analiza glavnih komponenti (PCA) pokazala je odsustvo značajnih razlika između tretmana, posebno na Per1 i Rev-erba. Ovakvi rezultati su potvrđeni ex vivo tretmanom Leydig-ovih ćelija hidrokortison-om i blokatorom glukokortikoidnog receptora. Rezultati ukazaju da uticaj glukokortikoidne komponente odgovora na stres na aktivnost Leydig-ovih ćelija, zavisi od vremena i tipa stresa, naglašavajući važnost cirkadijalne aktivnosti u podršci i održavanju homeostaze produkcije androgena, a samim tim i plodnosti kod muškaraca. Two systems, the circadian clock, and the stress response mechanism are key to adjusting and maintaining the body's dynamic balance. The influence of glucocorticoid-mediated stress in the circadian synchronization of the endocrine function of adult rats Leydig cells monitored through various in vivo and ex vivo experimental models. The experimental models included the application of stress by immobilization (IMO), at different circadian times, and with a different number of repetitions, as well as the treatment with the use of synthetic glucocorticoids. Specifically, these studies are designed to examine (1) are the effects of glucocorticoids on the pattern of clock gene expression, steroidogenesis-related genes, and other genes relevant to Leydig cell endocrine function time-dependent, and (2) are the effects dependent on the type of stressful event (acute and recurrent stress). In the analyzed experimental models, it was found that stress generally increased glucocorticoid levels and decreased blood testosterone levels. However, it was noticed that the effect of repeated stress on the level of testosterone in the blood was the least pronounced in the dark (active) phase of the day. Transcriptional analysis of genes revealed different susceptibility to stressful events depending on circadian time: most genes associated with steroidogenesis (Lhcgr, Nr3c1, Cyp11a1, Cyp17a1, Hsd3b1/2) showed reduced expression in the inactive (light) phase, while in the active dark) the phase of the day, their expression unchanged or even stimulated. Stress also affected the expression of clock genes and proteins and stimulated the expression of Bmal1/BMAL1, Per1/2/PER1. Treatment of rats with synthetic glucocorticoids showed a similar response as IMO treatment. Principal component analysis (PCA) showed no significant differences between treatments, especially on Per1 and Rev-erb. These results were confirmed by ex vivo treatment of Leydig cells with Hydrocortison and a glucocorticoid receptor blocker. The results indicate that the effect of the glucocorticoid component of the stress response on Leydig cell activity depends on the time and type of stress, emphasizing the importance of circadian activity in supporting and maintaining androgen homeostasis, and male fertility.

Published
2022

84. The role of glucocorticoids in circadian synchronization of endocrine functions of Leydig cells

Author

Medar, Marija, Kostić, Tatjana, Andrić, Silvana, Kaišarević, (Zorić) Sonja, Kojić, Zvezdana, and Đorđević, Ana

Subjects
Glukokortikoidi, testosteron, cirkadijalni ritam, stres, Leydigove ćelije, Glucocorticoids, testosterone,circadian rhythm, stress, Leydig cells
Abstract

