Acidosis defense mechanisms in the preimplantation stages of embryos in BALB/c strain mice.

Regulation of intracellular pH (pHi) is an important homeostatic function of cells. There are three major pHi regulatory mechanisms: the HCO3-/Cl- exchanger (AE), which alleviates alkalosis, and the Na+/H+ exchanger (NHE) and Na+,HCO3-/Cl- exchanger (NDBCE), both of which counteract acidosis. NHE activity, which is high at the germinal vesicle stage of oocyte, is inhibited during meiotic maturation, while this inhibition is abolished when the oocyte reaches the pronuclear (PN) stage of the zygote. On the other hand, we have previously found that NDBCE performs complementary regulation against acidosis during meiotic maturation. Additionally, we found that AE activity, which is a defense mechanism against alkalosis, gradually decreases during preimplantation period of embryonic development. Considering that NHE activity is inhibited during meiotic maturation and AE activity gradually decreases during embryonic development stages, we investigated whether NHE and NDBCE activities, both of which act against acidosis, functionally change from the PN zygote to the blastocyst stage of the embryo and identified these pH-regulating proteins at the molecular level in mice of the Balb/c strain. PN zygotes, two-cell (2-c), four-cell (4-c), morula and blastocyst stage embryos were obtained from 5-8-week-old, sexually mature female Balb/c mice by using the classical superovulation procedure. pHi was recorded by using the microspectrofluorometric technique on zygotes and embryos simultaneously loaded with the pH-sensitive fluorophore, 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The activities of NHE and NDBCE were determined from the recovery curve of induced-acidosis in bicarbonate-free and bicarbonate-containing media, respectively. Specific inhibitors such as cariporide (1 µM), S3226 (1 and 10 µM), EIPA (1, 5, and 25 µM), and amiloride (1 mM) were used to functionally identify NHE isoforms, and the nonspecific inhibitor 4,4'-diisocyanatostilbene-2,2' disulphonic acid, disodium salt (DIDS) was used to confirm NDBCE activity. The isoforms of the pHi-regulatory proteins were also identified by molecular biology using real-time PCR. We found that NHE activity was high at all embryonic stages, and differences between stages were not significant. Functional and molecular findings indicated that isoforms of NHE 1 and 5 are present in the blastocyst, whereas isoforms of NHE 1, 3, and 4 are functional at earlier embryonic stages. Although the contribution of NDBCE activity to recovery from induced-acidosis was detected at all embryonic stages, it was significant only in the PN zygote and the 2-c embryo. This finding was confirmed by molecular analysis, which detected the expression of SLC4A8 encoding NDBCE at all embryonic stages. In conclusion, NHE is an active and important defense mechanism against acidosis and is encoded by at least two protein isoforms in all stages of the Balb/c strain of mice. NDBCE has a supportive function in all embryonic stages, especially in the PN zygote and the 2-c embryo. Preimplantation stage embryos have effective mechanisms to defend against acidosis in response to their metabolic end products (increased acid load) and the acidic environment in utero.

Investigation the effects of 2-aminoethoxydiphenyl borate (2-APB) on aminoglycoside nephrotoxicity.

Investigation the protective effect of transient receptor potential channel modulator 2-Aminoethoxydiphenyl Borate (2-APB) on aminoglycoside nephrotoxicity caused by reactive oxygen species, calcium-induced apoptosis and inflammation was aimed. Forty Wistar rats were divided (n=8) as follows: Control group; DMSO group; 2-APB group; Gentamicin group (injected 100 mg/kg gentamicin intramuscularly for 10 days); Gentamicin+ 2-APB group (injected 2 mg/kg 2-APB intraperitoneally, then after 30 minutes 100 mg/kg gentamicin was injected intramuscularly for 10 days). Blood samples were collected for biochemical analyses, kidney tissue samples were collected for light, electron microscopic and immunohistochemical investigations. In gentamicin group glomerular degeneration, tubular dilatation, vacuolization, desquamation of tubular cells and hyaline cast formation in luminal space and leukocyte infiltration were seen. Disorganization of microvilli of tubular cells, apical cytoplasmic blebbing, lipid accumulation, myelin figure like structure formation, increased lysosomes, mitochondrial swelling and disorganization of cristae structures, apoptotic changes and widening of intercellular space were found. TNF-α, IL-6 and caspase 3 expressions were increased. BUN and creatinine concentrations were increased. Increase in MDA levels and decrease in SOD activities were determined. Even though degeneration still continues in gentamicin+2-APB treatment group, severity and the area it occupied were decreased and the glomerular and tubule structures were generally preserved. TNF-α, IL-6, caspase 3 immunoreactivities and BUN, creatinine, MDA concentrations were reduced and SOD activities were increased markedly compared to gentamicin group. In conclusion, it has been considered that 2-APB can prevent gentamicin mediated nephrotoxicity with its anti-oxidant, anti-apoptotic and anti-inflammatory effects.

