Gama amino butirik asit (GABA) reseptörlerinin genetik polimorfizmleri ile idiyopatik jeneralize epilepsi arasındaki ilişkinin araştırılması

TÜBİTAK TBAG Proje01.03.2012Epilepsy is one of the most common neurological diseases worldwide. Gamma amino butyric acid (GABA), the most important inhibitory neurotransmitter of the central nervous system, and its receptors are commonly mentioned in the pathophysiology of epilepsies. Some of the single nucleotide polymorphisms in the genes encoding GABA receptors have been reported to increase susceptibility to temporal lobe epilepsy. There are a limited number of studies analyzing the relationship of GABA receptors and idiopathic epilepsy, which is the most widespread (65%) when etiologically classified. In Turkish population, genetic polymorphisms of GABAA, GABAB1 and GABAB2 receptors have not been studied before. For these reasons, in this study we aimed to determine the frequencies of genetic polymorphisms of GABA receptors A, B1 and B2 in Turkish population by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method, and to investigate the correlation between these polymorphisms and idiopathic generalized epilepsy (IGE). A second aim of this study was to analzye the role of GABA receptor polymorphisms in differential diagnosis of pschogenic non-epileptic seizure (PNES). In this context, 196 IGE patients, 107 PNES subjects and 109 controls were inculded in this study. Polymorphic allele frequencies were very close to each other in the three groups (0.457 in IGE patients, 0.453 in PNES patients and 0.427 in controls) for the GABAA α1 rs2279020 polymorphism. A statistically significant relationship was not found between this polymorphism and IGE. We did not observe polymorphic alleles in Turkish population for GABAA 2 rs17852044, GABAA 3 rs79829085 and GABAB1 G1465A. For GABAB1 C59T polymorphism, when IGE patients and PNES subjects were compared, C59T polymorphism was found to increase IGE risk 1,5 times, but this result was not significant (P=0.164). The frequency of C59T polymorphism in control population was found as 0.110 in this study. We observed that four polymorphisms found on GABAB2 gene (rs3780428-rs1999501- rs967932-rs94688) did not constitute a significant risk for IGE when considered alone. When the genotype combinations were taken into account, we concluded that having polymorphic homozygote genotype for rs944688 and wild type genotype for rs967932 and rs1999501 had 15 times protective effect against IGE for PNES subjects (P=0.001). The same genotype combination constituted 12 times protective effect against IGE in control subjects (P=0.009). This triple combined genotype can be used to differentiate subjects who have psychogenic seizures (PNES) from those having epileptic seizures. All of the studied polymoprhisms were studied for the first time in Turkish populatio

Potential role of calcifying nanoparticles in the etiology of multiple sclerosis

Nanobacteria or calcifying nanoparticles are 80–500 nm sized nano-organisms that are physically associated with carbonate apatite mineral formations. They have been indicated in various diseases, including kidney stone formation, Alzheimer's disease, and atherosclerosis. Nanoparticles contain calcium and apatite-binding protein fetuin-A, a calcification inhibitor. However, recent evidence indicates that fetuin-A can form nucleation seeds or nidi that grow in size through ion sedimentation to become larger amorphous nanoparticles in the presence of excess calcium and apatite ions. Fetuin-A also functions as an inhibitor of meprin, a metalloproteinase implicated in inflammation and neurodegenerative diseases. During inflammation, meprin functions to regulate chemokine activity of monocyte chemotactic protein 1, which is associated with chronic inflammatory diseases, including atherosclerosis, renal inflammatory diseases, and multiple sclerosis (MS). In addition, calcium phosphate nanocrystals that contain fetuin-A are pro-inflammatory to macrophages and promote vascular smooth muscle cell mineralization, potentiating a vicious cycle of inflammation and calcification. Thus, mineral stress and inflammation appear to be associated with each other. Furthermore, fetuin-A deficient mice exhibited reduced experimental autoimmune encephalomyelitis severity. Thus, fetuin-A plays a direct role in the neuroinflammatory response. Indeed, the level of fetuin-A in cerebrospinal fluid has been defined as a biomarker of disease activity in MS. MS is a chronic, inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) with an unknown etiology. The “inside-out” model of MS, supported by recent data, states that the initial axonal degeneration in the CNS occurs before demyelination, which then stimulates an auto-immune attack. It was shown very recently that influx of calcium from the extracellular space through nanoscale ruptures of the axonal plasma membrane predict axon degeneration in neuroinflammation. Calcium is an activator of calpains, proteases that function to break down the cytoskeleton, leading to neurodegeneration. Nanoruptures of the plasma membrane were suggested to occur at the early stages of axon damage, especially at nodes of Ranvier, which are devoid of myelin. Here, I propose that calcifying nanoparticles may have a role in the etiology and/or pathophysiology of MS. The initial event causing neurodegeneration may be due to the nanoparticles that have been suggested to easily cross the blood-brain barrier. Following this, the nanoparticles may create nanoruptures in the axonal membrane and also increase the calcium concentration around and within the neurons by forming nidi for calcification, eventually causing neurodegeneration. Nanoparticles can self-replicate; hence, they may represent an infectious causative agent for the development of MS

