The Expression Levels of MicroRNAs Associated with T and B Cell Differentiation/stimulation in Ankylosing Spondylitis.

Spondyloarthropathies (SpAs), are a group of chronic inflammatory diseases with a number of genetic, physiopathological, clinical and radiological features. Ankylosing spondylitis (AS) is the most common type of spondylo-arthropathies, and >90.0% of patients with ankylosing spondylitis are human leukocyte antigen-B27 (HLA-B2 7)-positive. In recent years, non-HLA genetic factors have been reported to have an effect on ankylosing spondylitis. MicroRNAs (miRNAs), are endogenous non coding RNA molecules containing 18-23 nucleotides that play a role in the post-transcriptional regulation of gene expression. In this study, we aimed to determine the expression levels of miRNAs associated with T- and B-cell differentiation/stimulation in peripheral blood mononuclear cells and their relationship with the etiology of the AS in patients and healthy controls. In a molecular study, peripheral blood mononuclear cell isolation, and total RNA isolation were performed first. In the second step, cDNA synthesis and quantitative real-time PCR (qPCR) expression analysis were completed. Ultimately, in the patient and control group, the expression levels of miR-142-5p and miR-143 were found to be significantly different (p <0.05). According to current knowledge, miR-142-5p andmiR-143 expressions were found to be important for those diseases that share similar etiology with AS. We suggest that miR-142-5p and miR-143 may play a role in the pathogenesis, especially miR- 142-5p may be a potential biomarker and a target molecule for the treatment.

Effects of thymoquinone on liver miRNAs and oxidative stress in Ehrlich acid mouse solid tumor model.

We investigated the effects of thymoquinone (TQ) on the expression of liver microRNAs (miRNAs), liver histopathology and oxidative stress in Ehrlich acid solid tumor model induced mice. We used 24 male BALB/c mice divided randomly into three groups. Control (C) group mice were injected intraperitoneally (i.p.) with 0.5 ml saline for four weeks. Tumor (T) group mice were injected i.p. with 0.5 ml saline for four weeks, then Ehrlich acid tumor cells were injected subcutaneously into the neck to induce solid tumor formation. TQ (T + Tq) group mice injected i.p. with 10 mg/kg TQ for four weeks, then Ehrlich acid tumor cells were injected subcutaneously into the neck of the mice in this group to induce solid tumor formation. At the end of the study, liver from all groups were removed for histopathological and miRNAs analysis, and oxidative stress measurement. We found that the expression of miR-206b-3p was up-regulated and the oxidative stress and necrosis increased in the liver tissue of mice with Ehrlich acid solid tumor. TQ application decreased the oxidative stress, prevented necrosis, increased regeneration and down-regulated the expression of miR-206b-3p in the liver tissue.

PROTECTIVE EFFECT OF INSULIN TREATMENT ON EARLY RENAL CHANGES IN STREPTOZOTOCIN-INDUCED DIABETIC RATS.

OBJECTIVES: Chronic kidney disease is a progressive complication of diabetes mellitus (DM). This study aimed to analyse early renal changes in streptozotocin induced diabetic rats and demonstrate the effect of early treatment with insulin on kidney's histology. METHODS: 30 male-adult Sprague-Dawley rats were included in the study. Diabetes was induced in 24 of the rats by a single injection of 65 mg/kg streptozotocin dissolved in saline. 5 units/day NPH insulin injection was started to 10 rats as treatment group and 11 rats were followed untreated. 6 rats constituted the control group. Induction of diabetes failed in 3 animals and 3 untreated rats died during the study. After 21 days, all rats were sacrificed and their kidneys were removed to obtain histological sections to be evaluated by light microscopy. RESULTS: Ten treated and 8 untreated diabetic rats and 6 healthy controls, totally 24 rats completed the study. There was a significant weight loss in treated and untreated diabetic groups and a weight gain in the control group (p<0.05). Final blood glucose levels were significantly higher in untreated diabetic group when compared to treated diabetic and control groups and higher in treated diabetic group when compared to control group. Histological analysis of kidney sections showed normal morphology in control group. Changes like increased mesangium, tubular atrophy and tubules with dilated lumen and irregular cell shapes were found in the untreated group whereas, glomerulus and mesangium showed similar morphology with control group with a few changes in tubules, in insulin-treated group. CONCLUSION: In DM, renal changes start at an early point and it is possible to prevent/delay those changes at this point with early intervention of insulin treatment.

miRNA and mRNA expression profiling in rat brain following alcohol dependence and withdrawal.