Dva sistema, cirkadijalni časovnik i mehanizam odgovora na stres, ključni su za prilagođavanje i održavanje dinamičke ravnoteže organizma. Uticaj stresa, posredovan glukokortikoidima, u cirkadijalnoj sinhronizaciji endokrinih funkcija Leydig-ovih ćelija odraslih pacova praćen je kroz različite in vivo i ex vivo eksperimentalne modele. Eksperimentalni modeli obuhvatali su primenu stresa imobilizacije (IMO), u različitim cirkadijalnim vremenima, i različitom broju ponavljanja, kao i tretman primene sintetičkih glukokortikoida. Preciznije, ove studije su dizajnirane da ispitaju (1) da li je efekat glukokortikoida na obrazac ekspresije gena časovnika, gena povezanih sa steroidogenezom, kao i drugih gena od značaja za endokrinu funkciju Leydig-ovih ćelija, vremenski zavisan, kao i (2) da li je zavisan od tipa stresnog događaja (akutni i ponovljeni stres). U analiziranim eksperimentalnim modelima, utvrđeno je da je stres generalno povećao nivo glukokortikoida i smanjio nivo testosterona u krvi. Međutim, zapaženo je da je efekat ponovljenog stresa na nivou testostorena u krvi najmanje bio izražen u tamnoj (aktivnoj) fazi dana. Transkripciona analiza gena je otkrila različitu osetljivost na stresne događaje u zavisnosti od cirkadijalnog vremena: većina gena povezanih sa steroidogenezom (Lhcgr, Nr3c1, Cyp11a1, Cyp17a1, Hsd3b1/2) pokazala je smanjenu ekspresiju u neaktivnoj (svetloj) fazi, dok je u tamnoj fazi dana, njihova ekspresija nepromenjena ili čak stimulisana. Stres je takođe uticao i na ekspresiju gena i proteina časovnika i stimulisao ekspresiju Bmal1/BMAL1, Per1/2/PER1. Tretman pacova primenom sintetičkog glukokortikoida pokazao je sličan odgovor kao i IMO tretman. Analiza glavnih komponenti (PCA) pokazala je odsustvo značajnih razlika između tretmana, posebno na Per1 i Rev-erba. Ovakvi rezultati su potvrđeni ex vivo tretmanom Leydig-ovih ćelija hidrokortison-om i blokatorom glukokortikoidnog receptora. Rezultati ukazaju da uticaj glukokortikoidne komponente odgovora na stres na aktivnost Leydig-ovih ćelija, zavisi od vremena i tipa stresa, naglašavajući važnost cirkadijalne aktivnosti u podršci i održavanju homeostaze produkcije androgena, a samim tim i plodnosti kod muškaraca., Two systems, the circadian clock, and the stress response mechanism are key to adjusting and maintaining the body's dynamic balance. The influence of glucocorticoid-mediated stress in the circadian synchronization of the endocrine function of adult rats Leydig cells monitored through various in vivo and ex vivo experimental models. The experimental models included the application of stress by immobilization (IMO), at different circadian times, and with a different number of repetitions, as well as the treatment with the use of synthetic glucocorticoids. Specifically, these studies are designed to examine (1) are the effects of glucocorticoids on the pattern of clock gene expression, steroidogenesis-related genes, and other genes relevant to Leydig cell endocrine function time-dependent, and (2) are the effects dependent on the type of stressful event (acute and recurrent stress). In the analyzed experimental models, it was found that stress generally increased glucocorticoid levels and decreased blood testosterone levels. However, it was noticed that the effect of repeated stress on the level of testosterone in the blood was the least pronounced in the dark (active) phase of the day. Transcriptional analysis of genes revealed different susceptibility to stressful events depending on circadian time: most genes associated with steroidogenesis (Lhcgr, Nr3c1, Cyp11a1, Cyp17a1, Hsd3b1/2) showed reduced expression in the inactive (light) phase, while in the active dark) the phase of the day, their expression unchanged or even stimulated. Stress also affected the expression of clock genes and proteins and stimulated the expression of Bmal1/BMAL1, Per1/2/PER1. Treatment of rats with synthetic glucocorticoids showed a similar response as IMO treatment. Principal component analysis (PCA) showed no significant differences between treatments, especially on Per1 and Rev-erb. These results were confirmed by ex vivo treatment of Leydig cells with Hydrocortison and a glucocorticoid receptor blocker. The results indicate that the effect of the glucocorticoid component of the stress response on Leydig cell activity depends on the time and type of stress, emphasizing the importance of circadian activity in supporting and maintaining androgen homeostasis, and male fertility.

Published
2022

85. Mitohondrijalna biogeneza kao mehanizam adaptacije specifičnih tipova ćelija reproduktivne osovine

Author

Starovlah, Isidora, Andrić, Silvana, Korać, Bato, Kostić, Tatjana, Korać, Aleksandra, and Kaišarević, Sonja

Subjects
mitohondrijalna dinamika, mitohondrijalna biogeneza, mitofuzija, mitofisija, mitofagija, cirkulišući androgeni, spermatozoidi, Lajdigove ćelije, hipotalamus, adenohipofiza, mitochondrial dynamics, mitochondrial biogenesis, mitofusion, mitofision, mitophagy, circulating androgens, spermatozoa, Leydig cells, hypothalamus, adenohypophysis
Abstract