A literature review of biosensors for multiple sclerosis: Towards personalized medicine and point-of-care testing

Multiple sclerosis (MS) is a chronic neuroinflammatory disease of the central nervous system that leads to severe motor and sensory deficits in patients. Although some biomolecules in serum or cerebrospinal fluid have been suggested as biomarkers for MS diagnosis, following disease activity and monitoring treatment response, most of these potential biomarkers are not currently in clinical use and available for all patients. The reasons behind this are generally related to insufficient robustness of biomarker or technical difficulties, high prices, and requirements for technical personnel for their detection. Point-of-care testing (POCT) is an emerging field of healthcare that can be applied at the hospital as well as at home without the need for a centralized laboratory. Biosensor devices offer a convenient means for POCT. A biosensor is a compact analytical device that uses a bioreceptor, such as an antibody, enzyme, or oligonucleotide, to capture the analyte of interest. The interaction between the analyte and the bioreceptor is sensed and transduced into a suitable signal by the signal transducer. The advantages of using a biosensor for detecting the biomolecule of interest include speed, simplicity, accuracy, relatively lower cost, and lack of requirements for highly qualified personnel to perform the testing. Owing to these advantages and with the help of innovations in biosensor development technologies, there has been a great interest in developing biosensor devices for MS in recent years. Hence, the purpose of this review was to provide researchers with an up-to-date summary of the literature as well as to highlight the challenges and opportunities in this translational research field. In addition, because this is a highly interdisciplinary field of study, potentially concerning MS specialists, neurologists, biomedical researchers, and engineers, another aim of this review was to bridge the gap between these disciplines

Paraoksonaz 1 aktivite ve genetik polimorfizmleri ile inme riski arasındaki ilişkinin araştırılması.

Stroke is the third leading cause of death. Atherosclerosis in the carotid arteries is a risk factor for ischemic stroke. Oxidized low density lipoprotein (LDL) plays a central role in the progression of atherosclerosis. Human paraoxonase 1 (PON1), a high-density lipoprotein (HDL) associated serum esterase/lactonase, protects HDL and LDL from oxidative modifications. Thus, PON1 is protective against the development of atherosclerosis. PON1 gene has two functional coding region (192Q/R and 55L/M) and one promoter region (107T/C) polymorphism that affect the catalytic efficiency and levels of the enzyme, respectively. In this study, the aim was to determine the importance of PON1 genetic polymorphisms and activity as risk factors for ischemic stroke. The study population was comprised of 172 unrelated adult Caucasian patients with acute hemispheric ischemic stroke and 105 symptom-free controls. Serum and total blood samples were obtained from Gülhane Military Medical Academy Hospital Neurology Department, Ankara. Hypertension and diabetes were 2 times more common and HDL-C was significantly lower among patients compared to controls. Logistic regression analysis revealed hypertension and smoking to be significant predictors of stroke. Serum PON1 activities towards three substrates, paraoxon (paraoxonase activity; PON), phenyl acetate (arylesterase activity; ARE) and diazoxon (diazoxonase activity), which were measured by spectrophotometric methods, were found to be lower in stroke patients compared to controls. PON and PON/ARE were negatively associated with ischemic stroke by use of logistic regression analysis. PON/ARE was 1.26 times protective against stroke. The frequencies of the risky alleles 192R, 55L and 107T were increased in the patient group. Frequency of the 55L allele of PON1 was significantly increased among patients (0.690) compared to controls (0.628; P=0.003). Logistic regression analysis revealed PON1 55LL genotype to be associated with a 1.8-fold increase in the risk of ischemic stroke versus control status. Prevalence of triple combined haplotype QRLMTC was significantly lower in stroke patients (4.1%) when compared to controls (11.4%; P=0.019). The combined heterozygote haplotype had around 7 times increased protective effect against stroke in the overall population and 10 times protective effect in the elderly population. The low expressor genotype 107TT was associated with almost 2 times increased risk for stroke in elderly. 192R allele of PON1 represented 1.554 times increased risk for ischemic stroke in hypertensives relative to normotensives. Furthermore, the risk of hypertensive individuals having ischemic stroke was highest in the 192RR group (Odds Ratio; OR=7), followed by 192QR heterozygotes (OR=2.18), and the risk decreased to insignificant levels in 192QQ individuals. 192R allele constituted a 1.55 times increased risk in diabetics. 55L allele was associated with a 1.66 times increased risk of stroke in hypertensives and a 2.6 times increased risk for stroke in diabetics relative to non-diabetics. PON1 107T allele also represented a 1.35 times risk for stroke in hypertensives.Ph.D. - Doctoral Progra

