Structural changes of DNA mediated by HsOrc4 protein

У ћелијама свих живих организама репликација ДНК се одвија у неколико сложених и међусобно веома сличних фаза, а започиње тек када иницијаторни протеински комплекс препозна специфичне секвенце у геному. И поред великог биолошког значаја процеса репликације мало се зна о механизмима који регулишу иницицијацију синтезе ДНК вишећелијских еукариота. Ори секвенце ових организама не садрже консензус секвенце, али су богате АТ базним паровима груписаним у кратке, наизменичне или хомогене низове. Овакви елементи могу да формирају алтернативне структуре, а оне могу бити значајне за интеракцију ори секвенци и иницијаторног комплекса, или могу настати током интеракције иницијационог комплекса и ДНК и бити значајне за наредне фазе иницијације. У овом раду су испитиване интеракције рекомбинантног протеина HsOrc4 са фрагментом изолованим из хуманог репликатора LMNB2 и различитим синтетским ДНК које саджре А и Т елементе. Протеин је изабран за анализу јер чини део комплекса ORC (eng. Origin Recognition Complex), показује самосталну везивну активност и припада фамилији ААА+ суперфамилије макромолекулских шаперона. У раду је потврђено да се HsOrc4 преференцијално везује за ДНК богату АТ базним паровима и ТАТ елементима, и показано да стимулише некакнонску ренатурацију комплементарних фрагмената у специфичну троланчану структуру. Такође је показано да овај протеин стимулише и формирање ТАТ триплекса од одговарајућих олигонуклеотида. У раду је описана и сасвим неочекивана способност протеина да катализује асоцијацију хомоаденинских олигонуклеотида у раније непознате дволанчане и, вероватно, четвороланчане структуре. Хомоаденинске структуре биле су зависне од магнезијумових јона, термостабилне и повезане Хугстиновим везама. Судећи према деловању мутираних форми протеинa HsOrc4 неактивних у везивању или хидролизи АТР-а, за каталитичку активност HsOrc4 неопходно је везивање, али не и хидролиза АТР-а. Будући да је основна улога иницијационог комплекса да ремоделује ори секвенцу и припреми је за наредне фазе иницијације, описано деловање протеина HsOrc4 могло би да допринесе формирању комплексне архитектуре ори репликације и да има значајну улогу у иницијацији репликације.In multicellular eukaryotes DNA replication initiates at fixed chromosomal sites called origins of replication (ori). They host initiation start sites and binding sites for universal eukaryotic initiator called origin recognition complex (ORC), but do not share conserved consensus sequences. All origins are AT rich and contain homopurine/homopyrimidine stretches that can form alternative DNA structures, potentially important for origin function. In order to understand alternative structures of origins and the manner in which specific proteins influence DNA structure, we have investigated interactions of recombinant protein HsOrc4 with various origin or synthetic DNAs. HsOrc4 was chosen as DNA-binding component of ORC and a member of the AAA+ superfamily of macromolecular chaperones. HsOrc4 preferentially recognized double-stranded and triple-stranded AT rich DNA and stimulated renaturation of the origin fragment lboI into specific triple stranded structure, or renaturation of complementary oligonucleotides into intermolecular TAT triplex. An unexpected feature of HsOrc4 shown in our study was its ability to catalyse self-association of adenines creating homoadenine duplexes and possibly quadruplexes. The action of HsOrc4 was dependant on the presence of magnesium ions and ATP. ATP had a role as a cofactor, because HsOrc4 did not require its hydrolysis for activity. The mutated form HsOrc4 WA, which could not bind ATP, was inactive in restructuring reactions, whereas mutated form HsOrc4 WB, unable to hydrolyse ATP, showed similar activity to the wild type HsOrc4. Ability to form Hoogsteen bonds was essential for substrates in restructuring reactions, as these bonds held newly formed structures together. Substrates containing 7-Deaza-ATP, dATP analogue unable to form Hoogsteen bonds, were inactive in these assays. The features of HsOrc4 shown in this study are potentially biologically important, because its action could expand structural repertoire of genomes by producing energetically unfavourable DNA structures. These structures may be involved in determination of origin specificity

