Loop‐mediated isothermal amplification of single pollen grains

The polymerase chain reaction (PCR) has been a reliable and fruitful method for many applications in ecology. Nevertheless, unavoidable technical and instrumental requirements of PCR have limited its widespread application in field situations. The recent development of isothermal DNA amplification methods provides an alternative to PCR, which circumvents key limitations of PCR for direct amplification in the field. Being able to analyze DNA in the pollen cloud of an ecosystem would provide very useful ecological information, yet would require a field‐enabled, high‐throughput method for this potential to be realized. Here, we demonstrate the applicability of the loop‐mediated DNA amplification method (LAMP), an isothermal DNA amplification technique, to be used in pollen analysis. We demonstrate that LAMP can provide a reliable method to identify species from the pollen cloud, and that it can amplify successfully with sensitivity down to single pollen grains, thus opening the possibility of field‐based, high‐throughput analysis.     

Effect of transport and storage temperature of ovaries on in vitro maturation of bitch oocytes

In this study, the effects of ovary transport and storage temperature on in vitro maturation of bitch oocytes were investigated. Ovaries were collected from 23 mature bitches and one randomly selected ovary of each pair (n=23 pairs) was transported in physiologic saline at 4 degrees C, while the other one at 35-38 degrees C for 2-4h. A total of 316 cumulus oocyte complexes (COCs) were obtained from the 4 degrees C group and 301 COCs from the 35-38 degrees C group. All COCs were matured in modified synthetic oviduct fluid (mSOF) supplemented with follicle stimulating hormone (FSH), essential and non-essential amino acids at 38 degrees C in a humidified 5% CO2, 5% O2, and 90% N2 atmosphere for 72 h. At the end of the in vitro maturation period, nuclear maturation of oocytes was classified as germinal vesicle (GV), germinal vesicle breakdown (GVBD), metaphase I (MI), metaphase II (MII), undetermined nuclear maturation (UDNM), and MI+MII. The nuclear maturation rates to MI, MII, and MI+MII stages were 60.44%, 10.75%, and 71.20% in the 4 degrees C group and 37.20%, 7.64%, and 45.85% in the 35-38 degrees C group, respectively. The data demonstrated that oocytes obtained from ovaries transported at 4 degrees C had higher maturation rates than from the ones transported at 35-38 degrees C (p<0.001).     

Boron regulates mineralized tissue-associated proteins in osteoblasts (MC3T3-E1)

The aim of this study was to determine the effects of boron (B) on the cell-survival, proliferation, mineralization and mRNA expression of mineralized tissue-associated proteins. Additionally, determination of the effects of B on the BMP-4, -6 and -7 protein levels of pre-osteoblastic cells (MC3T3-E1) was also intended. The effects of B (pH 7.0) concentrations (0, 0.1, 1, 10, 100, 1000, 2000, 4000, 8000 and 10,000 ng/ml) on the survival of the cells were evaluated at 24 and 96 hrs with MTT assay. To evaluate the proliferation in long term, MC3T3-E1 cells were treated with different concentrations of B (0, 0.1, 1, 10, 100 and 1000 ng/ml) and were counted on days 2, 5, and 14. While in short term, decreased cell survival rate was observed at 1000 ng/ml and above, at long term no statistically significant difference was detected in different B concentrations applied. Slight decreases at the proliferation of the B-treated groups were determined on days 5 and 14 but one-way analysis of variance revealed that the difference was statistically insignificant. In mineralization assay, increased mineralized nodules were apparently observed in B treatment (1 and 10 ng/ml concentrations) groups. Based on quantitative RT-PCR results, remarkable regulation in favor of osteoblastic function for Collagen type I (COL I), Osteopontin (OPN), Bone Sialoprotein (BSP), Osteocalcin (OCN) and RunX2 mRNA expressions were observed in B treatment groups in comparison with untreated control groups. Increased BMP-4, -6 and -7 protein levels were detected at 0.1, 1, 10 and 100 ng/ml B concentrations. Results of the study suggest that at the molecular level B displays important roles on bone metabolism and may find novel usages at the regenerative medicine.     

A new species of trout from the northern drainages of Euphrates River,Turkey (Salmoniformes: Salmonidae)

Salmo fahrettini, a new species, is distributed in the northern tributaries of the Euphrates River. It differs from other Salmo species in adjacent waters by a combination of the following characters: a greyish body; one black spot behind the eye and on the cheek; three to six black spots on the opercle; numerous black spots on the back (missing on the predorsal area), flank and middle part of body, surrounded by a roundish white ring; red spots in the median part of the body, surrounded by a roundish white ring; short and narrow maxilla; increase in the number of black and red spots with an increase in size; adipose fin medium size, no or rarely one red spot at its posterior edge; 109–116 lateral line scales; 27–30 scale rows between dorsal-fin origin and lateral line; 20–23 scale rows between the lateral line and anal-fin origin; maxilla length 8.8–10.0% standard length in males, 8.8–9.6 in females.     