Koyun karaciğer mikrozomal flavin-monooksijenazlar: Karakterizasyon ve bazı modülatörlerin etkileri

The flavin-containing monooxygenases (also called flavin-monooxygenases and abbreviated to FMO; E.C.I. 14.13.8) catalyze the NADPH and oxygen-dependent oxidation of a wide range of nucleophilic nitrogen-, sulfur-, phosphorus-, and selenium heteroatom-containing chemicals, drugs, and agricultural agents. FMOs have been found in various organisms from bacteria to humans and five mammalian FMO genes have been identified, FM01-FM05. Sheep liver microsomal FMO enzyme activity was characterized using methimazole as substrate, which is a highly specific substrate for FMO enzymes. The average specific activity of sheep liver microsomal FMO was found to be 3.8 ±1.5 nmol/min/mg protein (Mean ± SE, n=7) when no detergent was added to the reaction 111medium, and 6.1 + 1.4 nmol/min/mg protein (Mean ± SE, n=6) in the presence of 0.1 % Triton X-100 in the reaction mixture. The rate of reaction was linear up to 500 |j.g of sheep liver microsomal protein and for 27 minutes of incubation period. The maximum FMO enzyme activity was detected at 37 °C, at pH 8.0. Effects of detergents; Triton X-100, Cholate, and Emulgen 913, on FMO activity were determined and found that enzyme activity increased by the addition of either detergent at all concentrations. The apparent Vmax and Km values of sheep liver microsomal FMO for methimazole substrate were found as 12.3 nmol/min/mg protein and 0.133 mM, respectively. Thermostability of sheep liver FMO was studied at four different temperatures; 24 °C, 37 °C, 50 °C, and 65 °C. The incubation time required for the complete loss of enzyme activity was 5 minutes at 65 °C, 1.5 hours at 50 ° C, 2 days at 37 °C, and 20 days at 24 ° C. Effects of two metal ions, Hg+2 and Mg+2, on sheep liver FMO activity was studied using HgCh and MgCİ2. Sheep liver microsomal FMO activity towards two drug substrates, imipramine and chlorpromazine, was also determined and found to be 10.7 and 12.3 nmol NADPH oxidized/min/mg protein, respectively. Effects of imipramine, chlorpromazine, and n-octylamine on methimazole oxidation activity of sheep liver microsomal FMO was studied and found that the two drug substrates and n-octylamine inhibited FMO-catalyzed methimazole oxidation activity when they are used at high concentrations. Western blot-immunochemical analysis revealed the presence of FMO 3 in sheep liver, sheep lung, and bovine liver microsomes. The immunoblot analysis suggested that FM03 expression is higher in sheep lung, compared to sheep and bovine liver.Flavin-içeren monooksijenazlar (flavin-monooksijenazlar da denir ve FMO şeklinde kısaltılır; E.C.l. 14.13.8), birçok değişik nükleofilik nitrojen, sülfür, fosfor ve selenyum heteroatomu içeren kimyasallar, ilaçlar ve tarımsal ajanların, NADPH ve oksijene bağımlı oksidasy onunu katalize ederler. FMO' lar bakterilerden insanlara kadar birçok değişik organizmada bulunmuş ve bugüne kadar 5 tane memeli FMO geni belirlenmiştir; FM01-FM05. Koyun karaciğer mikrozomal FMO enzim aktivitesi, FMO enzimleri için çok spesifik bir substrat olan metimazol kullanılarak karakterize edilmiştir. Ortalama FMO spesifik aktivitesi, reaksiyon ortamında deterjan kullanılmadığında3.8 ±1.5 nmol/dak/mg protein (Ortalama ± SS, n=7), reaksiyon ortamına % 0.1 Triton X-100 eklendiğinde ise 6.1 ± 1.4 nmol/dak/mg protein (Ortalama ±1.5, n=6) olarak bulundu. Reaksiyon hızı 500 jig protein ve 27 dakika inkübasyon süresine kadar doğru orantılı arttı. En yüksek FMO enzim aktivitesi 37 °C ve pH 8.0 de tespit edildi. FMO aktivitesi üzerine Triton X-100, Kolat ve Emulgen 913 gibi deterjanların etkileri çalışıldı ve enzim aktivitesinin bu deterjanların herbirinin reaksiyon ortamına eklenmesi sonucu arttığı gözlendi. Koyun karaciğer mikrozomal FMO enzimin metimazol substratı için görünen Km ve VmaX değerleri sırasıyla 0.133 mM ve 12.3 nmol/dak/mg protein olarak bulundu. Koyun karaciğer mikrozomal FMO enziminin ısıya dayanıklılığı dört değişik ısıda çalışıldı; 24 °C, 37 °C, 50 °C ve 65 °C. Enzim aktivitesinin tamamen kaybolması için gereken inkübasyon süresi 65 °C'de 5 dakika, 50 °Cde 1.5 saat, 37 °CTde 2 gün ve 24 °CTde 20 gün olarak gözlendi. Enzim aktivitesine iki metal iyonunun, Hg+2 ve Mg+2, etkileri HgCİ2 ve MgCİ2 kullanılarak çalışıldı. Koyun karaciğer mikrozomal FMO enziminin imipramin ve klorpromazin gibi iki ilaç substratına karşı aktivitesi de ölçüldü ve sırasıyla 10.7 ve 12.3 nmol NADPH oksitlenmiş/dak/mg protein olarak tespit edildi. İmipramin, klorpromazin ve n- oktilamin'in koyun karaciğer mikrozomal FMO enziminin metimazol oksidasyon aktivitesi üzerine etkileri çalışıldı. Bu iki ilaç substratın ve n-oktilaminin FMO enzim aktivitesini yüksek konsantrasyonlarda kullanıldıkları zaman inhibe ettikleri bulundu. Western blot immünokimyasal analizi, koyun karaciğer, koyun akciğer ve dana karaciğerinde FM03 izoziminin varlığını ortaya koydu. İmünoblot analizi FM03 ekspresyonunun koyun akciğerinde koyun ve dana karaciğerine göre daha yüksek seviyede olduğunu gösterdi