Analysis of CFTR Gene Variants in Idiopathic Bronchiectasis in Serbian Children

This study has investigated a potential role of common Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene variants in the etiology of noncystic fibrosis bronchiectasis in Serbian children. The study has encompassed 48 patients (19 male and 29 female, aged between 5 and 18 years, median age 10.6 +/- 3.3), diagnosed with idiopathic bronchiectasis based on high-resolution computed tomography of thorax and pathologic examination of lobectomy materials. The CFTR gene analysis was performed on genomic DNA extracted from peripheral blood samples of patients by polymerase chain reaction (PCR)-Mediated Site-Directed Mutagenesis method, Denaturing Gradient Gel Electrophoresis method, and DNA sequencing. Mutation c.1521_1523delCTT (F508del) was detected with an allelic frequency of 1.0%, and c.224G gt A (R75Q) variant. Carriers of c.1210-12T[5] (IVS8-5T) allele were significantly more common than in the general population (10.4% vs. 5.0%, P = 0.0302). The frequency of homozygotes for Met 470 allele was higher in patients than in the general population (33% vs. 20%), while heterozygotes for p. Met470Val were less frequent (31% vs. 50%), and this difference was statistically significant (P = 0.0222). The results obtained in this study indicate involvement of 2 common CFTR variants, c. 1210-12T[5] and c. 1408A, in idiopathic bronchiectasis in children, but this observation should be further confirmed by more extensive analysis of the CFTR gene in a larger group of patients

CFTR gene analysis in patient with atypical cystic fibrosis

U ovom radu je prikazan slučaj atipične cistične fibroze sa graničnom vrednošću znojnog testa. U većini slučajeva znojni test je glavni dijagnostički parametar za dijagnostikovanje cistične fibroze, ali se dijagnoza može potvrditi samo na osnovu rezultata molekularno-genetičkog testiranja. Utvrđeno je da je pacijent složeni heterozigot za dve CFTR mutacije, F508del i D1152H. Prisustvo mutacije F508del detektovano je PSM metodom, dok je za analizu prisustva druge mutacije korišćena DGGE metoda. Strategija detekcije mutacija kod pacijenata sa cističnom fibrozom, naročito onih sa atipičnim prezentacijama bolesti koji nose ređe mutacije, trebalo bi da uključuje i direktne i indirektne metode molekularne dijagnostike.This paper reports a case of a patient presenting with atypical cystic fibrosis whose sweat test shows borderline values. In vast majority of cases the sweat test is essential diagnostic tool for establishing the diagnosis of cystic fibrosis, but only after the molecular genetic testing the diagnosis can be confirmed. The patient was found to be compound heterozygote for two CFTR mutations, F508del and D1152H. The presence of F508del mutation was analyzed by PSM method, while the screening for the second mutation was performed using DGGE. The strategy of mutation detection in cystic fibrosis patients, especially those with atypical presentations who carry less frequent mutations, should include both direct and indirect methods of molecular diagnostics

Evaluation of toxicity and antioxidative effects of Tussilago farfara and Verbascum thapsus water extracts in zebrafish and in bronchial epithelial cells

Tussilago farfara (coltsfoot) and Verbascum thapsus (mullein) have been used as folk remedies for treating respiratory disorders. The aim of this study was to test the toxicity of the water extracts of T. farfara and V. thapsus in vivo in zebrafish and in vitro in BEAS 2B epithelial bronchial cells. To the best of our knowledge, this is the first study to investigate the antioxidative properties of T. farfara and V. thapsus extracts in cell culture. Our results show that the T. farfara leaf extract does not produce toxic effects on zebrafish embryos or BEAS 2B cells, and that it has a protective effect in BEAS 2B after induction of oxidative stress. The water extract from V. thapsus displayed pronounced toxic effects on zebrafish embryos and BEAS 2B cells and did not exhibit a significant antioxidative effect on BEAS 2B cells exposed to oxidative stress. Our results suggest that the use of T. farfara water leaf extract is potentially safe and effective in treating respiratory disorders, whereas the use of V. thapsus needs further investigation

Analysis of Y chromosome microdeletions and CFTR gene mutations as genetic markers of infertility in Serbian men

