Effect of melatonin on torsion and reperfusion induced pathogenesis of rat uterus

ABSTRACT

We investigated the use of melatonin to improve fertility and reduce uterine damage caused by torsion of the uterus in pregnant rats. We used 35 pregnant rats at gestational age 18 days. The animals were randomized into five groups. Group 1 was anesthetized only. Group 2 was subjected to experimental uterine torsion of 360° and the torsion was corrected after 6 h. Group 3 was subjected to uterine torsion of 360°, the torsion was corrected after 6 h and melatonin was administered at the time of correction. Group 4 rats were subjected to 360º uterine torsion and melatonin was administered 6 h later at the time of correction. Group 5 was administered melatonin followed by uterine torsion of 360 degrees followed by correction of torsion 6 h later. Samples were obtained from the uterine horns on the day 1 postpartum. We used Bax, Bcl-2 and caspase 3 staining to measure apoptosis in the uterine tissues. The mRNA levels of Rho-associated, coiled-coil containing protein kinases 1 (ROCK1), homeobox D10 (Hox4 HoxD10), TLR4, NFκB1, caveolin 1 (Cav1) heat shock protein 90 alpha (cytosolic), class B member 1 (Hsp90ab1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using quantitative real-time polymerase chain reaction analysis (qRT-PCR). Bax, Bcl-2 and caspase 3 were detected using immunohistochemistry. No difference was observed among groups with respect to abortion, neonatal mortality or congenital abnormalities. Compared to the control group, the mRNA levels of Rock1, Hox4, TLR4, NFκB1, Cav1 and Hsp90 genes were decreased significantly in the study groups; the decrease was greater in groups 3 and 4, which were treated with melatonin. The greatest amount of Bax staining was found in group 1 and the least amount of Bcl-2 staining was found in groups 4 and 5; the greatest amount of caspase 3 staining was found in group 2. Our findings indicate that melatonin reduced uterine torsion related tissue damage and that its application during torsion was more effective than application following removal of torsion.     

Functional characterization of the S41Y (C2755A) polymorphism of tryptophan hydroxylase 2

Human TPH2 (hTPH2) catalyzes the rate‐limiting step in CNS serotonin biosynthesis. We characterized a single‐nucleotide polymorphism (C2755A) in the hTPH2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased V_max with unchanged K_m values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37°C) for the S41Y enzyme (as compared to the wild‐type enzyme). The S41Y polymorphism decreased cyclic AMP‐dependent protein kinase A‐mediated phosphorylation ~ 50% relative to wild‐type hTPH2, suggesting that the S41Y mutation may disrupt the post‐translational regulation of this enzyme. Transfected PC12 cells expressed hTPH2 mRNA, active protein, and synthesized and released serotonin. Paradoxically, while S41Y‐transfected PC12 cells expressed higher levels of hTPH2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression.

     

Valproic acid inhibits the proliferation of SHSY5Y neuroblastoma cancer cells by downregulating URG4/URGCP and CCND1 gene expression

Valproic acid (VPA), used for the treatment of epilepsy and bipolar disorder, regulates several signaling pathways in brain cells. The up-regulated gene 4 (URG4/URGCP) is a novel gene located on 7p13. URG4/URGCP stimulates cyclin D1 (CCND1) mRNA expression, and URG4/URGCP silencing diminishes CCND1 mRNA expression in HepG2 cells. This study was performed to investigate the anti-cancer mechanism of action of VPA by analyzing the expression of novel gene URG4/URGCP, CCND1, p21, p53, p65 (RelA), Bax, and Bcl-2 in SHSY5Y neuroblastoma (NB) cancer cells. Cytotoxic effects of VPA in SHSY5Y were noticed in time and dose dependent manner with the IC₅₀ doses within the range of 0.5–10 mM. IC₅₀ doses in the SHSY5Y were detected as 7.5 mM. Expression profiles were determined by semi quantitative RT-PCR and URG4/URGCP protein change by western blot analysis. Our results suggest that VPA induces cell cycle arrest in SHSY5Y due to the decrease in URG4/URGCP, CCND1 gene expression and the increase in p65. To conclude, VPA may be a prospective agent for the treatment of NB as a single agent or in combination with other drugs. Thus, more studies should be designed to find a safe dose with the best effects of VPA.     

