Sex hormone binding globulin gene polymorphisms and metabolic syndrome in postmenopausal Turkish women

Background: Insulin resistance is associated with obesity, glucose intolerance or diabetes, hypertension, dyslipidemia, and cardiovascular disease. Constellation of these risk factors iscalled metabolic syndrome (MetS). MetS is common among postmenopausal women. Low sex hormone binding globulin (SHBG) levels associate with an increased risk of MetS in postmenopausal women. Variations in SHBG gene associate with low levels of circulating SHBG levels. We aim to study the association between SHBG gene polymorphisms — rs1799941 (A/G) and rs6257 (T/C) — with MetS among postmenopausal women.Methods: The study population consisted of 182 postmenopausal women with MetS and119 control subjects. We analyzed the allele frequencies of SHBG gene polymorphisms in relationto the risk components of MetS.Results: MetS patients displayed significantly lower SHBG levels compared to the lean controlsubjects (p = 0.036). rs1799941 A allele was associated with high SHBG levels (p = 0.031), low blood pressure, body mass index and waist circumference. The number of ‘high risk’ alleles (G allele of the rs1799941 and T allele of rs6257) correlated positively with waist circumference (r = 0.203, p = 0.006) and negatively with SHBG levels (r = –0.291, p = 0.024).Conclusions: SHBG gene polymorphisms associate with SHBG levels and MetS risk components among postmenopausal women. Hence, A allele (rs1799941) may have a protectiveeffect for MetS through its association with high SHBG levels among postmenopausal women

Propensities of Amino Acid Pairings in Secondary Structure of Globular Proteins

A class of secondary structure prediction algorithms use the information from the statistics of the residue pairs found in secondary structural elements. Because the protein folding process is dominated by backbone hydrogen bonding, an approach based on backbone hydrogen-bonded residue pairings would improve the predicting capabilities of these class algorithms. The reliability of the prediction algorithms depends on the quality of the statistics, therefore, of the data set. In this study, it was aimed to determine the propensities of the backbone hydrogen-bonded residue pairings for secondary structural elements of alpha-helix and beta-sheet in globular proteins using a new and comprehensive data set created from the peptides deposited in Worldwide Protein Data Bank. A master data set including 4882 globular peptide chains with resolution better than 2.5 angstrom, sequence identity smaller than 25% and length of no shorter than 100 residues were created. Separate data sub sets also were created for helix and sheet structures from master set and each sub set includes 4594 and 4483 chains, respectively. Backbone hydrogen-bonded residue pairings in helices and sheets were detected and the propensities of them were represented as odds ratios (observed/[random or expected]) in matrices. Propensities assigned by this study to the residue pairings in secondary structural elements (as helix, overall strands, parallel strands and antiparallel strands) differ from the previous studies by 19 to 34%. These dissimilarities are important and they would cause further improvements in secondary structure prediction algorithms

Go proteininin alfa altbiriminin üçüncül yapısının deneysel ve kuramsal olarak incelenmesi

Go PROTEİNİNİN ALFA ALTBİRİMİNİN ÜÇÜNCÜL YAPISININ DENEYSEL VE KURAMSAL OLARAK İNCELENMESİ Bu çalışmada, merkezi sinir sistemi ve beyinde yaygın bulunan Go proteininin a altbiriminin yapısı deneysel ve teorik yöntemlerle incelendi. Goa’nın üçüncül yapısı ve işlevi tam olarak bilinmediğinden, benzeşim modellemisi çalışmalarıyla yapısı ve işlevi aydınlatılmaya çalışıldı. Ekspresyon deneylerine pGEX-4T2/BL21(DE3)pLysS sisteminde başlandı. IPTG’ye bağlı yüksek ekspresyon düzeyine ulaşılarak Go protein büyük miktarlarda elde edilmesine karşın, protein çözünür olarak elde edilemedi. Proteinin çözünür kılmak için birinci deneysel strateji olarak farklı kültür koşulları denendi. Çözünürlük sorunu aşılamadığından ikinci deneysel stratejisi olarak protein inklüzyon cisimcikleri olarak saflaştırılıp 1 x PBS’e karşı diyaliz edildi. Çözünürlük sorununun devam etmesi üzerine NDSB ajanı, pET-28a ve pQE-80 vektör sistemleri kullanılarak yeni girişimlerde bulunuldu. NDSB ajanı ve pET-28a sistemi ile çözünürlük sorunu aşılamamasına karşın pQE-80 vektör sistemi ile çözünür protein elde edilmiştir. Teorik çalışmada, yapısı bilinen proteinlerin dizileri kalıp olarak kullanılarak Goa’nın üçüncül yapısı modellendi. Genel G-proteini topolojisi elde edildi. Ancak 115-122. amino asitleri kapsayan bölgedeki halkanın yapısı dizi benzerliğinin yetersizliği nedeniyle son şekline kavuşturulamadı. Modele ilişkin moleküler dinamik çalışmaları sürmektedir. STUDIES ON THE TERTIARY STRUCTURE OF ALPHA SUBUNIT OF Go PROTEIN BY EXPERIMINTAL AND THEORETICAL METHODS In this study, the tertiary structure of a-subunit of Go (Goa) which is the most abundant type of G-protein in central nervous system was studied by both experimental and theoretical methods. Because the tertiary structure of Goa is unknown and the function of is not clear yet, in context of the structure-to-function paradigm it was aimed to propose arguments for the function of Goa using homology modelling. Expression experiments were initiated using pGEX-4T2/BL21(DE3)pLysS system. Due to IPTG dependent overexpression, large amounts of Go protein was obtained but the protein was mostly insoluble. To overcome the solubility problem, different incubation temperatures and IPTG concentrations were tried as the first experimental strategy. This did not result in increase of soluble protein. As the second experimental strategy, the protein was purified as inclusion bodies and then dialysed against 1xPBS buffer. The soluble protein could not be obtained by this method, either. Two other attempts, one using NDSB, a mild solubilizing agent, and the other, cloning to the pET-28 vector system, did not work. Finally, Go protein was purified from cytoplasmic fraction using pQE-80 vector system. In theoretical studies, the tertiary structure of Goa was modelled by using known structures that have high sequence similarity to our sequence as a template. Overall G-protein topology was obtained except for a loop encompassing residues 115-122. Molecular dynamical and molecular mechanics studies to refine the modelled structure are in progress