Long- lasting alterations in brain gene expression in alcohol addiction have been determined although no clear mechanism has yet been elucidated. There exist many factors regulating the mechanism of gene expression. We aimed in this study to detect miRNA (microRNA) and mRNA expression profile at the specific brain regions regarding ethanol exposure and withdrawal. Rats were exposed to liquid alcohol consumption for 21 days. Oligonucleotides microarrays and bioinformatics analyses were used to identify gene expression, miRNA expression and their functions in the Prefrontal cortex, Hippocampus and Corpus striatum of wistar rats.  A bioinformatics strategy with microarray analysis, quantitative real time PCR, bioinformatics and mRNA (messenger RNA) miRNA- miRNA integrative analyses revealed that expression models interact with neuroplasticity and synaptic processes. Those significantly changed after ethanol exposure and withdrawal processes included 160 mRNAs and 29 rat-miRNAs at prefrontal cortex, 142 mRNAs and 26 rat-miRNAs at hippocampus, and 143 mRNAs and 30 rat-miRNAs at corpus striatum. Gene ontology and ingenuity pathway analyses revealed that most of the altered genes were responsible for synaptic plasticity, neuron differentiation, chromatin organization and some certain important signaling pathways. In conclusion, consistent and integrated variations in miRNA expression and in their focus mRNAs in rat brain were noted after alcohol exposure and withdrawal. Besides, understanding the molecular mechanisms of alcohol abuse will no doubt guide to development of significant cure methods for addiction. We are of the opinion that our findings may shed light on classification of novel biomarkers.

IS SALUSIN-ALPHA A NEW MARKER OF CARDIOVASCULAR DISEASE RISK IN HYPOTHYROIDISM?

INTRODUCTION: Salusins are multifunctional endogenous peptides shown in human and rat tissues. Serum salusin α level is decreased in coronary artery disease and lack of salusin α enhances coronary atherosclerosis. Hypothyroidism is a chronic inflammatory disease that has a high risk of developing cardiovascular disease. Here we aimed to search the relationship of overt hypothyroidism and subclinical hypothyroidism with salusin α and other inflammatory markers, also the effect of L-thyroxine treatment on these findings. MATERIAL AND METHODS: 32 patients with overt hypothyroidism taking L-thyroxine treatment, 18 patients with subclinical hypothyroidism without treatment and 25 healthy patients as control group were included in the study. Serum salusin α, TNF α, sCRP, glucose, insulin and lipid levels were tested for all groups and results were evaluated with SPSS statistical analysis method. RESULTS: HDL, sCRP, salusin mean values were statistically significantly different in all 3 groups. HDL level was statistically significantly higher in control group compared to treatment group. sCRP level was higher and salusin level was lower in both treatment and non-treatment hypothyroidism groups compared to control group. When treatment and non-treatment hypothyroidism groups were compared, there was no statistically significant difference for salusin α, but HDL level was high and insulin level was low statistically significant in treatment group. CONCLUSIONS: Salusin α that is shown to be protective for coronary artery disease and hypertension, is found to be significantly low in hypothyroidism, thus it is a marker that increases the cardiovascular disease risk in this specific patient group.

THE EFFECT OF SHORT TERM ALPHA LIPOIC ACID ADMINISTRATION ON ADIPONECTIN AND BODY WEIGHT IN TYPE 2 DIABETES MELLITUS PATIENTS.