EFHC1 gene mutation profile of Turkish JME patients and its association with disease risk.

OBJECTIVES: Juvenile myoclonic epilepsy (JME) is a common form of generalized epilepsy with an important genetic component. This cohort study aimed to examine the frequency of EFHC1 gene variants in Turkish JME patients and a healthy control group and evaluate the association between these mutations and disease risk. METHODS: We screened 72 JME patients with a mean age of 31.8 ± 9.9 (20-65) years and 35 controls with a mean age of 29.1 ± 7.6 (17-50) years from southern Turkey using direct sequencing analyses. RESULTS: EFCH1 single nucleotide variants were detected in 24 of 72 JME patients and 3 of 35 controls. The most common mutations were R182H in JME patients (p = 0.010) and 3'UTR in the control group (p < 0.001). The R182H mutation is a common variant in JME (95 % CI: 1.232-76.580, p = 0.031) and the 3'UTR mutation may be associated with lower risk of JME in the Turkish population (95 % CI: 13.89-166.67, p < 0.001). SIGNIFICANCE: Our results indicate that EFHC1 gene variants carry a risk for JME and the 3'UTR variant may have a protective role against JME in the Turkish population. Screening for other genes is needed to further clarify the genetic inheritance of JME in Turkish patients.

Cord Blood Hematological Parameters of Fetuses Detected Different Thalassemia Genotypes in the Second Trimester of Pregnancy

Background: Hemoglobinopathies are the most common inherited diseases in humans resulting from impaired globin chain synthesis of hemoglobin. The progression of thalassemia rates is prevented with prenatal screening methods. Aims: To evaluate the hematological parameters of α- and ß-thalassemia and normal fetuses aged 17-25 weeks of gestation. Study Design: A cross-sectional study. Methods: Pregnant women who underwent cordocentesis in the second trimester because of the risk of having a baby with thalassemia were included in the study. Hematological indices and molecular DNA methods were analyzed from the cord blood samples of 129 women who were 17-25 weeks into pregnancy. The HPLC method was used for Hb fraction analysis. Amplification refractory mutation system, restriction enzyme analysis, multiplex polymerase chain reaction, and sequencing methods were used for the molecular analysis. Maternal contamination was eliminated by the short tandem repeat method. Results: In total, 112 of the fetuses carry α- and ß-thalassemia heterozygous or homozygous (α: 37, ß: 58, mixed: 17) and 17 fetuses had a normal genotype for thalassemia. Significant differences in adult hemoglobin (HbA), fetal hemoglobin (HbF), Hb Barts, MCV, MCH, and RDW were detected in three groups compared with the normal group (p < 0.001, except for RBC, Hb, HCT, and MCHC). Differences in HbF, Hb Barts, MCV, MCH, and RDW were observed in the α-thalassemia groups compared with the normal group (p < 0.001). Among the five ß-thalassemia subgroups, only HbA and RDW were different from the normal group (p < 0.001). Conclusion: This study could be a good reference for future studies and prenatal diagnostic applications in emphasizing the importance of changes in the blood parameters of fetuses before molecular genotyping. These hematological data give valuable information to clinicians about the fetus to enlighten families in making appropriate decisions during prenatal diagnosis.