Uvod/Cilj. Poremećena plodnost muškog partnera je glavni uzrok infertiliteta kod polovine neplodnih parova. Na genetskom nivou infertilitet kod muškaraca mogu uzrokovati hromozomske aberacije ili genske mutacije. U ovoj studiji analizirano je prisustvo i tip mikrodelecija Y hromozoma i mutacija u genu za regulator transmembranske provodljivosti u cističnoj fibrozi (CFTR) kao genetska osnova infertiliteta kod muškaraca u Srbiji. Cilj studije je bio da se analiziraju mutacije u CFTR genu i mikrodelecije Y hromozoma, kao potencijalni uzroci infertiliteta kod muškaraca u Srbiji, kao i da se testira hipoteza da su CFTR mutacije kod infertilnih muškaraca predominantno locirane u nekoliko poslednjih egzona. Metode. Studija je obuhvatila 33 muškarca sa oligo ili azospermijom. Detekcija mikrodelecija Y hromozoma u regionu faktora azospermije (AZF) vršena je pomoću multipleks PCR metode. Pretraživanje CFTR gena vršeno je metodom elektroforeze u gelu sa gradijentom denaturišućeg agensa (DGGE). Rezultati. Delecije Y hromozoma su detektovane kod četiri bolesnika, predominantno u AZFc regionu (četiri od ukupno šest). Mutacije u CFTR genu su detektovane na osam od 66 analizovanih hromozoma infertilnih muškaraca. Najčešće detektovana CFTR mutacija je F508del (šest od osam). Zaključak. Ova studija je potvrdila da mikrodelecije Y hromozoma i mutacije u CFTR genu igraju važnu ulogu u etiologiji infertiliteta kod muškaraca u Srbiji. Genetsko testiranje koje obuhvata detekciju mikrodelecija Y hromozoma i mutacija u CFTR genu uvedeno je u rutinsku dijagnostičku praksu i ponuđeno je parovima koji pristupaju asistiranoj reprodukciji. S obzirom da tip mikrodelecija Y hromozoma i tip mutacija u CFTR genu imaju prognostički značaj, preporuka je da se genotipizacija AZF regiona i CFTR gena ne vrši samo kod bolesnika sa umanjenim kvalitetom sperme pre pristupanja asistiranoj reprodukciji, već i u svrhe preimplantacione i prenatalne dijagnostike kod parova kod kojih je uspešno izvršena in vitro fertilizacija.Background/Aim. Impaired fertility of a male partner is the main cause of infertility in up to one half of all infertile couples. At the genetic level, male infertility can be caused by chromosome aberrations or gene mutations. The presence and types of Y chromosome microdeletions and cystic fybrosis transmembrane conductance regulator (CFTR) gene mutations as genetic cause of male infertility was tested in Serbian men. The aim of this study was to analyze CFTR gene mutations and Y chromosome microdelations as potential causes of male infertility in Serbian patients, as well as to test the hypothesis that CFTR mutations in infertile men are predominantly located in the several last exons of the gene. Methods. This study has encompassed 33 men with oligo- or azoospermia. The screening for Y chromosome microdeletions in the azoospermia factor (AZF) region was performed by multiplex PCR analysis. The screening of the CFTR gene was performed by denaturing gradient gel electrophoresis (DGGE) method. Results. Deletions on Y chromosome were detected in four patients, predominantly in AZFc region (four of total six deletions). Mutations in the CFTR gene were detected on eight out of 66 analyzed chromosomes of infertile men. The most common mutation was F508del (six of total eight mutations). Conclusion. This study confirmed that both Y chromosome microdeletions and CFTR gene mutations played important role in etiology of male infertility in Serbian infertile men. Genetic testing for Y chromosome microdeletions and CFTR gene mutations has been introduced in routine diagnostics and offered to couples undergoing assisted reproduction techniques. Considering that both the type of Y chromosome microdeletion and the type of CFTR mutation have a prognostic value, it is recommended that AZF and CFTR genotyping should not only be performed in patients with reduced sperm quality before undergoing assisted reproduction, but also for the purpose of preimplantation and prenatal diagnostics in couples in which in vitro fertilization has been performed successfully

Noncanonical DNA elements in the lamin B2 origin of DNA replication

DNA replication origins of eukaryotes lack linear replicator elements but contain short (dT)(n) (dA)(n) sequences that could build mutually equivalent unorthodox structures. Here we report that the lamin B2 origin of DNA replication adopts an alternative form characterized by unpaired regions CTTTTTTTTTTCC/GGAAAAAAAAAAG (3900-3912) and CCTTTTTTTTC/GAAAAAAAAGG (4141-4151). Both unpaired regions are resistant to DNase and except in central parts of their homopyrimidine strands are sensitive to single strand-specific chemicals. Interactions that protect central pyrimidines probably stabilize the bubble-like areas. Because DNA fragments containing either one or both bubbles migrate in TBM (89 mM Tris base, 89 mM boric acid, and 2 mM MgCl2) PAGE even faster than expected from their linear size, interacting regions are expected to belong to the same molecule. In an origin fragment containing a single bubble, free homopyrimidine strand can only interact with Hoogsteen hydrogen bonding surfaces from a complementary double stranded sequence. Indeed, this origin fragment reacts with triplex preferring antibody. In competition binding experiments control double stranded DNA or single stranded (dT) 40 do not affect origin-antibody interaction, whereas TAT and GGC triplexes exert competitive effect. Because the chosen fragment does not contain potential GGC forming sequences, these experiments confirm that the lamin B2 origin adopts a structure partly composed of intramolecular TAT triads