BACKGROUND: Alpha lipoic acid (ALA) acts as essential co-factor for mitochondrion respiratory enzymes. It has an increasing importance in diabetic neuropathy treatment. Its positive effects on weight gain and metabolic parameters have also been discussed. In this study, we aimed to search for the effect of ALA on weight, appetite, adiponectin and metabolic parameters in type 2 diabetes mellitus patients. METHODS: This study is designed as a randomised, double-blind, placebo controlled, prospective study. 23 type 2 diabetes mellitus patients with peripheral neuropathy (6 normal weight, 17 obese) and 21 normal weight control group were included in the study. Patients were given 600mg/day oral ALA for 6 weeks, added to their routine therapy. Body mass index (BMI), adiponectin, fasting plasma glucose, HbA1C, lipid parameters and CRP levels were tested before and after ALA treatment. Results were evaluated using SPSS 15.0 for Windows. RESULTS: Adiponectin levels were statistically significantly lower and CRP levels were higher in diabetes group when compared to control group. Although ALA treatment caused a slight weight loss, it was not statistically significant. Appetite scores were decreased in the diabetes group but it did not cause statistically significant weight loss. There was no significant change in metabolic parameters or adiponectin after the treatment. CONCLUSIONS: 600mg/dL ALA treatment for 6 weeks did not favor for metabolic parameters in type 2 diabetes patients. This result might be due to the dose or the duration of the treatment, genetic predisposition or dietery habits. Trial of higher doses for long terms might be needed for recovery.

The effects of mitochondrial DNA deletion and copy number variations on different exercise intensities in highly trained swimmers.

It has been suggested that heavy exercise might increase oxidative stress, causing mitochondrial DNA (mtDNA) mutations as well as DNA mutations and changes in the mtDNA copy number in cells. mtDNA4977 deletion is one of the most common deletions seen on mitochondria. We hypothesize association between exercise induced oxidative stress and mtDNA damage in peripheral blood lymphocytes (PBLs) of highly trained swimmers. Therefore we studied the mtDNA4977 deletion level, mtDNA copy number and their relationship with cellular ATP and oxidative stress status in PBLs of swimmers. 8 highly trained and 8 normal trained swimmers and 8 non-athlete subjects were included in the study. The mtDNA4977 deletion and amount of mtDNA were measured using RT-PCR method whereas dichlorohydrofluoroscein (DCF) assay method was used to assess cellular oxidative stress and ATP levels were measured using bioluminescence method. Even though an increase in mtDNA4977 deletion was found in all study groups, the difference was not statistically significant (p=0.98). The mtDNA copy numbers were found to be surprisingly high in highly trained swimmers compared to normal trained swimmers and non-athlete subjects by 4.03 fold (p= 0.0002) and 5.58 fold (p=0.0003), respectively. No significant differences were found between groups by means of intracellular ATP levels (p=0.406) and oxidative stress (p=0.430).  No correlation was found between mtDNA copy number and intracellular ATP content of the PBLs (p=0.703). Our results suggest that heavy training does not have a specific effect on mtDNA4977 deletion but it may be affecting mitochondrial copy numbers which may act as a compensatory mechanism related to ATP levels in blood.

Determination of effective miRNAs in wound healing in an experimental Rat Model.

The larvae of Lucilia sericata have been used for centuries as medicinal maggots in the healing of wounds. The present study aimed to screen potential microRNAs related to ES-induced wound healing in rat skin wounds and to investigate the potential mechanisms contributing to accelerated wound healing. Healthy, male, 12 weeks old Wistar albino rats weighing 250-300 g were supplied by the Animal Experimental Center. All animal studies were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Wistar albino rats were treated by ES after post wounding and the differentially expressed miRNAs in wound biopsies were screened by microarray analysis at the end of treatments for 4,7 and 10 days. In addition, bioinformatics approaches were used to identify the potential target genes of differentially expressed miRNAs and the functions of their target genes. We found a significant up-regulation of rno-miR-99a* and rno-mir-877 in response to ES treatment. Further investigation of rno-miR-99a* and rno-mir-877 and their target genes (TGFa, TNF, TAGLN, MAPK1, MMP-9) implicated in present study could provide new insight for an understanding lead to the development of new treatment strategies. The identified miRNAs can be new biomarkers for ES- induced wound healing.

Identification of a differential expression signature associated with tumorigenesis and metastasis of laryngeal carcinoma.