The effect of Vitamin D on testosterone and uncarboxylated osteocalcin levels in aged male rats.

There is a well-established complex interaction between vitamin D metabolism and bone and gonad functions. In this study, we aimed to investigate the potential effects of vitamin D therapy on testosterone and osteocalcin (OC) levels in aged male rats. Forty-five adult male rats were divided into three groups in this study. Unlike the control group, the two experimental groups received 50 IU/kg/day and 100 IU/kg/day of vitamin D3 (cholecalciferol), respectively, for a 4-week period using the gavage method. Testicular tissue and blood samples from rats were collected under general anesthesia at the end of the 4-week period. Testicular tissue samples were examined using light and electron microscopy. Additionally, serum testosterone and OC levels were measured in blood samples. The 50 IU/kg dose of cholecalciferol increased testosterone and OC levels, which were lower than normal due to aging, and regulated the organization of the seminiferous tubule epithelium and interstitium more effectively than the 100 IU/kg dose of cholecalciferol. Male fertility functions and bone health, which degrade due to aging, were increased due to the use of exogenous vitamin D, although the higher dose was not associated with more effective results.

The effect of different intravenous lipids on free bilirubin levels in premature infants.

BACKGROUND: Intravenous lipid emulsions are commonly used as a part of parenteral nutrition in premature infants. The potential bilirubin-displacing effects of high free fatty acid (FFA) levels during lipid infusions are known. Levels of free bilirubin (FB) predict the risk of bilirubin neurotoxicity more accurately than indirect serum bilirubin levels. In the present study, we decided to compare the effect of two different lipid solutions on free bilirubin and free fatty acids levels in premature infants. METHODS: Infants were grouped into two groups according to intravenous lipid preparations: Infants in Group 1 received lipid emulsion containing olive oil + soybean oil and Group 2 received containing olive oil + soybean oil + fish oil. The blood samples were gained when lipid intake was 3.5 g/kg/day and FFA and FB levels were analyzed. RESULTS: Serum FFA and FB levels were similar in groups (p = 0.26 and 0.69 respectively). There were significant correlation between serum FFA and FB levels in Group 1 (r = 0.74, p < 0.001) and in Group 2 (r = 0.92, p < 0.001). CONCLUSION: Both lipid preparations seem to have similar effects on free bilirubin and free fatty acid levels in premature newborns.

Bisphenol A decreases expression of Insulin-like factor 3 and induces histopathological changes in the Testes of Rats.

Bisphenol A (BPA) is a chemical agent known to have detrimental reproductive and developmental effects. The tissue-specific impacts of BPA exposures and target tissues sensitiveness to BPA are still unclear. The aim of this study was to determine the short- and long-term dose-dependent toxic effects of BPA on rat testes. Forty-eight Wistar albino male rats were divided into four groups each containing 12 rats. To induce toxicity, BPA was administered orally at three different dosages (50, 100, and 200 mg/kg) for 14 and 28 days, respectively. Testis tissues were examined using light and electron microscopy, immunohistochemistry, and biochemical methods. Serum testosterone (T) and luteinizing hormone (LH) levels were measured. Additionally, insulin-like factor 3 (INSL3) as a marker of Leydig cell function was evaluated immunohistochemically. Groups administered high doses of BPA showed severe degenerations such as testicular atrophy, spermatogenic arrest, and interstitial edema in testis. Also, a significant decrease in INSL3 immunoreactivity and serum LH and T levels was found. The results indicated that both increased exposure time and dosage of BPA caused more serious detrimental effects on testes in the rat. Decreased INSL3 and T levels was evidence of Leydig cell function impairment due to BPA.

Ultrastructural effects of nerve growth factor and betamethasone on nerve regeneration after experimental nerve injury.

