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).     

Cytopathologic features of pituitary carcinoma with cervical vertebral bone metastasis: a case report

BACKGROUND: Pituitary carcinomas are extremely rare tumors of the adenohypophysis. The presence of craniospinal and/or systemic extracranial metastases is the only reliable criterion for the diagnosis of pituitary carcinoma. To date, only 2 cases have been reported correctly by fine needle aspiration biopsy (FNAB). We present an additional case of pituitary carcinoma with FNAB features. CASE: A 60-year-old woman presented with clinical features of Cushing's disease and a pituitary tumor. She underwent transsphenoidal resection of the tumor. The initial diagnosis was an adrenocorticotrophic hormone (ACTH)-producing invasive pituitary adenoma. The patient presented again with neck pain 6 years after the operation. Magnetic resonance imaging revealed metastatic tumor masses at the level of C5-C6 of the cervical vertebrae. Intraoperative fine needle aspiration and incomplete excision of metastatic tumors were performed. Cytologically, tumor cells were composed of a combination of loose groups and single cells. Neoplastic cells had a relatively monotonous appearance and displayed characteristic neuroendocrine tumor features. Immunocytochemistry from cell block sections revealed AE1/ AE3, synaptophysin chromogranin A and ACTH positivity in the tumor cells. CONCLUSION: Pituitary carcinoma with extracranial systemic metastases demonstrates typical neuroendocrine features on fine needle aspiration. In the differential diagnosis, metastatic neuroendocrine carcinomas should be kept in mind. In the absence of sufficient clinical data, these 2 entities cannot be distinguished correctly through the cytologic features.     

Identification of adsorption inhibition, restriction/modification and abortive infection type phage resistance systems in Lactococcus lactis strains

98 Lactococcus lactis strains were isolated from traditional fermented milk products in Turkey tested against 60 lactococcal lytic phages to determine their resistance levels. While 82 L. lactis strains were sensitive against lactic phages at different levels, 16 L. lactis strains showed resistance to all phages tested. Types of phage resistance among 16 L. lactis strains were identified as phage adsorption inhibition in eight strains, restriction/modification in six strains and abortive infection (heat sensitive phage resistance) in two strains, using three broad-spectrum phages phi pll 98-32, phi pld 67-42 and phi pld 67-44.     

Nitric oxide levels in patients with psoriasis treated with methotrexate

Psoriasis is a chronic, recurrent, inflammatory, and hyperproliferative disease. Recently there have been studies regarding increases in the levels of NO in inflammatory dermatoses including psoriasis. In this study, 22 patients with psoriasis were scored with PASI (psoriasis area and severity index) and the levels of serum nitrite-nitrate were evaluated before and after therapy with methotrexate (Mtx). The results were compared with age- and sex-matched healthy volunteers. The relation of the results with the clinical severity and the cumulative Mtx dose were also evaluated. The serum levels of nitrite-nitrate of the psoriatic patients with active lesions were found to be significantly higher than the levels of the healthy volunteers and the patients after therapy. The elevated nitrite-nitrate serum levels in the inflammatory period may suggest the possible role of this mediator in the etiopathogenesis of psoriasis and the potential future use of NO inhibitors in the treatment of psoriasis.     

Reproductive Organ and Vascular Specific Promoter of the Rice Plasma Membrane Ca2+ATPase Mediates Environmental Stress Responses in Plants

Background

Plasma membrane Ca²⁺ATPase is a transport protein in the plasma membrane of cells and helps in removal of calcium (Ca²⁺) from the cell, hence regulating Ca²⁺ level within cells. Though plant Ca²⁺ATPases have been shown to be involved in plant stress responses but their promoter regions have not been well studied.

Results

The 1478 bp promoter sequence of rice plasma membrane Ca²⁺ATPase contains cis-acting elements responsive to stresses and plant hormones. To identify the functional region, serial deletions of the promoter were fused with the GUS sequence and four constructs were obtained. These were differentially activated under NaCl, PEG cold, methyl viologen, abscisic acid and methyl jasmonate treatments. We demonstrated that the rice plasma membrane Ca²⁺ATPase promoter is responsible for vascular-specific and multiple stress-inducible gene expression. Only full-length promoter showed specific GUS expression under stress conditions in floral parts. High GUS activity was observed in roots with all the promoter constructs. The −1478 to −886 bp flanking region responded well upon treatment with salt and drought. Only the full-length promoter presented cold-induced GUS expression in leaves, while in shoots slight expression was observed for −1210 and −886 bp flanking region. The −1210 bp deletion significantly responded to exogenous methyl viologen and abscisic acid induction. The −1210 and −886 bp flanking region resulted in increased GUS activity in leaves under methyl jasmonate treatments, whereas in shoots the −886 bp and −519 bp deletion gave higher expression. Salicylic acid failed to induce GUS activities in leaves for all the constructs.

Conclusions

The rice plasma membrane Ca²⁺ATPase promoter is a reproductive organ-specific as well as vascular-specific. This promoter contains drought, salt, cold, methyl viologen, abscisic acid and methyl jasmonate related cis-elements, which regulated gene expression. Overall, the tissue-specificity and inducible nature of this promoter could grant wide applicability in plant biotechnology.     

Current techniques in reprogramming cell potency

The first successful attempt to reprogram somatic cell into embryonic‐like stem cell was achieved on 2006. Since then, it had sparked a race against time to bring this wonderful invention from bench to bedside but it is not easily achieved due to severe problems in term of epigenetic and genomic. With each problem arise, new technique and protocol will be constructed to try to overcome it. This review addresses the various techniques made available to create iPSC with problems hogging down the technique. J. Cell. Biochem. 114: 1230–1237, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

Reduced catalytic activity of human CYP2C9 natural alleles for gliclazide: molecular dynamics simulation and docking studies

Amongst sulfonylureas, gliclazide is one of the mostly prescribed drugs to diabetic patients and is metabolized extensively by P450 CYP2C9. Among 24-CYP2C9 alleles, the *2/*2 and *3/*3 genotypes showed significantly lower gliclazide clearances with reductions of 25 and 57%, respectively. However, the reason for the change in drug-metabolizing activity induced by these natural alleles is unknown. In the present study, we used molecular dynamics simulation and autodocking studies to provide models for gliclazide-bound complexes of CYP2C9*2, *3 and *2/*3 mutants, which give insight into CYP2C9-gliclazide interactions and explain the reduced enzymatic activity seen in these variants. Our data shows that the size of the substrate-access entry site is significantly reduced in mutants, which limits the access of gliclazide to heme and the active site. The distance from the substrate oxidation site and heme is >5 Å in *3 and *2/*3. Therefore, the addition of an active oxygen molecule by heme-Fe is hindered. The absence of F100, F114 and F476 in the interacting amino acid pocket in *3 reduces catalytic efficiency toward gliclazide. In *1, gliclazide is stabilized by the formation of two hydrogen bonds with R108 while it is absent in mutants. Further in *3 and *2/*3, the key heme-stabilizing residue, R97 stabilization is greatly reduced. Therefore, the decreased catalytic activity of these variants can be explained from the reduced access of the gliclazide to heme, and the interaction between heme and substrate is affected due to their instability in the active site.