OBJECTIVES: Metastasis is the most significant prognostic factor for laryngeal carcinoma which necessitates the identification of molecular alterations associated with metastasis. The identification of such molecular alterations will not only prove useful in treatment but also provide insight into mechanisms of cancer metastasis. The studies conducted so far have not specifically focused on metastasis or invasion pathways. Therefore we investigated the expression profiles with a pathway focused approach. MATERIALS AND METHODS: Total RNA was extracted from 36 laryngeal tumors and paired cancer free tissue. Expression levels of 88 genes were determined using a PCR array system following cDNA synthesis. Obtained data was used for the calculation of altered expression levels, facilitating relevant algorithms. Significant alterations were determined according to their p-value obtained by Student's t-test. RESULTS: Sixteen genes have shown altered expression when compared with adjacent cancer-free tissue. 2 of these 16 genes have shown differential expression in tumors with neck metastasis in respect to non-metastatic tumors. CONCLUSION: We found that TGFB1, TIMP1, c-Myc, SPARC, COL4A2 and SOX4 show altered expression in laryngeal tumors. c-Myc and SOX4 expression is decreased as laryngeal tumors switch to metastatic phenotype.

Boric acid increases the expression levels of human anion exchanger genes SLC4A2 and SLC4A3.

Boron is an important micronutrient in plants and animals. The role of boron in living systems includes coordinated regulation of gene expression, growth and proliferation of higher plants and animals. There are several well-defined genes associated with boron transportation and tolerance in plants and these genes show close homology with human anion exchanger genes. Mutation of these genes also characterizes some genetic disorders. We investigated the toxic effects of boric acid on HEK293 cells and mRNA expression of anion exchanger (SLC4A1, SLC4A2 and SLC4A3) genes. Cytotoxicity of boric acid at different concentrations was tested by using the methylthiazolyldiphenyl-tetrazolium bromide assay. Gene expression profiles were examined using quantitative real-time PCR. In the HEK293 cells, the nontoxic upper concentration of boric acid was 250 µM; more than 500 µM caused cytotoxicity. The 250 µM boric acid concentration increased gene expression level of SLC4A2 up to 8.6-fold and SLC4A3 up to 2.6-fold, after 36-h incubation. There was no significant effect of boric acid on SLC4A1 mRNA expression levels.

A critical review of the cannabinoid receptor as a drug target for obesity management.

The discovery of cannabinoids, with the well-known stimulatory effect of Cannabis sativa on appetite, has offered a new drug target for obesity treatment. Cannabinoids act on two different receptors: CB1 receptors which are sited in the brain and many peripheral tissues, and CB2 receptors which are primarily found in immune system cells. Cannabinoid receptor antagonists act centrally by blocking CB1 receptors, thereby reducing food intake. Moreover, they probably also act peripherally by increasing thermogenesis and therefore energy expenditure, as has been suggested by animal experiments. Despite these promising mechanisms of action, recent clinical studies examining the effect of the two CB1 receptor antagonists rimonabant and taranabant showed that the attained weight loss did not exceed that attained with other currently approved anti-obesity medications. Moreover, potentially severe psychiatric adverse effects limit their clinical use. As several new CB1 receptor antagonists are presently undergoing development, it remains to be elucidated to what extent they differ in terms of efficacy and safety. This review primarily discusses how close cannabinoid receptor antagonists are to the ideal anti-obesity drug, with respect to their mechanisms of action, clinical effectiveness and safety.

A novel protein involved in heart development in Ambystoma mexicanum is localized in endoplasmic reticulum.

The discovery of the naturally occurring cardiac non-function (c) animal strain in Ambystoma mexicanum (axolotl) provides a valuable animal model to study cardiomyocyte differentiation. In homozygous mutant animals (c/c), rhythmic contractions of the embryonic heart are absent due to a lack of organized myofibrils. We have previously cloned a partial sequence of a peptide cDNA (N1) from an anterior-endoderm-conditioned-medium RNA library that had been shown to be able to rescue the mutant phenotype. In the current studies we have fully cloned the N1 full length cDNA sequence from the library. N1 protein has been detected in both adult heart and skeletal muscle but not in any other adult tissues. GFP-tagged expression of the N1 protein has revealed localization of the N1 protein in the endoplasmic reticulum (ER). Results from in situ hybridization experiments have confirmed the dramatic decrease of expression of N1 mRNA in mutant (c/c) embryos indicating that the N1 gene is involved in heart development.

The presence of erythropoietin receptor in parathyroid cells.