The role of Wnt pathway antagonists in early-stage lung adenocarcinoma

Molecular Biology Reports - We aimed to examine the expression levels of the genes encoding adenomatous polyposis coli (APC) 1, APC-2, Dickkopf related protein (DKK)-1, DKK-3, secreted...     

Lymphocyte-mediated lysis of sheep chorion: susceptibility of chorionic cells to third-party and maternal cytotoxic lymphocytes and presence of cells in the endometrium exhibiting cytotoxicity toward natural-killer cell targets

In several species, the trophoblast is resistant to lysis by cytotoxic lymphocytes. Such resistance is believed to contribute to survival of the semiallogenic conceptus. We tested whether ovine chorionic cells are susceptible to lysis by specific and nonspecific cytotoxic lymphocytes in peripheral blood (PBL) and whether cytotoxic cells that can lyse target cells for natural-killer cells are present in the endometrium. Primary chorionic cells from pregnant ewes at Days 51-91 of gestation were labeled with 51Cr and incubated for 20 h at 50:1 and 100:1 ratios with PBL from the pregnant mother or from a third-party ewe. In the absence of interleukin-2 (IL-2), there was no killing of primary chorionic cells by third-party PBL even after infection of chorionic cells with bovine herpes virus-1. Incubation with IL-2-induced cytotoxic action in third-party PBL towards one of six primary chorionic cell preparations only. Primary chorionic cells from two of four placentae were lysed by maternal PBL. Luminal epithelial cells from cyclic ewes and from the pregnant and nonpregnant uterine horns of unilaterally-pregnant ewes were evaluated for the presence of cells capable of killing D17 target cells (a natural-killer cell target). Killing was observed but there was no difference in activity between physiological stages. In contrast, there was intense immunochemical localization of perforin in glandular and luminal endometrial epithelial cells in pregnant ewes, and less intense staining in nonpregnant animals. It is concluded that ovine chorionic cells are generally resistant to killing by natural-killer-like cells and lymphokine-activated killer cells. Generation of maternal cytotoxic lymphocytes against trophoblast can occur in some cases and may contribute to pregnancy loss.     

GJB2 mutations and degree of hearing loss: a multicenter study

Hearing impairment (HI) affects 1 in 650 newborns, which makes it the most common congenital sensory impairment. Despite extraordinary genetic heterogeneity, mutations in one gene, GJB2, which encodes the connexin 26 protein and is involved in inner ear homeostasis, are found in up to 50% of patients with autosomal recessive nonsyndromic hearing loss. Because of the high frequency of GJB2 mutations, mutation analysis of this gene is widely available as a diagnostic test. In this study, we assessed the association between genotype and degree of hearing loss in persons with HI and biallelic GJB2 mutations. We performed cross-sectional analyses of GJB2 genotype and audiometric data from 1,531 persons, from 16 different countries, with autosomal recessive, mild-to-profound nonsyndromic HI. The median age of all participants was 8 years; 90% of persons were within the age range of 0-26 years. Of the 83 different mutations identified, 47 were classified as nontruncating, and 36 as truncating. A total of 153 different genotypes were found, of which 56 were homozygous truncating (T/T), 30 were homozygous nontruncating (NT/NT), and 67 were compound heterozygous truncating/nontruncating (T/NT). The degree of HI associated with biallelic truncating mutations was significantly more severe than the HI associated with biallelic nontruncating mutations (P<.0001). The HI of 48 different genotypes was less severe than that of 35delG homozygotes. Several common mutations (M34T, V37I, and L90P) were associated with mild-to-moderate HI (median 25-40 dB). Two genotypes--35delG/R143W (median 105 dB) and 35delG/dela(GJB6-D13S1830) (median 108 dB)--had significantly more-severe HI than that of 35delG homozygotes.