The Effects of Levosimendan Exposure on Oxidant/Antioxidant Status and Trace Element Levels in the Pulmonary Artery of Rats

We investigated both the effect of levosimendan and the role of oxidant/antioxidant status and trace element levels in the pulmonary artery of rats. Fourteen male Wistar albino rats were randomly divided into two groups of seven animals each. Group 1 was not exposed to levosimendan and served as a control. Levosimendan (12 μg/kg) diluted in 10 ml 0.9 % NaCl was administered intraperitoneally to group 2. Animals of both groups were killed after 3 days, and their pulmonary arteries were harvested to determine changes in tissue oxidant/antioxidant status and trace element levels. The animals in both groups were killed 72 h after the levosimendan exposure treatment, and pulmonary arteries were harvested to determine levels of the lipid peroxidation product MDA and the antioxidant GSH as well as the decreased activity of antioxidant enzymes such as SOD, GSH-Px and CAT. It was found that MDA levels increased in pulmonary artery tissues of rats after levosimendan administration. The GSH level decreased in the pulmonary artery of rats after levosimendan treatment. Co, Mn, Fe, Cd and Pb levels were significantly higher (P < 0.001) and Mg, Zn and Cu levels significantly lower (P < 0.001) in the levosimendan group compared to the control group. These results suggest that levosimendan treatment caused an increase in free radical production and a decrease in antioxidant enzyme activity in the pulmonary artery of levosimendan-treated rats. It also caused a decrease or increase in the levels of many minerals in the pulmonary artery, which is an undesirable condition for normal pharmacological function.     

Association between fibromyalgia syndrome and polymorphism of the IL-4 gene in a Turkish population

Purpose

Fibromyalgia (FM) syndrome is a form of non-articular rheumatism characterized by long term and widespread musculoskeletal pain, morning stiffness, sleep disturbance, paresthesia, and pressure hyperalgesia at characteristic sites, called soft tissue tender points. The etiology of FM is still obscure. Genetic factors may predispose individuals to FM. Cytokines may play a role in the pathophysiology of FM. The aim of this study was to investigate the interleukin-4 (IL-4) 70 bp VNTR variations in Turkish patients with FM and evaluate if there was an association with clinical features, especially between these polymorphisms.

Methods

The study included 300 patients with FM and 270 healthy controls. Genomic DNA was isolated and genotyped using polymerase chain reaction (PCR) for the IL-4 gene 70 bp VNTR polymorphisms.

Results

There was statistically significant difference between the groups with respect to IL-4 genotype distribution and allele frequencies (p < 0.0001). The homozygous P₁P₁ genotype and P₁ allele were significantly higher in FM patients than in healthy controls (p = 0.04; OR: 3.25, 95% CI: 1–10, p < 0.0001; OR:4.84, 95% CI:3–7.7). There was not any difference between the groups respect to IL-4 genotype distribution and allele frequencies (p > 0.05) and clinical characteristics.

Conclusion

Our findings suggest that there is an association of IL-4 gene 70 bp VNTR polymorphism with susceptibility of a person for development of FM. As a result, further studies are necessary to determine whether IL-4 may be a genetic marker for FM in the Turkish population.     

Basal damage and oxidative DNA damage in children with chronic kidney disease measured by use of the comet assay

One consequence of chronic kidney disease (CKD) is an elevated risk for cancer. There is sufficient evidence to conclude that there is an increased incidence of at least some cancers in kidney-dialysis patients. Cancer risk after kidney transplantation has mainly been attributed to immunosuppressive therapy. There are no data evaluating DNA damage in children with CKD, in dialysis patients, or following kidney transplantation. In this study, the comet assay and the enzyme-modified comet assay - with the use of endonuclease III (Endo III) and formamidopyrimidine glycosylase (FPG) enzymes - were conducted to investigate the basal damage and the oxidative DNA damage as a result of treatment in peripheral blood lymphocytes of children. Children at various stages of treatment for kidney disease, including pre-dialysis patients (PreD) (n=17), regular hemodialysis patients (HD) (n=15), and those that received kidney transplants (Tx) (n=17), comprised the study group. They were compared with age- and gender-matched healthy children (n=20) as a control group. Our results show that the %DNA intensity, a measure of basal damage, was significantly increased in children with CKD (mean ± SD) (5.22 ± 1.57) and also in each of the PreD, HD, and Tx groups [(4.92 ± 1.23), (4.91 ± 1.35), and (5.79 ± 1.94), respectively, vs the healthy children (2.74 ± 2.91) (p<0.001). Significant increases in oxidative DNA damage were only found in the FPG-sensitive sites for the PreD and Tx groups, compared with control and HD groups (p<0.05), suggesting that basal DNA damage was more evident for the PreD, HD, and Tx groups. The findings of the present study indicate a critical need for further research on genomic damage with different endpoints and also for preventive measures and improvements in treatment of pediatric patients, in order to improve their life expectancy.     

Differential changes in Substance P, VIP as well as neprilysin levels in patients with gastritis or ulcer

The protective effect of capsaicin-sensitive sensory nerve (CSSN) activation was recently demonstrated in human gastric mucosa. We here examined changes in neuropeptides, specifically Substance P (SP), calcitonin-gene related peptide (CGRP) and vasoactive intestinal peptide (VIP) in patients with chronic gastritis or ulcer. Furthermore changes in neprilysin levels, which hydrolyse these neuropeptides, were determined. Gastric biopsies were obtained from both lesion- and normal-appearing mucosa of 57 patients. The presence of H. pylori infection was verified with rapid urease assay. Neuronal and non-neuronal levels of SP, VIP, CGRP and neprilysin activity were determined in freshly frozen biopsies. Immunohistochemical localization of neprilysin was performed in 30 paraffin embedded specimens. We here found that neuronal SP levels decreased significantly in normally appearing mucosa of patients with gastritis while levels of non-neuronal SP increased in diseased areas of gastritis and ulcer. The presence of H. pylori led to further decreases of SP levels. The content of VIP in both disease-involved and uninvolved mucosa, and expression of neprilysin, markedly decreased in patients with gastritis or ulcer. Since VIP, as well as SP fragments, formed following hydrolysis with neprilysin is recognized to have gastroprotective effects, decreased levels of VIP, SP and neprilysin may predispose to cellular damage.