G protein mutations in pituitary tumors: A study on Turkish patients

Activating mutations of the G proteins, G8α (gsp) and Gi2a. (gip) have been reported in subsets of pituitary tumors. The objective of the study was to assess the frequency of gsp and gip mutations in pituitary tumors from Turkish patients and to investigate the possibility of mutations of protein kinase A catalytic subunit (PKAC) that activates the downstream effectors of adenylyl cyclase. PCR-amplified genomic DNA was analyzed for the presence of mutations in codons 201 and 227 of Gsα, codon 179 and 205 of Gi2α and codon 196 of PKAC, by single strand conformation polymorphism analysis, allele-specific oligonucleotide hybridization and DNA sequencing. Twenty-two patients from Turkey, 15 females and 7 males were investigated; 7 somatotroph adenomas, 7 clinically non-functioning tumors, 7 prolactinomas and 1 corticotroph adenoma. G protein mutations were identified in 6 of 22 (27.3%) pituitary tumors. Four tumors (3/7 somatotroph adenomas, 43%, 1/7 clinically non-functioning tumor) demonstrated gsp mutations at codon 201 arginine to cysteine and one recurrent somatotroph adenoma demonstrated a mutation of the Gi2α, gene at codon 193 lysine to arginine. One tumor exhibited a C to T variation in the intervening sequence between codons 179 and 205 of the Gi2α gene. No mutations at codon 227 of Gsα, codons 179 and 205 of Gi2α, and codon 196 of the PKAC gene were identified

Over Tümörlerinde Gip Mutasyonlarının Hibritleme ve SSCP Yöntemiyle İncelenmesi

Bu çalışmada sinyal iletim sisteminde önemli rolü olan G-proteinlerinin a-altbirimlerindeki gsp ve gip mutasyonlarının over ve paratiroid tümörlerindeki olası rolü araştırıldı. a-altbirimlerindeki olası mutasyonları incelemek için taze doku ve parafine gömülü doku örneklerinden DNA izole edildi. Elde edilen DNA'dan Gas proteininin 201, Gai2 proteininin 179 ve 205 numaralı kodonları içeren bölgeler, özgül primerler kullanılarak polimeraz zincir reaksiyonu ile çoğaltıldı. PZR ürünlerindeki mutasyonlar SSCP (Tek Sarmal Konformasyon Polimorfizmi) yöntemiyle incelendi. Örnekler, gliserollü MDE ve denatüre edici olmayan poliakrilamid jellerinde elektroforeze tabi tutulduktan sonra gümüş nitratla boyandı. Ayrıca, nokta-emdirme düzeneği kullanılarak naylon filtreye emdirilen PZR ürünleri radyoaktif işaretlenmiş allel seçici problarla hibritleştirildi. SSCP analizi sonuçları ile uyumlu şekilde, hiçbir örnekte gsp ve gip mutasyonlarına rastlanmadı. SUMMARY The overexpression of G-proteins a-subunits as a result of mutations has been associated with growth and neoplasia. In this study, gsp and gip mutations in 12 ovarian tumors and 6 parathyroid tumors were studied. DNA was isolated from both fresh tissue and paraffin-embedded tissue and regions encompassing codon 201 of Gsa and codons 179 and 205 of Gi2a were amplified using the polymerase chain reaction. To identify the role of gsp and gip mutations in these neoplasias, DNA samples were denatured and subjected to SSCP analysis in non-denaturing polyacrylamide gels and MDE gels containing 6% glycerol. Comparison of the mobility of the variously folded single-strand conformers of tumor and normal DNA revealed no differences in these samples. The same DNA samples were dot blotted onto nylon membranes and hybridized with radiolabeled allele specific oligonucleotide probes. Consistent with SSCP analysis, no gsp and gip mutations were detected in either of the tumor samples