İskemik inme riski ile kolesterolü metabolize eden sitokrom P450lerin genetik polimorfizimleri arasındaki ilişkinin incelenmesi

İskemik inme, beyindeki damarların tıkanması sonucu gerekli olan oksijen ve besinin dokulara ulaşamamasıyla oluşan inme türüdür. Beyin damarlarının tıkanması, beyindeki veya boyundaki damarlarda pıhtı oluşması veya vücudun herhangi bir yerindeki pıhtının dolaşım yoluyla beyin damarına ulaşması ve burayı tıkayarak beyne kan iletimini engellemesi sonucu oluşabilir. Beyindeki sinir hücreleri oksijensiz ve besinsiz kaldığında fonksiyonlarını yerine getiremez. Bu inme türü hipertansiyon ve diyabet gibi bazı kronik hastalıklar, genetik ve çevresel faktörler tarafından tetiklenir. İskemik inme dünyada ölüme ve sakatlanmaya yol açan en önemli nedenlerden biridir. Araşidonik asitten eikosanoid üretimi, beyin bölgesindeki kan akışını düzenlemede ve nörovasküler eşleşmede önemli bir rol oynamaktadır. Epoksieikosanoid asitler iltihap söktürücü, ateş düşürücü, antitrombotik ve damar oluşumu başlatıcı özelliklere sahiplerdir. Hücreler siklooksijenaz, lipoksijenaz ve sitokrom P450 reaksiyonları olmak üzere üç ayrı şekilde eikosanoid üretimi yapabilir. Sitokrom P450 reaksiyonlarıyla üretilen epoksieikosatrionik asitlerin beyindeki kan akışını düzenlemenin yanı sıra yukarıda bahsedilen özelliklerinden dolayı iskemik beyin hasarlarını önleyici bir role de sahip oldukları bilinmektedir. İnsanda sitokrom P450 gen ailesi 57genden oluşmaktadır.Bu genler ilaç , yabancı kimyasallar,araşidonik asit ,eikosanoid ve kolesterol metabolizmalarında rol alan enzimleri kodlarlar.Kolesterolden safra as