Peripheral nerve injuries (PNI) are an important health problem in the world. In this study, the effects of nerve growth factor (NGF) and betamethasone on nerve regeneration after sciatic nerve crush injury were examined by footprint analysis, electron microscopic, histomorphometric, and biochemical methods. Fifty Wistar rats were divided into five groups as intact control, experimental control, NGF, betamethasone, and NGF+betamethasone combined treatment groups. After the injury, betamethasone was subcutaneously injected into the lesion area of the treatment groups three times during the first day. NGF was subcutaneously injected into the lesion area of treatment groups for 14 days. Footprint analysis was made on 7, 14, 21, 28, and 35 days and after 6 weeks, tissue samples were obtained from all groups. In the experimental control group, there were severe degenerative changes in most of the axons and myelin sheaths of the nerve fibers. Moreover, an increase of MDA levels and a decrease in SOD activities were found in this group. On the other hand, malondialdehyde (MDA) levels decreased, superoxide dismutase (SOD) activities increased and significant motor functional recovery were found in the combined treatment group. The number of axons, axon diameters, and myelin thickness were significantly greater in the combined treatment group when compared with experimental control and other treatment groups. It was thought that nerve regenerative effects of NGF and anti-inflammatory and/or anti-edematous effects of betamethasone could induce functional recovery in the combined treatment group. In conclusion, combined therapy of NGF and betamethasone may be an effective approach for the treatment of PNI.

Effects of combined treatment of minocycline and methylprednisolone on the expression of tumor necrosis factor alpha and interleukine-6 in experimental spinal cord injury: a light and electron microscopic study.

Spinal cord injury (SCI) is an important health problem, and there is no universal treatment protocol for it today. Following SCI pro-inflammatory mediators such as tumor necrosis factor- alpha (TNF-α) and interleukin-6 (IL-6) increase at the lesion site and play important roles in secondary tissue damage. Methylprednisolone (MP) is a glucocorticoid, and minocycline is a tetracycline-derived antibiotic both with neuroprotective effects on central nervous system trauma. However, there are limited studies on their effects on SCI. In this study, we aimed to evaluate effects of MP+minocycline combined treatment on cellular distribution and localization of TNF-α And IL-6 after SCI. Eighty Wistar rats were divided into three main groups as the intact control group, sham operation group, and experimental control group that received spinal cord compression injury. Following the injury, the experimental control group was subdivided into four groups as control, methylprednisolone treatment, minocycline treatment and, MP+minocycline combined treatment groups. Tissue samples were obtained from all groups at 24 hours and 72 hours after the injury. We found a significant decrease in TNF-α And IL-6 expressions in combined treatment group at 24 hours after injury. Also, there was a significant decrease in MDA and increase in SOD levels in this group. Furthermore, decreased lipid peroxidation and neuronal and glial cell death were also observed in combined treatment group. These results suggest that MP+minocycline combined treatment promotes functional recovery and, it should be considered as an effective treatment protocol following SCI.

21-Hydroxylase deficiency: Mutational spectrum and Genotype-Phenotype relations analyses by next-generation sequencing and multiplex ligation-dependent probe amplification.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) is autosomal recessive disorder of cortisol biosynthesis. Genetic defects in CYP21A2 cause 21OHD. The aim of this study was to determine spectrum of mutations in CYP21A2 in a large cohort and analyze the genotype-phenotype correlation to assess predictive characteristics of genotype. We investigated a total of 113 patients with 21OHD. Next-generation sequencing and Multiplex ligation-dependent probe amplification of the CYP21A2 gene were performed in patients and their parents. The genotypes were categorized into Groups 0, A, B, and C according to the residual 21-hydroxylase activities. In this study, the group A was divided into two subgroups as A1 and A2. Three novel variants were found. The genotype-phenotype correlation of the mutation classification was 91.5%. Positive predictivity of subgroups A1 was higher than groups A and subgroups A2. Our study reports genotype-phenotype correlations in the largest 21OHD cohort in Turkey. This correlation sustained when we analyzed our data in combination with metadata from other published studies. This study confirms that CYP21A2 genotyping with next-generation sequencing and MLPA can accurately and reliably confirm the diagnosis of 21OHD. We propose a new classification by dividing group A into two new subgroups to better predict the phenotype. In light of this very high genotype-phenotype correlation, with their ever-increasing availability, declining cost, and turnaround time, we propose that molecular genetic studies can be more economical and practical alternative to the current initial diagnostic laboratory studies based on assays of intermediary steroid metabolites.