Sažetak: U potrazi za ulogom markera mitohondrijalne dinamike u adaptaciji ćelija reproduktivne osovine, sa posebnim fokusom na spermatozoide, dizajnirani su razliĉiti in vivo pristupi koji mimikriraju situacije vezane za poremećenu homeostazu cirkulišućih androgena (stres, hipogonadizam) u adultnoj humanoj populaciji. Rezultati pokazuju da ponavljani psihofiziĉki stres povećava transkripciju 82% ispitivanih markera mitohondrijalne dinamike i funkcionalnosti, kao i 73% transkripata za markere cAMP i MAPK signalinga koji regulišu homeostazu spermatozoida i mitohondrijalnu dinamiku/funkcionalnost u spermatozoidima. Oporavak organizma od akutnog psihofiziĉkog stresa dovodi do promene transkripcije 91% markera mitohondrijalne dinamike i funkcionalnosti, kao i 91% markera cAMP i MAPK signalinga. Ponavljani psihofiziĉki stres primenjen u razliĉitim vremenskim taĉkama u toku dana menja transkripciju 91% markera mitohondrijalne dinamike/funkcionalnosti, kao i 86% transkripata za elemente cAMP i MAPK signalinga. Hipogonadizam menja transkripciju 61% markera mitobiogeneze, fuzije/arhitekture i funkcionalnosti mitohondrija, kao i 59% transkripata za elemente cAMP i MAPK signalinga. Starenje u kombinaciji sa Sildenafilom menja ekspresiju 56% markera mitobiogeneze, fuzije/arhitekture i mitofunkcionalnosti, i ekspresiju 29% transkripata za elemente cAMP i MAPK signalinga. Starenje u kombinaciji sa Metforminom menja ekspresiju 75% markera mitobiogeneze, fuzije/arhitekture i mitofunkcionalnosti, i ekspresiju 77% markera cAMP i MAPK signalinga. Fiziološki znaĉaj se ogleda u promeni funkcionalnosti spermatozoida. Sumirano, promena ekspresije markera mitohondrijalne dinamike moţe biti jedan od adaptacionih mehanizma spermatozoida za oĉuvanje funkcionalnosti u uslovima poremećene homeostaze cirkulišućih androgena. Abstract: In search of the role of mitochondrial dynamics markers in the adaptation of reproductive axis cells, with a special focus on spermatozoa, various in vivo approaches have been designed related to impaired circulating androgen homeostasis (stress, hypogonadism) that mimic situations in the adult human population. The results show that repeated psychophysical stress increases the transcription of 82% of the examined markers for the mitochondrial dynamics and functionality, as well as 73% of transcripts for markers of cAMP and MAPK signaling that regulate spermatozoa homeostasis and mitochondrial dynamics/functionality in spermatozoa. Recovery of the organism from acute psychophysical stress leads to a change in the transcription of 91% of mitochondrial dynamics and functionality markers, as well as 91% of markers of cAMP and MAPK signaling. Repeated psychophysical stress applied at different time points during the day alters 91% of transcripts of mitochondrial dynamics/functionality markers, as well as 86% of transcripts for cAMP and MAPK signaling elements. Hypogonadism alters the transcription of 61% of mitobiogenesis, fusion/architecture markers, and mitochondrial functionality markers, as well as 59% of transcripts for cAMP and MAPK signaling elements. Ageing in combination with Sildenafil alters the expression of 56% of mitobiogenesis, fusion/architecture and mitofunctionality markers, as well as the expression of 29% of transcripts for cAMP and MAPK signaling elements. Ageing in combination with Metformin alters the expression of 75% of mitobiogenesis, fusion/architecture, and mitofunctionality markers, and the expression of 77% of cAMP and MAPK signaling markers. Physiological significance is reflected in the changes in spermatozoa function. In summary, the change in the expression of mitochondrial dynamics markers may be one of the adaptive mechanisms of spermatozoa for the preservation of the functionality in the conditions of disturbed homeostasis of circulating androgens.

Published
2022

86. The role of insulin and IGF1 receptors in regulation of teroidogenesis and mitochondrial biogenesis in Leydig cells

Author

Radović, Sava, Andrić, Silvana, Kostić, Tatjana, Matić, Gordana, Kojić, Zvezdana, and Kaišarević, Sonja

Subjects
Leydig-ove ćelije, steroidogeneza, steroidogenesis, androgeni, mitochondrial biogenesis, androgens, cell diferentiation, diferencijacija ćelija, mitochondria, mitohondrijalna fuzija, Leydig cell, mitochondrial fusion, insulinski receptor, mitohondrijalna biogeneza, insulin receptor, IGF1 receptor, mitohondrije
Abstract