Evaluation of tear and aqueous humor level, and genetic variants of connective tissue growth factor as biomarkers for early detection of pseudoexfoliation syndrome/glaucoma

Pseudoexfoliation syndrome (PEX) may lead to the development of pseudoexfoliative glaucoma (PEG), a potential cause of irreversible blindness, if left untreated. This type of glaucoma often presents with much higher intraocular pressure (IOP) values than observed in primary open angle glaucoma, and patients are often unaware of their condition. Therefore, early diagnosis is of utmost importance in PEX and PEG. Unfortunately, no valid objective biomarkers are available that can be used for this purpose. The excessive synthesis and deposition of elastic microfibrillar pseudoexfoliation material is observed in the pathophysiology of PEX, therefore, growth factors may play roles in this pathology. Thus, in this study, we sought to determine the roles of phenotypes and genotypes of connective tissue growth factor (CTGF) as objective biomarkers for early diagnosis of PEX and PEG. Thus, we investigated possible associations involving tear and aqueous humor CTGF concentrations and four single nucleotide polymorphisms (SNPs) of the CTGF gene in PEX and PEG. The study was designed as a 2-year case-control study in the Turkish population. Study population was composed of 214 patients with PEG, 214 patients with PEX, and 214 age-matched controls for CTGF SNP analysis. Tear fluid study group consisted of 78 patients with PEG, 77 patients with PEX, and 78 controls. Aqueous humor analysis included 8 patients with PEG, 17 patients with PEX, and 23 controls. Tear fluid was collected using Schirmer strips, and aqueous humor samples were taken during cataract surgery. CTGF concentration was determined by ELISA, and total protein concentration was determined by Bradford assay in tear and aqueous humor samples. PCR followed by restriction fragment length polymorphism analysis was used for genotyping of rs6918698 G/C and rs9399005 C/T, while real-time PCR was used for rs9402373 C/G and rs12526196 T/C. Intraocular pressure, visual field score, mean deviation, and pattern standard deviation parameters were also evaluated. CTGF concentration in tear fluid was significantly higher in PEG patients compared with controls (P = 0.001), while it was lower in PEX patients. Similarly, total protein concentration in tear fluid was significantly increased in PEG patients relative to PEX patients (P = 0.026) and controls (P = 0.004). CTGF concentration in aqueous humor did not differ markedly between the groups, whereas total protein was significantly higher in the PEG group compared with the PEX group (P = 0.012) and controls (P = 0.003). Receiver operating characteristic analysis revealed that total protein in aqueous humor was a robust classifier for evaluating the presence of PEG against controls (Area under the curve = 0.897, P = 0.001). The genotypes of the studied SNPs were not significantly correlated with CTGF concentration in aqueous humor or tear fluid, and did not exhibit significant association with PEG or PEX. In conclusion, this was the first study to investigate tear fluid CTGF concentration in PEX and PEG, which came out not to be a good classifier for PEG or PEX. Total protein level in tear fluid and CTGF SNPs also did not predict PEG or PEX status successfully

Importance of NOS3 Genetic Polymorphisms in the Risk of Development of Ischemic Stroke in the Turkish Population

In the present study, we aimed to investigate the relationship between endothelial nitric oxide synthase 3 (NOS3) G894T, T-786C, and intron 4 variable number of tandem repeat (VNTR) variants, alone or in combination, and the risk of incidence of ischemic stroke in the Turkish population. The genotypes for all polymorphisms were determined by polymerase chain reaction/restriction fragment length polymorphism techniques on 245 ischemic stroke patients and 145 controls. In the case-control analysis, no significant difference was observed between stroke patients and controls with respect to NOS3 G894T, T-786C, and intron 4 VNTR polymorphisms genotype and allele frequency distribution. However, the copresence of G894T and intron 4 VNTR risk-elevating genotypes in the same individual increased the risk of stroke seven times (odds ratio=7.083, 95% confidence interval=0.866-57.963, p=0.029)