Effects of Osteocalcin on Synthesis of Testosterone and INSL3 during Adult Leydig Cell Differentiation.

Proliferation and differentiation of adult Leydig cells are mainly completed in puberty. In many studies, apart from normal postnatal development process, it is widely indicated that, through administrating EDS, Leydig cell population is eliminated and regenerated. It is believed that osteocalcin released from osteoblasts, which is responsible for modulating bone metabolism, induces testosterone production in Leydig cells, independent of the HPG axis. In addition, INSL3 produced by Leydig cells, such as testosterone, plays a critical role in bone metabolism and is known to reflect the development process and functional capacities of Leydig cells. This study is aimed at investigating OC-mediated testosterone regulation and INSL3 synthesis during differentiation of adult Leydig cells that are independent of LH. For this purpose, male rats were divided into 2 groups: prepubertal normal rats and adult EDS-injected rats. Each group was divided into 4 subgroups in which GnRH antagonist or OC was applied. After adult Leydig cells completed their development, testicular tissue samples obtained from the sacrificed rats were examined by light-electron microscopic, immunohistochemical, and biochemical methods. Slight upregulation in 3ßHSD, INSL3, and GPRC6A expressions along with the increase in serum testosterone levels was observed in groups treated with osteocalcin against GnRH antagonist. In addition, biochemical and microscopic findings in osteocalcin treated groups were similar to those in control groups. While there was no significant difference in the number of Leydig cells reported, the presence of a significant upregulation in INSL3 and GPRC6A expressions and the increase in serum testosterone and ucOC levels were observed. After evaluation of findings altogether, it is put forward that, for the first time in this study, although osteocalcin treatment made no significant difference in the number of Leydig cells, it increased the level of testosterone through improving the function of existing adult Leydig cells during normal postnatal development process and post-EDS regeneration. This positive correlation between osteocalcin-testosterone and osteocalcin-INSL3 is concluded to be independent of LH at in vivo conditions.

Characterization CYP1A2, CYP2C9, CYP2C19 and CYP2D6 Polymorphisms Using HRMA in Psychiatry Patients with Schizophrenia and Bipolar Disease for Personalized Medicine.

BACKGROUND: The interindividual genetic variations in drug metabolizing enzymes effects the impact and toxicity in plenty of drugs. OBJECTIVE: CYP1A2, CYP2C9, CYP2C19 and CYP2D6 gene polymorphisms were characterized using high resolution melting analysis (HRMA) in follow-up patients in psychiatry clinic as a preliminary preparation for personalized medicine. METHOD: Genotyping of CYP1A2*1F, CYP2C9 *2, *3, CYP2C19 *2, *3 and *17 and CYP2D6 *3, *4 was conducted in 101 patients using HRMA. Genotype and allele frequencies of the CYP variants were found to be in equilibrium with the Hardy-Weinberg equation. RESULTS: The frequency of the CYP1A2*1F allele in schizophrenia and bipolar disease was 0.694 and 0.255, respectively. The CYP2C9 allele frequencies were 0.087 (CYP2C9*2), and 0.549 (CYP2C9*3) for bipolar; 0.278 (CYP2C9*2) and 0.648 (CYP2C9*3) in schizophrenias. The CYP2C19*2 and *17 allele frequencies was 0.111 and 0.185 in schizophrenia and variant *2 was 0.117 and variant *17 was 0.255 in bipolar group. The frequency of the CYP2D6*3 allele was 0.027 in schizophrenias. The frequencies for the CYP2D6*4 variant were 0.092 and 0.096 in schizophrenia and bipolar groups, respectively. CONCLUSION: The knowledge in pharmacogenomic and also the developments in molecular genetics are growing rapidly. In future, this can be expected to provide new methodologies in the prediction of the activity in drug metabolizing enzymes. The HRMA is a rapid and useful technique to identify the genotypes for drug dosage adjustment before therapy in psychiatry patients.

Therapeutic Potential of Pterostilbene and Resveratrol on Biomechanic, Biochemical, and Histological Parameters in Streptozotocin-Induced Diabetic Rats.