Functional analysis of novel alpha-1 antitrypsin variants G320R and V321F

Alpha-1 antitrypsin (AAT) gene is highly polymorphic, with a large number of rare variants whose phenotypic consequences often remain inconclusive. Studies addressing functional characteristics of AAT variants are of significant biomedical importance since deficiency and dysfunctionality of AAT are associated with liver and lung diseases. We report the results of the functional analysis of two naturally occurring AAT variants, G320R and V321F, previously identified in patients with lung disease. Neither of variants has been fully functionally characterized. In order to perform their functional analysis both variants were expressed in prokaryotic and eukaryotic systems and their intracellular localization, activity, stability, and polymerization were determined. The results of this study demonstrated that variants G320R and V321F have neither impaired activity against porcine pancreatic elastase nor propensity to form polymers. However, both variants had altered electrophoretic mobility and reduced thermostability when compared to M variant of the protein, indicating a slightly impaired secondary or tertiary structure

Tumor necrosis factor alpha and alpha-1 antitrypsin gene variants in Serbian pediatric arterial ischemic stroke patients

Etiologija arterijskog ishemijskog moždanog udara (AIS) kod dece je veoma kompleksna i razlikuje se od one kod odraslih. Iako je redak, moždani udar kod dece predstavlja značajan uzrok mortaliteta i morbiditeta. Sve je više podataka o ulozi genetičkih faktora, uključujući tu i medijatore inflamacije, u nastanku i ishodu moždanog udara. U ovoj studiji, odabrali smo da ispitamo ulogu polimorfizma -308G/A u genu za faktor nekroze tumora-alfa (eng. Tumor Necrosis Factor α -TNFα), kao i mutacija S i Z u genu za alfa1-antitripsin (AAT)u etiologiji moždanog udara kod dece. Polimorfizam -308G/A u genu za TNFα dovodi do povećanja koncentracije ovog proteina u plazmi, što bi moglo da doprinese patologiji moždanog udara. Pokazano je i da povišena koncentracija AAT može da predstavlja rizik za nastanak moždanog udara kod dece. S obzirom da mutacije S i Z u genu za AAT dovode do smanjenja koncentracije ovog proteina u plazmi, one bi mogle da imaju protektivnu ulogu kada je u pitanju moždani udar.Genske varijante TNFα (-308G/A) i AAT (S i Z) su ispitivane u grupi od 26 dece sa AIS i 100 odraslih osoba PCR/RFLP metodom.Nije nađena statistički značajna razlika u učestalosti -308G/A TNFα polimorfizma između pacijenata i kontrola, tako da u našoj grupi pacijenata TNFα najverovatnije nije imao značajnu ulogu u razvoju bolesti. Ni kod jednog pacijenta nije pronađena nijedna od ispitivanih mutacija u genu za AAT, što je bilo u skladu sa potencijalnom protektivnom ulogom ovih varijanti.AIS je multifaktorijalno oboljenje, u čijoj patologiji veliki broj gena ima -ulogu, tako da je potrebna dalja analiza zajedničkog delovanja većeg broja genskih varijanti da bi se rasvetlila njihova uloga kao genetičkih faktora rizika i njihovog uticaja na razvoj i ishod moždanog udara.The etiology of arterial ischemic stroke (AIS) in children is complex, and different from that in adults. Although rare, stroke in children is an important cause of mortality and morbidity. There is increasing evidence that genetic factors, including inflammation mediators, have a role in occurrence and outcome of stroke. We have chosen to assess the role of polymorphism -308G/A in the promoter of tumor necrosis factor α (TNFα) gene and S and Z mutations in alpha 1-antitrypsin (AAT) gene in the etiology of stroke in children. TNFα polymorphism affects plasma levels of this proinflamatory cytokine, and this could contribute to stroke pathology. It has been shown that increased AAT concentration may present a risk for AIS in children. Since S and Z mutations in AAT gene reduce its levels in plasma they could have a protective role in pediatric stroke. In this study twenty six children with AIS and 100 unrelated individuals from Serbian general population were investigated by PCR/RFLP for these gene variations. No statistically significant difference was observed between patients and general population in distribution of genotypes for -308G/A TNFα polymorphism, so its contributory role in the etiology of stroke was not evident in our group of patients. None of the tested AAT gene mutations were found in patients, which is in concordance with the proposed protective role of deficient AAT variants. AIS is a multifactorial disease, with many genes having a modest role in its pathophysiology, so further analyses of their combined effect are needed to elucidate genetic risk factors in the etiology and outcome of stroke in pediatric patients