Leydig-ove ćelije testisa su primarno mesto sinteze muških polnih hormona. Ovi hormoni su neophodani za reproduktivno, ali i za opšte zdravlje budući da su ozbiljni zdravstveni problemi često povezani sa njihovom smanjenom produkcijom. Insulin i insulinu sličan faktor rasta 1, IGF1 (engl. insulin like growth factor 1), i signalizacija koju pokreću preko svojih receptora (INSR i IGF1R), su jedan od ključnih faktora koji regulišu specifični razvoj tkiva, pa i samih gonada. Ipak, uloga i mehanizmi delovanja ovih receptora u steroidogenim tkivima nisu u potpunosti poznati. Stoga je istraživanje uokviru ove doktorske disertacije koncipirano sa ciljem da se, na modelu prepubertalnih (P21) i adultnih (P80) mužjaka miševa sa kondicionalnom delecijom Insr i Igf1r gena u steroidogenim ćelijama (Insr/Igf1r-DKO), definiše uloga INSR i IGF1R u regulisanju diferencijacije i steroidogene funkcije Leydig-ovih ćelija. Pored toga, mužjaci i ženke P21 miševa sa istom delecijom su korišćeni za praćenje ekspresije glavnih markera mitohondrijalne biogeneze i fuzije/arhitekture u Leydigovim ćelijama, ovarijumima i nadbubrežnim žlezdama. Rezultati su potvrdili da delecija Insr i Igf1r u steroidogenim tkivima utiče na diferencijaciju i funkcionalne karakteristike Leydig-ovih ćelija P21 i P80 miševa, upućujući na pojavu tzv. „feminizacije“. Broj Leydig-ovih ćelija izolovanih iz P21 i P80 Insr/Igf1rDKO miševa bio je smanjen, a morfologija i ultrastruktura ovih ćelija izmenjene kod P21 Insr/Igf1rDKO miševa. Steroidogeni kapacitet i aktivnost, kao i ekspresija glavnih elemenata steroidogene mašinerije (Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd3b1 i 6, Hsd17b3, Sf1) bili su smanjeni u Leydig-ovim ćelijama P21 i P80 Insr/Igf1r-DKO miševa, dok je ekspresija transkripcionih represora steroidogeneze (Arr19 i Dax1) bila povećana specifično u istim ćelijama, ali ne i u ostatku testisa. Transkripcioni profil markera muškog pola (Sry, Sox9, Amh) bio je izmenjen u Leydig-ovim ćelijama P21 i P80 Insr/Igf1r-DKO miševa. Transkripcija markera ženskog pola (Rspo1, Wnt4) u testisima, kao i ekspresija Cyp19a1 i produkcija estradiola (E2) u Leydig-ovim ćelijama, P21 i P80 Insr/Igf1r-DKO miševa bile su povećane. Transkripcija markera mitohondrijalne biogenze (Ppargc1a, Tfam, Mtnd1) bila je smanjena u Leydigovim ćelijama P21 Insr/Igf1r-DKO miševa, dok supromene ekspresije izostale u ovarijumima ženki istog genotipa. Isti markeri su bili povećani u nabdubrežnim žlezdama oba pola. Markeri mitohondrijalne fuzije/arhitekture (Mfn1 i Mfn2) bili su povećani u Leydig-ovim ćelijama P21 Insr/Igf1r-DKO miševa, što je praćeno i narušenom mitohondrijalnom fazom steroidogeneze (produkcija progesterona), kao i brojem i morfologijom ovim organela. Ekspresija istih markera u ovarijumima bila je nepromenjena. Sumirano, rezultati ovog istraživanja su pokazali da su INSR i IGF1R važni za diferencijaciju i steroidogenu funkciju Leydig-ovih ćelija P21 i P80 miševa. Takođe, ovi receptori su važni regulatori markera mitohondrijalne biogeneze i fuzije/arhiteture u steroidogenim ćelijama muških gonada P21 miševa, ali ne i u steroidogenim ćelijama ovarijuma. Leydig cells of testes are the primary site of the male sex hormones synthesis. These hormones are indispensable for both reproductive and general health since serious health problems are often associated with their reduced production. Insulin and insulin-like growth factor 1, IGF1 (insulin like growth factor 1), and signaling triggered through their receptors (INSR and IGF1R), are one of the key factors that regulate specific development of tissue including gonads. However, the role and mechanisms of these receptors action in steroidogenic tissues are not known enough. This study was designed to observe the role of INSR and IGF1R in regulating the differentiation and steroidogenic function of Leydig cells by using the model of prepubertal (P21) and adult (P80) male mice with the conditional deletion of the Insr and Igf1r genes in steroidogenic cells (Insr/Igf1r-DKO). In addition, male and female P21 mice with the samedeletion were used to monitor the expression of the main markers of mitochondrial biogenesis and fusion/architecture in Leydig cells, ovaries and adrenal glands. The results confirmed that deletion of Insr and Igf1r in steroidogenic tissues influences differentiation and functional characteristics of Leydig cells isolated from P21 and P80 mice, suggesting an appearance of "feminization". The number of Leydig cells isolated from both P21 and P80 Insr/Igf1r-DKO mice was reduced. Morphology and ultrastructure of Leydig cells were disturbed in P21 Insr/Igf1r-DKO mice. Steroidogenic capacity and activity, as well as expression of the main elements of steroidogenic machinery (Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd3b1 and 6, Hsd17b3, Sf1) were decreased in Leydig cells from P21 and P80 Insr/Igf1r-DKO mice, while the expression of transcriptional repressors of steroidogenesis (Arr19 and Dax1) was increased in the same cells, but not in the rest of the testes. Transcription profile of the male sex markers (Sry, Sox9, Amh) was altered in Leydig cells from P21 and P80 Insr/Igf1r-DKO mice. Transcription of the female sex markers (Rspo1, Wnt4) in the testes, as well as Cyp19a1 expression and estradiol (E2) production in Leydig cells, from P21 and P80 Insr/Igf1rDKO mice were increased. Transcription of mitochondrial biogenesis markers (Ppargc1a, Tfam, Mtnd1) was declined in Leydig cells from P21 Insr/Igf1r-DKO mice, while changes were absent in the ovaries of the same genotype. Transcription of the same markers was increased in the adrenal glands of both sexes. The mitochondrial fusion/architecture markers (Mfn1 and Mfn2) were increased in Leydig cells from Insr/Igf1r-DKO mice and followed by disturbedmitochondrial phase of steroidogenesis (progesterone production), as well as decreased number and disturbed morphology of mitochondria. Expression of the same markers in the ovaries was unchanged. In summary, results of this study showed that INSR and IGF1R are important in differentiation and steroidogenic function of Leydig cells from P21 and P80 mice. Also, these receptors are important regulators of mitochondrial biogenesis and fusion/architecture markers in steroidogenic cells of P21 male mice, but not in steroidogenic cells of ovaries.