AIMS: The aim of this study was to investigate the effects of pterostilbene (PTS) (trans-3,5-dimethoxy-4'-hydroxystilbene) and resveratrol (RSV) (trans-3,5,4' trihydroxystilbene) applied at different doses for the treatment of streptozotocin- (STZ-) induced diabetic rats. MATERIALS AND METHODS: At the end of the 5-week experimental period, the right gastrocnemius muscles of the rats were examined biomechanically, while the left ones were examined histologically. In addition, blood glucose, serum insulin, and malondialdehyde (MDA) levels were analyzed in blood samples taken from the rats. RESULTS: The skeletal muscle isometric contraction forces, which showed a decrease with diabetes, were observed to increase with antioxidant applications. Blood glucose, serum insulin, and MDA levels in diabetic rats approached normal levels after applying PTS. When the electron microscopic images of the rat skeletal muscle were examined, those in the combination treatment group were observed to show a better enhancement in the skeletal muscle morphological structure compared to the other diabetic and treatment groups. CONCLUSION: According to the findings, we suggest that these antioxidant treatments might have good therapeutic nutraceutical potential for some muscle diseases that coexist with diabetes. These treatments should be comprehensively investigated in the future.

A new biosensor for noninvasive determination of fetal RHD status in maternal blood of RhD negative pregnant women.

Prenatal detection of the fetal RHD status can be useful in the management of RhD incompatibility to identify fetuses at risk of hemolytic disease. Hemolytic disease causes morbidity and mortality of the fetus in the neonatal period. The routine use of antenatal and postnatal anti-D prophylaxis has reduced the incidence of hemolytic disease of the fetus and newborn. This study describe the detection of fetal RhD antigens in blood of RhD negative pregnant women using a nanopolymer coated electrochemical biosensor for medical diagnosis. Cell free fetal DNA in maternal plasma was also used to genotyping fetal RHD status using multiplex real-time PCR. Twenty-six RhD negative pregnant women in different gestational ages were included in the study. RhD positive fetal antibodies detected with a developed biosensor in maternal blood of RhD negative mothers. The electrochemical measurements were performed on a PalmSens potentiostat, and corundum ceramic based screen printed gold electrode combined with the reference Ag/AgCl electrode, and the auxiliary Au/Pd (98/2%) electrode. Fetal RHD genotyping performed using fluorescence-based multiplex real-time PCR exons 5 and 7 of the RHD gene. The fetal RHD status of 26 RhD negative cases were detected 21 as RhD positive and 5 as RhD negative with electrochemical biosensor. Fetal RHD status confirmed with extracted fetal DNA in maternal plasma using multiplex real-time PCR RHD genotyping and by serological test after delivery. The new method for fetal RhD detection in early pregnancy is useful and can be carry out rapidly in clinical diagnosis. Using automated biosensors are reproducible, quick and results can be generated within a few minutes compared to noninvasive fetal RHD genotyping from maternal plasma with real-time PCR-based techniques. We suggest the biosensor techniques could become an alternative part of fetal RHD genotyping from maternal plasma as a prenatal screening in the management of RhD incompatibility.

Beneficial effects of rolipram, a phosphodiesterase 4 specific inhibitor, on testicular torsion-detorsion injury in rats.