Propensities of Some Amino Acid Pairings in alpha-Helices Vary with Length

The results of secondary structure prediction methods are widely used in applications in biotechnology and bioinformatics. However, the accuracy limit of these methods could be improved up to 92%. One approach to achieve this goal is to harvest information from the primary structure of the peptide. This study aims to contribute to this goal by investigating the variations in propensity of amino acid pairings to alpha-helices in globular proteins depending on helix length. (n):(n + 4) residue pairings were determined using a comprehensive peptide data set according to backbone hydrogen bond criterion which states that backbone hydrogen bond is the dominant driving force of protein folding. Helix length is limited to 13 to 26 residues. Findings of this study show that propensities of ALA:GLY and GLY:GLU pairings to alpha-helix in globular protein increase with increasing helix length but of ALA:ALA and ALA:VAL decrease. While the frequencies of ILE:ALA, LEU:ALA, LEU:GLN, LEU:GLU, LEU:LEU, MET:ILE and VAL:LEU pairings remain roughly constant with length, the 25 residue pairings have varying propensities in narrow helix lengths. The remaining pairings have no prominent propensity to alpha-helices

GNAI2 Geninde İki Aday Polimorfizm

Amaç: GNAI2 geninin 193 numaralı kodonunda bir hipofiz tümöründe ilk kez saptanmış olan bir mutasyonun polimorfizm olma olasılığının incelenmesi. Hastalar ve Yöntem: Kandan elde edilen DNA’lar GNAI2 geninin beşinci ve altıncı eksonları ile aralarındaki intron bölgesini içerecek şekilde Polimeraz Zincir Tepkimesi (PZT) ile çoğaltıldı. PZT ürünleri Tek Sarmal Konformasyon Polimorfizmi (SSCP) yöntemiyle mutasyon belirleme (MDE) ve poliakrilamit jellerinde incelendi. Seçilen bazı örneklerin otomatik DNA analizi gerçekleştirildi. Bulgular: SSCP analizinde farklı örnekçe belirlenen iki örneğin otomatik DNA dizi analizlerinde, 193 numaralı kodonda mutasyon olmadığı ancak GNAI2 geninin beşinci ve altincı eksonları arasındaki intron bölgesinde bir C/T (C20151) değişikliğinin olduğu saptandı. Sonuç: Bulgularımız GNAI2 geninin 193 numaralı kodonunda daha önce belirlenen değişikliğin polimorfizm olma olasılığını desteklememektedir

The Role of G Protein beta 3 Subunit Polymorphisms C825T, C1429T, and G5177A in Turkish Subjects with Essential Hypertension

Hypertension is a multifactorial disorder that constitutes a major risk factor for the cardiovascular system. Heterotrimeric G-proteins, which couple receptors for diverse extracellular enzymes or ion channels, are correlated with disease mechanisms. Several studies have demonstrated an association between G protein polymorphisms and essential hypertension in some populations, although contradictive results also exist. In this study, we have investigated the potential role of the C825T, C1429T, and G5177A polymorphisms of the beta 3 subunit of G-proteins in essential hypertension in a group of Turkish subjects. Genomic DNA from 106 normotensive individuals (117.4 +/- 13.1, 75.2 +/- 10.5; systolic blood pressure (SBP) and diastolic blood pressure (DBP) levels, respectively) and 101 hypertensive subjects (152.3 +/- 18.0, 92.5 +/- 11.6; SBP and DBP levels, respectively) were studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and direct sequencing methods for these polymorphisms. Allele frequencies of the polymorphisms were consistent with Hardy Weinberg equilibrium, except for the C825T polymorphism (chi(2) = 7.8). The frequencies of the 825T and 1429T variants were higher in hypertensive subjects compared to those of controls. Differences between hypertensives and controls were not statistically significant, though difference was very close to significance for C825T (p = 0.056 and 0.099 for 825T and 1429T, respectively). T allele frequency in overall population showed significant association with hypertension for C825T (0.0134). The prevalence of the 5177A-variant was very low and all subjects carrying it were heterozygotes in both groups