Published
2019

87. Functionality and pattern of signaling pathways of Leydig cells in adult rats after administration of anabolic androgenic steroids

Author

Srbovan, Maja M., Andrić, Silvana, Jasnić, Nebojša, Kostić, Tatjana, and Đorđević, Jelena

Subjects
steroidogeneza, steroidogenesis, ARR19, Leydig cells, anabolic androgenic steroids, PRL-JAK-STAT signalizacija, androgeni receptor, membranski potencijal mitohodrija, ADCY-cAMP-PRKA signalizacija, mitochondrial membrane potential, HSD3B, androgen receptor, testosterone, Lajdigove ćelije, androgeni anabolički steroidi, testosteron, ADCY-cAMP-PRKA signaling, PRL-JAK-STAT signaling
Abstract

Lajdigove ćelije intersticijuma testisa su primarno mesto sinteze androgenih hormona, a dominantno testosterona (T). Ovaj hormon je zajedno sa svojim metabolitom dehidrotestosteronom (DHT) neophodan, kako za produženje vrste obezbeđivanjem pravilnog razvoja i funkcionisanja muškog reproduktivnog sistema, tako i za opšte zdravlje individue. U cilju tretiranja brojnih kliničkih poremećaja, kao i u svrhu kontracepcije, sintestisani su derivati androgena čija se primena u klinici zasniva na dejstvu koje ostvaruju posredstvom androgenih i/ili anaboličkih efekata, te su zajedničkim imenom nazvani anabolički androgeni steroidi (AAS). Nažalost, AAS se često zloupotrebljavaju, ne samo od strane profesionalnih i rekreativnih sportista, nego i velike populacije adolescenata, iako je dobro poznato da njihova upotreba u neterapeutske svrhe može izazvati niz neželjenih zdravstvenih posledica. Stoga su AAS svrstani u grupu farmakoloških preparata čija je upotreba strogo regulisana i nisu dostupni bez lekarskog recepta. Uprkos široko rasprostranjenoj kliničkoj upotrebi i zloupotrebi AAS, kao i velikom interesovanju naučne zajednice za ovu problematiku, nisu u potpunosti razjašnjeni molekulski događaji koji su posledica njihove kratkoročne i dugoročne primene. S obzirom na značaj T za reprodukciju i produženje vrste, ali i zdravlje i kvalitet života indivudue, kao i široku primenu i zloupotrebu T i njegovih derivata, neophodno je okarakterisati precizne molekulske događaje nastale kao posledica poremećene homeostaze T. Ovo je važno zbog toga što, prema trenutno dostupnoj literaturi, ne postoji dovoljan broj podataka o funkcionalnosti i obrascima signalnih puteva Lajdigovih ćelija, čija je osnovna uloga sinteza i sekrecija T. Stoga je glavni cilj ovog istraživanja bio da ispita funkcionalnost i obrasce signalnih puteva važnih za održavanje steroidogene funkcije Lajdigovih ćelija, narušene primenom egzogenih agonista i/ili antagonista T u in vivo ili in vitro uslovima. U tu svrhu primenjen je derivat T, testosteron-enantat (TE), koji se najčešće upotrebljava u kliničkoj praksi, ali se i u najvećoj meri zloupotrebljava i najprodavaniji je na tzv. “crnom” tj. ilegalnom tržištu. Ovakav model daje mehanistički pristup, ali ima i translacioni aspekt, s obzirom na to da su upotrebljavane doze/koncentracije T koje se koriste u kliničkoj praksi ili se zloupotrebljavaju. Rezultati su pokazali da in vivo aplikacija TE inhibira steroidogenu funkciju Lajdigovih ćelija odraslih pacova, kao i relativnu ekspresiju gena za komponente cAMP-PRKA signalizacije, kao glavnog regulatora steroidogeneze ovih ćelija... Leydig cells of testis interstitium represent the major site for synthesis of androgenic hormone, primarily testosterone (T). This hormone, together with its metabolite dihydrotestosterone (DHT), is required, not only for the continuation of the species by ensuring proper development and functioning of male reproductive system, but for overall health of an individual as well. For the purpose of treatment of multiple clinical disorders, as well as for contraception purposes, androgen derivatives have been synthesized, the clinical application of which is based on the influence they have through anabolic and/or androgenic effects, thus having a common name anabolic androgenic steroids (AASs). Unfortunately, AASs are often abused, not only by professional and recreational athletes, but by a large population of adolescents as well, although it is well known that non-therapeutic use thereof may cause a series of adverse health effects. Therefore, AASs are classified into a group of pharmacological preparations, the use of which is strictly regulated and which are not available without a medical prescription. Despite widespread clinical use and abuse of AASs, as well as the great interest shown by the scientific community in this field, molecular events resulting from their short-term and long-term use have not been fully clarified. Given the importance of T for reproduction and continuation of the species, but also for health and quality of life of an individual, and the widespread use and abuse of T and its derivatives, it is necessary to characterize precise molecular events resulting from disturbed T homeostasis. This is important because, according to the currently available literature, there is insufficient data on the functionality and patterns of signaling pathways of Leydig cells, the basic role of which is the synthesis and secretion of T. Therefore, the main aim of this study is to examine the functionality and patterns of signaling pathways relevant for maintaining the steroidogenic function of Leydig cells, impaired by the use of exogenous T agonists and/or antagonists under in vivo or in vitro conditions. To this end, a T derivative known as testosterone-enanthate (TE) is applied, which is most commonly used in clinical practice, but is largely abused and best-selling product in the so-called "black" or illegal market. Such model provides a mechanistic approach, but has also a translational aspect, since the applied T doses/concentrations are used in clinical practice or are abused. The results have shown that in vivo application of TE inhibits the steroidogenic function of Leydig cells in adult rats, as well as gene expression for cAMP-PRKA signaling components, being the main regulator of steroidogenesis of such cells...