INTRODUCTION: The aim of the study is to investigate the effect of Rolipram, a selective phosphodiesterase 4 inhibitor, on testicular torsion - detorsion injury. METHODS: Sixty young male rats were divided into five groups. In each group, the right testes of six rats were removed four hours after detorsion for biochemical analysis, and the right testes of the remaining six rats were removed 24 h after detorsion for pathological analysis. In group 1 (sham-operated) right orchiectomy was performed without torsion, and right testes were sent to the laboratory for biochemical and pathologic analyses. In group 2 (control) torsion was applied to the right testes for 60 min, and detorsion was performed without the administration of Rolipram. In group 3 torsion was applied to the right testes for 60 min. 1 mg/kg Rolipram was administered 30 min before detorsion. In group 4 torsion was applied to the right testes for 60 min, and 1 mg/kg Rolipram was administered during detorsion. In group 5 torsion was applied to the right testes for 60 min. 1 mg/kg Rolipram was administered 30 min after detorsion. The malondialdehyde and nitric oxide levels were determined. The rates of necrosis and apoptosis were evaluated by histopathological examination. RESULTS: The level of malondialdehyde was higher in the torsioned groups (Group 2, 3, 4, 5) than that in group 1 (p = 0.004). There was no statistically significant difference between the groups regarding the level of nitric oxide (p = 0.182). Apoptosis was higher in groups 2, 3 and 4 than in group 1; however, apoptosis was similar in group 1 and group 5 (p = 0.122). The level of necrosis in group 1 was similar to that in groups 4 and 5 (p = 0.194 and p = 0.847, respectively). CONCLUSION: We suggest that the administration of Rolipram can decrease the rate of necrosis and apoptosis in testicular ischaemia-reperfusion injury.

Synthesis, characterization and electrochemistry studies of iron(III) complex with curcumin ligand.

Iron overload is a serious clinical condition for humans and is a key target in drug development. The aim of this study was to investigate the coordination of iron(III) ions with curcumin ligand that may be used in the treatment of iron overload. Iron(III) complex of curcumin was synthesized and structurally characterized in its solid and solution state by FT-IR, UV-Vis, elemental analysis, and magnetic susceptibility. Electrochemical behaviour of the ligand and the complexes were examined using cyclic voltammetry. The cytotoxic activities of the ligand and the iron(III) complex were evaluated by the MTT assay. Curcumin reacted with iron in high concentrations at physiological pH at room temperature. Subsequently, a brown-red complex was formed. Data regarding magnetic susceptibility showed that the complexes with a 1:2 (metal/ligand) mole ratio had octahedral geometry. The complex showed higher anti-oxidant effect towards the cell line ECV304 at IC50 values of 4.83 compared to curcumin. The complex exhibited very high cytotoxic activity and showed a cytotoxic effect that was much better than that of the ligand. The potentials for redox were calculated as 0.180 V and 0.350 V, respectively. The electrochemistry studies showed that Fe3+ /Fe2+ couple redox process occurred at low potentials. This value was within the range of compounds that are expected to show superoxide dismutase activity. This finding indicates that the iron complex is capable of removing free radicals. The observed cytotoxicity could be pursued to obtain a potential drug. Further studies investigating the use of curcumin for this purpose are needed.

Effects of vitamin D on ovary in DHEA-treated PCOS rat model: A light and electron microscopic study.

AIM: The aim of this study was to investigate the effects of vitamin D treatment on ovary in experimentally designed polycystic ovary syndrome of female rats using light and electron microscopic techniques. METHODS: Twenty-four female pre-pubertal rats were divided into control, DHEA and DHEA+Vit.D groups. In DHEA group, the PCOS rat model was developed by 6mg/kg/day dehydroepiandrosterone administration as subcutaneously injections. In DHEA+Vit.D group, 6 mg/kg/day DHEA and 120ng/100g/week 1,25(OH)2D3 was performed simultaneously. Controls were injected with vehicle alone. At the end of the 28 days, blood samples were collected and the ovarian tissues were taken for histological examinations. RESULTS: FSH, LH levels, LH/FSH ratio, and testosterone levels showed a significant increase in DHEA group when compared with the control group. Moreover, these measurements were lower in the treatment group than the DHEA group. In DHEA group, increased number of atretic follicles and cystic follicles were seen with light microscopic analysis. Cystic follicles with attenuated granulosa cell layers and thickened theca cell layers and lipid accumulation in interstitial cells were observed by electron microscope. It is observed that atretic and cystic follicles were decreased as a result of vitamin D treatment. CONCLUSION: Our results indicate the curative role of vitamin D treatment on the androgen excess in PCOS rat model which causes abnormalities in ovarian morphology and functions. Vitamin D has positive effects on the hormonal and structural changes observed in PCOS, but it has been concluded that long-term use may be more beneficial.

Evaluation of intracellular pH regulation and alkalosis defense mechanisms in preimplantation embryos.