CONTEXT: Effects of erythropoietin on parathyroid cell function has not been studied before. OBJECTIVE: We aimed to demonstrate whether erythropoietin receptor present in parathyroid cells. DESIGN: The specimens of normal parathyroid gland, parathyroid adenoma and hyperplasia were retrieved from our pathology archives. The sections were stained immunohistochemically. Quantitative gene expression study was performed for erythropoietin and erythropoietin receptor. RESULTS: Erythropoietin receptors were detected by immunohistochemical staining and by its gene expression. Its density was higher in normal parathyroid, followed by parathyroid adenoma and hyperplasia. CONCLUSION: Erythropoietin receptor is present in normal parathyroid, parathyroid adenoma, and hyperplasia.

High-dose exposure to valsartan with suicidal intention.

Valsartan is a strong angiotensin receptor inhibitor specific for the angiotensin I receptor, which has been proven safe and well-tolerated in clinical trials. We were able to confirm its safety and tolerability in a case of high-dose exposure to valsartan with suicidal intention. A 25-year-old, fully conscious, female patient was brought to our hospital by relatives on July 24, 2001, at 9:15 p.m. following intake of a high dose of valsartan. It was established that she had taken 28 Diovan 80 mg tablets (2.24 g) 5 hours before admission to the hospital. Her clinical condition at the time of admission was good and did not deteriorate after admission. During the follow-up, her blood pressure never fell below 90/60 mmHg. The only complaint she had were painful muscle cramps which, with only supportive therapy, disappeared spontaneously over 2 days, and her blood pressure also returned to normal during this period. This report demonstrates the effect/side effect profile of valsartan when taken at a high dose, not achievable in a clinical trial.

Are individuals with glutathione S-transferase GSTT1 null genotype more susceptible to in vitro oxidative damage?

Recent epidemiological studies proposed that glutathione S-transferase (GST) T1 null genotype was correlated with an increased susceptibility to diseases associated with oxidative stress, including cancer. A comparative study using erythrocytes from individuals with GSTT1 null genotype was carried out to determine how resistance to oxidative stress is affected by lack of this gene, and whether the GST status of a person is an important factor in risk toward oxidant chemicals. Malondialdehyde and carbonyl levels and fluorescence and chemiluminescence formation were used as biomarkers of oxidative stress in erythrocytes exposed in vitro to cumene hydroperoxide (CumOOH), an oxidizing agent. When peroxidation-dependent changes in these parameters were compared between GSTT1 null genotype and controls, who are both GSTM1 and GSTT1 positive, no significant differences were found between the two genotypes, although the erythrocytes of the GSTT1 null group had lower GSTT1 activity toward CumOOH. Our results indicate that erythrocytes from individuals with GSTT1 null genotype are not abnormally susceptible to CumOOH-induced oxidant challenge.

GSTT1 null genotype frequency in a Turkish population.

In humans, glutathione-S-transferases (GSTs) are involved in the detoxification of xenobiotics. A deletion polymorphism in the glutathione S-transferase Theta 1 gene (GSTT1 null genotype) is associated with an increased risk of certain forms of cancer. The distribution of this polymorphism in 240 healthy individuals has been examined, using a polymerase chain reaction (PCR)-based method. The GSTT1 null genotype frequency was 20% in a Turkish Population.

Penetration of cyclosporin A into the rabbit cornea and aqueous humor after topical drop and collagen shield administration.

We used commercially available 12-hour collagen shields to deliver cyclosporin A (CsA) to the cornea and aqueous humor in rabbit eyes. Six New Zealand white rabbits were divided into three groups. The first group (four eyes) received 6 mg of CsA in castor oil and the second group (four eyes) received 6 mg of CsA in olive oil applied as topical drops to rabbit eyes within 12 hours. In the third group (four eyes) 12-hour collagen shields soaked in 6 mg CsA in olive oil were applied to rabbit eyes. The amount of CsA in corneal and aqueous samples from eyes treated with CsA castor oil and CsA olive oil were compared with each other and with collagen shield treated eyes. CsA concentrations were measured by radioimmunoassay. After the total dose of 6 mg CsA, percentage penetration was measured as follows: CsA castor oil--0.51% in aqueous and 20.75% in cornea; CsA olive oil--0.17% in aqueous and 11.13% in cornea; and with collagen shields--0.44% in aqueous and 11.84% in cornea. These results show that the CsA levels of castor oil drops were higher than those obtained with olive oil drops. In eyes with collagen shields, CsA levels were higher than olive oil drops but nearly equal to the castor oil drops. Collagen shields may be useful as an ocular delivery system for CsA.