Published
2018

88. Mehanizmi uključeni u regulaciju smanjene endokrine funkcije Lajdigovih ćelija tokom starenja

Author

Sokanović, Srđan J., Kostić, Tatjana, Jasnić, Nebojša, and Andrić, Silvana

Subjects
hipogonadizam, Aging, steroidogenesis, Lajdigove ćelije steroidogeneza, cAMP, NO-cGMP, energetska homeostaza, hypogpnadism, PDE5, Leydig cells, MAPK, energy homeostasis, Starenje
Abstract

Reproduktivno starenje muškaraca razvija se uporedo sa starenjem organizma, a manifestuje se značajnim promena u funkciji endokrinog sistema i smanjenim fekunditetom. U kontekstu izmenjene funkcionalnosti endokrinog sistema ističe se smanjen nivo testosterona koji zajedno sa brojnim psiho-fizičkim promenama smanjuje kvalitet života jedinke. Konstantno produženje životnog veka i potencijalno neželjena dejstva zamenske terapije androgenima usmeravaju buduća istraživanja ka boljem razumevanju molekularnih osnova muškog hipogonadizma i pospešivanju endogene produkcije androgena u starijem životnom dobu muškaraca. U skladu sa iznetim činjenicama i usmerenjma postavljeni su sledeći ciljevi istraživanja: 1. Karakterizacija staračkog hipogonadizma kod pacova soja Wistar kao modela za istraživanje reproduktivnog starenja; 2. Ispitivanje udela promena cAMP- i cGMP-signalizacije u formiranju starog fenotipa Lć. Najvažnijim rezultatima ustanovljeno je da starenje Wistar pacova prati pojava primarnog hipogonadizma koji je iskazan smanjenom produkcijom testosterona i smanjenim eksprimiranjem steroidogenih gena i proteina od dvanaestog meseca. Zajedno sa smanjenim androgenim kapacitetom poremećena je i cAMP, MAPK i NO-cGMP signalizacija kao i energetska homeostaza u Lć starog fenotipa. Tokom istraživanja ustanovljeno je da smanjen nivo cAMP nije jedini uzročnik smanjenog androgenog kapaciteta, a akutna i hronična inhibicija PDE5 povećava steroidogeni kapacitet i normalizuje energetsku homeostazu u Lć starog fenotipa. Takođe je ustanovljeno da cAMP i cGMP različito regulišu energetsku homeostazu Lć, pri čemu cGMP normalizuje parametre funkcionalnosti mitohondrija i eksprimiranje regulatora energetske homeostaze. Reproductive aging of males develops along with the aging of the organism, and it is manifested by a significant change in the endocrine system and decreased fecundity. The decreased level of testosterone stands out in the context of the changed functionality of the endocrine system which together with numerous psycho-physical changes, reduces the quality of life of the individual. In respect with extended human life and undesirable effects of androgens replacement therapy, further investigations has been dedicated to better understanding of male hypogonadism by the molecular approach as well as endogen testosterone production during the aging. According to the presented facts we defined the two aims of the study: 1. Characterization of the age-related hypogonadism in the male Wistar rats as a model for reproductive aging, and 2. Investigation of the impact of a cAMP and a cGMP disturbances in development of aged Leydig cells. Our results showed appearance of primary hypogonadism during the aging of male Wistar rats, including impair testosterone production and impaired expression of steroidogenic genes and proteins from the 12th month of age. Beside that cAMP, MAPK and NO-cGMP signaling has been disturbed in aged Lc as well as energy homeostasis. We also showed that less production of cAMP is not the only cause of aged Lc sub-functionality and that acute and chronic inhibition of the PDE5 showed positive effect at steroidogenesis and energy homeostasis in aged Lc. Further, cAMP and cGMP differently regulated energy homeostasis of aged LC, with positive impact of the cGMP treatment on mitochondrial functionality and energy homeostasis regulators.