Intracellular pH (pHi) regulation is an important homeostatic function of cells. There are three major pHi-regulatory mechanisms: HCO3(-)/Cl(-) exchanger (anion exchanger [AE]), which alleviates alkalosis, and the Na(+)/H(+) and Na(+),HCO3(-)/Cl(-) exchangers, both of which alleviate acidosis. We hypothesized that there would be developmental changes in pHi-regulatory activity in preimplantation embryos as conditions in the oviduct are alkaline but acidic in the uterus. This study focused on the AE mechanism in pronuclear (PN) zygotes, two-cell (2-c), four-cell (4-c), morula, and blastocyst stage embryos from Balb/c mice. Microspectrofluorometry was used to monitor changes in pHi in embryos subjected to Cl(-)-free media in presence or absence of an AE inhibitor, DIDS, and in embryos recovering from NH4Cl-induced alkalosis. Real-time polymerase chain reaction was used to identify AE isoforms. The pHi changes were greatest in PN zygotes (0.086 ± 007 pHU/min) but fell as embryos developed to the 2-c, 4-c, morula, and blastocyst stages (0.063 ± 006; 0.035 ± 007; 0.024 ± 004, and 0.014 ± 004 pHU/min, respectively). DIDS significantly reduced the rise in pHi caused by Cl(-) removal in all embryos; the finding pointed out that this pHi changes are due to AE activity. But DIDS only inhibited the recovery responses of PN zygote, 2-c and 4-c embryos but not morula or blastocyst stage embryos. In bicarbonate-containing medium, all embryos recovered from induced alkalosis but only the morula and blastocyst stages could fully compensate from ammonium induced-alkalosis in bicarbonate-free medium. The finding showed that commonly used ammonium pulse method to investigate AE function against alkalosis is not suitable for morula and blastocyst embryonic stages. All embryos expressed SLC4A2 and SLC4A4 coding for AE-2 and AE-4, but none expressed either AE-1 or AE-3. The gradual change in the response to alkalosis in preimplantation embryos may be adaptations to their normal in vivo environment, where the early embryos are located in the alkaline oviduct, whereas the morula and blastocyst move into the acidic uterus.

Noninvasive prenatal diagnosis experience in the Çukurova Region of Southern Turkey: detecting paternal mutations of sickle cell anemia and ß-thalassemia in cell-free fetal DNA using high-resolution melting analysis.

OBJECTIVE: This study used a high-resolution melting (HRM) technique to detect paternal mutations for the noninvasive prenatal diagnosis (NIPD) of ß-thalassemia and sickle cell anemia (HbS). We also determined the levels of cell-free fetal DNA and total cell-free DNA. METHODS: We used the HRM technique for fetal genotyping of paternal mutations in maternal plasma from 32 pregnancies at risk of ß-thalassemia and 57 pregnancies at risk of HbS. The DNA levels in maternal plasma were measured using real-time quantitative PCR. Multiples of the median (MoM) values were calculated in women at risk for ß-thalassemia or HbS. RESULTS: Twenty-two paternal mutations were detected in 89 pregnant women. Although we were successfully able to detect the paternal ß-thalassemia mutations, the mutant HbS fetuses could not be distinguished from maternal background in the early weeks of pregnancy. The detection of DYS14 in male fetuses was 100%. The MoM values of women at high risk of having HbS-affected fetuses were higher than those for the other groups. CONCLUSION: High-resolution melting is a useful method for NIPD of ß-thalassemias by detecting paternal mutations in the maternal plasma. Cell-free fetal DNA quantification and MoM values were not informative for HbS or ß-thalassemias in early pregnancy.

Identification of two rare ß-globin gene mutations in a patient with ß-thalassemia intermedia from Azerbaijan.

ß-Thalassemias are an inherited group of disorders of hemoglobin (Hb) and comprise the most common monogenic disorders in Azerbaijan. They are extremely heterogeneous at the molecular level. Here we report the first identification of a patient who is a compound heterozygote for two rare ß-thalassemia (ß-thal) mutations, IVS-I-130 (G>C) and codon 37 (TGG>TGA).