Alpha-1-Antitrypsin Deficiency in Serbian Adults with Lung Diseases

Aim: Alpha-1-antitrypsin (A1AT) is the main inhibitor of neutrophil elastase, and severe alpha-1-antitrypsin deficiency (A1ATD) is a genetic risk factor for early-onset emphysema. Despite the relatively high prevalence of A1ATD, this condition is frequently underdiagnosed. Our aim was to determine the distribution of the A1ATD phenotypes/alleles in patients with lung diseases as well as in the Serbian population. Methods: The study included the adults with chronic obstructive pulmonary disease (COPD) (n = 348), asthma (n = 71), and bronchiectasis (n = 35); the control was 1435 healthy blood donors. The A1ATD variants were identified by isoelectric focusing or polymerase chain reaction-mediated site-directed mutagenesis. Results: PiMZ heterozygotes, PiZZ homozygotes, and Z allele carriers are associated with significantly higher risk of developing COPD than healthy individuals (odds ratios 3.43, 42.42, and 5.49 respectively). The calculated prevalence of PiZZ, PiMZ, and PiSZ was higher in patients with COPD (1:202, 1:8, and 1:1243) than in the Serbian population (1: 5519, 1: 38, and 1:5519). Conclusion: The high prevalence of A1ATD phenotypes/allele in our population has confirmed the necessity of screening for A1ATD in patients with COPD. On the other hand, on the basis of the estimated number of those with A1ATD among the COPD patients, it is possible to assess the diagnostic efficiency of A1ATD in the Serbian population

Prevalence of S and Z alpha 1-antitrypsin mutations in patients with pancreatic diseases in Serbian population

Jedan od osnovnih izazova u proučavanju patologije bolesti pankreasa predstavlja dalje razjašnjavanje uloge proteaza i antiproteaza, zbog toga što poremećena ravnoteža između njih može dovesti do oštećenja pankreasa. Alfa 1-antitripsin (AAT) je jedan od najvažnijih inhibitora proteolitičkih enzima u serumu, među kojima su i enzimi pankreasa: tripsin, himotripsin i elastaza. Pretpostavlja se da mutacije u AAT genu mogu da utiču na pojavu i razvoj bolesti pankreasa. Prisustvo najčešćih mutacija u AAT genu, označenih kao Z i S, analizirano je u 160 pacijenata sa bolestima pankreasa (50 pacijenata sa kancerom pankreasa, 50 pacijenata sa hroničnim pankreatitisom i 60 pacijenata sa dijabetesom tipa 2) i u 129 zdravih osoba. Prisustvo mutacija detektovano je analizom dužina restrikcionih fragmenata. Jedan pacijent sa kancerom pankreasa je bio heterozigotni nosilac Z mutacije, kao i jedan pacijent sa dijabetesom tipa 2. Jedan pacijent sa hroničnim pankreatitisom je bio heterozigotni nosilac S mutacije. Dve najčešće mutacije u AAT genu su bile statistički značajno učestalije kod pacijenata sa bolestima pankreasa (3 / 160 pacijenata, alelska frekvencija 0,9%) nego u kontrolnoj grupi (1 / 129 osoba, alelska frekvencija 0,4%). Rezultati ove studije, koje ukazuju na moguću povezanost Z i S mutacija sa umerenim povećanjem rizika za razvoj bolesti pankreasa.One of the key points in research of pancreatic disease pathology is further elucidation of the role of proteases and antiproteases, since their imbalance can lead to pancreatic injury. Alpha 1-antitrypsin (AAT) is one of the most important serum inhibitors of proteolytic enzymes, including pancreatic enzymes trypsin, chymotrypsin and elastase. It is speculated that mutations in the AAT gene may influence the onset and the development of pancreatic disease. The presence of the most common AAT mutations Z and S was analyzed in 160 patients with pancreatic diseases (50 patients with pancreatic cancer, 50 patients with chronic pancreatitis and 60 patients with type 2 diabetes mellitus) and 129 healthy individuals by PCR-mediated site-directed mutagenesis (PSM) method. One patient with pancreatic cancer was found to be a carrier of Z mutation, as well as one patient with type 2 diabetes mellitus. One patient with chronic pancreatitis was found to be a carrier of S mutation. The common AAT mutations were statistically significantly over-represented in patients with pancreatic diseases (3 of 160 patients, allelic frequency 0.9%) than in the control group (1 of 129 individuals, allelic frequency 0.4%). The results of this study, requiring confirmation, suggest that common AAT mutations Z and S may be associated with a modest increase in susceptibility to the development of pancreatic disease