Published
2017

89. Characterization and pathways of synhronization of peripheral biological clock and steroidogenesis in rat Leydig cells

Author

Baburski, Aleksandar Z., Kostić, Tatjana, Matić, Gordana, and Andrić, Silvana

Subjects
steroidogeneza, steroidogenesis, aging, melatonin, Leydig cells, cGMP, hipogonadizam, cAMP, pacov, circadian clock, testosterone, Lajdigove ćelije, starenje, hypogonadism, rat, testosteron, cirkadijalni časovnik
Abstract

Biološki časovnik organizuje metabolizam i fiziološke procese u cirkadijalne ritmove. Na nivou ćelije, on se sastoji od grupe gena koji preko negativnih povratnih sprega održavaju ritam sopstvene transkripcije, ali regulišu i ritmičnost transkripcije mnogih drugih gena. Iako je poznato da su određeni geni časovnika neophodni za sintezu testosterona i fertilnost mužjaka, još uvek nema preciznih podataka o cirkadijalnoj fiziologiji testosteron produkujućih Lajdigovih ćelija. Ova studija je dizajnirana da definiše (1) cirkadijalni obrazac endokrine funkcije Lajdigovih ćelija uključujući i eksprimiranje gena perifernog časovnika i (2) upletenost LH-cAMP signalizacije u sinhronizaciju ritma Lajdigovih ćelija korišćenjem in vivo modela poremećene cAMP homeostaze (hipogonadotropni hipogonadizam, starački hipogonadizam, pinealektomija) i in vitro stimulacije Lajdigovih ćellija. Rezultati su pokazali cirkadijaln ritam funkcije Lajdigovih ćelija koji se ogleda u vremenski koordinisanoj oscilatornoj produkciji testosterona i intracelularnog cAMP, cirkadijalnom eksprimiranju regulatora (Nur77 i Arr19), steroidogenih elementa (Star/StAR, Cyp11a1 i Cyp17a1), kao i elementa časovnika (Bmal1/BMAL1, Per1/2/3, Cry1/2, Rev-erba/b/REV-ERBA, Rorb, Dec1/2, Dbp i E4bp4). Ritam transkripcije osnovnih gena časovnika kao i ključnog elementa steroidogeneze (Star) se održava i u primarnoj kulturi ovih ćelija. Redukcija cAMP detektovana u Lajdigovim ćelijama pacova sa hipogonadotropnim hipogonadizmom stimuliše transkripciju većeg broja gena časovnika: Per2, Rorb, Rev-erbb, Dec1/2, E4bp4, Ck1e/d, i inhibiše Npas2. Sa druge strane, in vitro stimulacija cAMP-signalizacije povećava transkripciju Per1, Dec1/2, Rorb, Npas2 i E4bp4, i smanjuje transkripciju Rev-erba. Starenje, dovodi do opadanja robusnosti cirkadijalne funkcije Lajdigovih ćelija koja se ogleda u smanjenju oscilacija intracelularnog cAMP, smanjenja amplitude eksprimiranja najvažnijih gena časovnika (Bmal1/BMAL1, Per1/2, Rev-erba/REV-ERBA), gena uključenih u metabolizam holesterola (Lipe, Soat2, Scarb1) i steroidogenih gena, (Star/StAR, Cyp11a1, Cyp17a1, Hsd3b1/2/HSD3B, Hsd17b4)... Biological clock organizes metabolic and physiological processes in circadian rhythms. At cell level, it consists of group of genes that regulate its own transcription by negative feedback loop, also regulating transcription rhythmicity of other genes. Although, it is known that some clock genes are necessary for testosterone synthesis and male fertility, there is no precise data about circadian physiology of testosterone-producing Leydig cells. This thesis was design to define (1) circadian pattern of endocrine function of Leydig cells, including expression of clock genes, and (2) involvement of LH-cAMP signaling in synchronization of Leydig cells rhythm using in vivo model of disturbed cAMP homeostasis (hypogonadotropic hypogonadism, hypogonadism in aging, pinealectomy) and in vitro Leydig cell stimulation. Results confirmed circadian rhythmicity of Leydig cell function represented by temporal coordination of cyclic testosterone production and intracellular cAMP, circadian expression of regulators (Nur77, Arr19), steroidogenic (Star/StAR, Cyp11a1 i Cyp17a1) and clock elements (Bmal1/BMAL1, Per1/2/3, Cry1/2, Rev-erba/b/REV-ERBA, Rorb, Dec1/2, Dbp, E4bp4). Rhythm in transcription of core clock genes as well as key steroidogenic element (Star) was preserved in primary Leydig cell culture. Reduction in cAMP, detected in Leydig cells from hypogonadotropic hypogonadal rats, stimulated transcription of some clock genes: Per2, Rorb, Rev-erbb, Dec1/2, E4bp4, Ck1e/d, but inhibited Npas2. On the other hand, in vitro stimulation of cAMP signaling increased transcription of Per1, Dec1/2, Rorb, Npas2 and E4bp4, and reduced transcription of Rev-erba. Aging dulled robustness of circadian function of Leydig cells, represented by decline in intracellular cAMP oscillations and amplitude of expression of core clock genes (Bmal1/BMAL1, Per1/2, Rev-erba/REV-ERBA), genes involved in cholesterol metabolism (Lipe, Soat2, Scarb1) and steroidogenic genes (Star/StAR, Cyp11a1, Cyp17a1, Hsd3b1/2/HSD3B, Hsd17b4). Abolishment of melatonin, a main cue that spread information of light regime via cAMP signaling, stimulated expression of clock (Bmal1/BMAL1, Per1/2) and steroidogenic (Star/StAR, Hsd3b/HSD3B) elements...

Published
2017

90. HLA genotyping in pediatric celiac disease patients.

Author

Stanković B, Radlović N, Leković Z, Ristić D, Radlović V, Nikčević G, Kotur N, Vučićević K, Kostić T, Pavlović S, and Zukic B

Subjects
Adolescent, Adult, Alleles, Case-Control Studies, Child, Child, Preschool, Female, Gene Frequency, Genetic Predisposition to Disease, Genotype, HLA-DQ alpha-Chains genetics, Haplotypes, Homozygote, Humans, Infant, Inflammation, Male, Young Adult, Celiac Disease genetics, HLA-DQ Antigens genetics
Abstract

Celiac disease (CD) is a chronic inflammatory disease in the small intestine triggered by gluten uptake that occurs in genetically susceptible individuals. HLA-DQ2 protein encoded by HLA-DQA1*05 and DQB1*02 alleles is found in 90-95% of CD patients. All of the remaining patients carry HLA-DQ8 protein encoded by HLA-DQA1*03 and DQB1*03:02 alleles. Specific HLA-DQ genotypes define different risk for CD incidence. Presence of susceptible HLA-DQ genotypes does not predict certain disease development, but their absence makes CD very unlikely, close to 100%. Here we presented for the first time the distribution of HLA-DQ genotypes in the group of pediatric celiac patients from the University Children's Hospital, Belgrade, Serbia and estimated risk for CD development that these genotypes confer. Seventy three celiac disease patients and 62 healthy individuals underwent genotyping for DQA1, DQB alleles and DRB1 allele. 94.5% of patients carried alleles that encode DQ2 protein variant and 2.7% carried alleles that encode DQ8 protein variant. Two patients carried single DQB1*02 allele. No patients were negative for all the alleles predisposing to CD. The highest HLA-DQ genotype risk for CD development was found in group of patients homozygous for DQ2.5 haplotype, followed by the group of heterozygous carriers of DQ2.5 haplotype in combination with DQB1*02 allele within the other haplotype. The lowest risk was observed in carriers of a single copy of DQB1*02 or DQA1*05 allele or other non-predisposing alleles. HLA genotyping, more informative than serological testing commonly used, proved to be a useful diagnostic tool for excluding CD development.

Published
2014
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91. [JAK2-V617F mutation in patients with myeloproliferative neoplasms: association with FLT3-ITD mutation].

Author

Spasovski V, Tosić N, Kostić T, Pavlović S, and Colović M

Subjects
Cell Proliferation, Humans, Myeloproliferative Disorders pathology, Janus Kinase 2 genetics, Mutation, Myeloproliferative Disorders genetics, fms-Like Tyrosine Kinase 3 genetics
Abstract

Introduction: An acquired somatic mutation V617F in Janus kinase 2 gene (JAK2) is the cause of uncontrolled proliferation in patients with myeloproliferative neoplasms. It is known that uncontrolled myeloid cell proliferation is also provoked by alteration in other genes, e.g. mutations in receptor tyrosine kinase FLT3 gene. FLT3 represents the most frequently mutated gene in acute myeloid leukaemia. Interestingly, mutated FLT3-ITD (internal tandem duplication) protein is a member of the same signalling pathway as JAK2 protein, the STATS signalling pathway. STAT5 activation is recognized as important for self-renewal of haematopoetic stem cells., Objective: The aim of this study was the detection of JAK2-V617F mutation in patients with myeloproliferative neoplasms. Additionally, we investigated the presence of FLT3-ITD mutation in JAK2-V617F-positive patients in order to shed the light on the hypothesis of a similar role of these two molecular markers in haematological malignancies., Methods: Using allele-specific PCR, 61 patients with known or suspected diagnosis of myeloproliferative neoplasms were tested for the presence of JAK2-V617F mutation. Samples that were positive for JAK2 mutation were subsequently tested for the presence of FLT3-ITD mutation by PCR., Results: Eighteen of 61 analysed patients were positive for JAK2-V617F mutation. Among them, 8/18 samples were diagnosed as polycythaemia vera, and 10/18 as essential thrombocythaemia. None of JAK2-V617F-positive patient was positive for FLT3-ITD mutation., Conclusion: This study suggests that one activating mutation is sufficient for aberrant cell proliferation leading to malignant transformation of haematopoetic stem cell.

Published
2010
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