Mutation analysis of mitogen activated protein kinase 1 gene in Indian cases of 46,XY disorder of sex development.

BACKGROUND: Determination of sex is the result of cascade of molecular events that cause undifferentiated bipotential gonad to develop as a testis or an ovary. A series of genes such as SRY, steroidogenic factor‑1 (SF1), AR, SRD5 α, Desert hedgehog (DHH) etc., have been reported to have a significant role in development of sex in the fetus and secondary sexual characteristics at the time of puberty. Recently, mitogen activated protein kinase kinase kinase 1 (MAP3K1) gene was found to be associated with 46, XY disorders of sex development (DSD). AIM: The present study is focused to identify mutations in MAP3K1 gene in the cohort of 10 Indian patients with 46,XY DSD including one family with two affected sisters. These patients were already screened for SRY, SF1 and DHH gene, but no mutation was observed in any of these genes. MATERIALS AND METHODS: The entire coding regions of MAP3K1 were amplified and sequenced using the gene specific primers. RESULTS AND DISCUSSIONS: Sequence analysis of MAP3K1 gene has revealed four variants including one missense, two silent and one deletion mutation. The missense mutation p.D806N was observed in four patients with hypospadias. Two patients showed the presence of silent mutation p.Q1028Q present in exon 14. Another silent mutation p.T428T was observed in a patient with gonadal dysgenesis. We have also observed one deletion mutation p. 942insT present in two patients. The pathogenicity of the missense mutation p.D806N was carried out using in‑silico approach. Sequence homology analysis has revealed that the aspartate at 806 was found to be well‑conserved across species, indicated the importance of this residue. The score for polyphen analysis of this mutation was found to be 0.999 indicating to be pathogenic mutation. Since, p.D806N mutation was found to be important residue; it might contribute to sexual development. We have reported the presence of mutations/polymorphism in MAP3K1 gene. All the mutations were found to be polymorphism upon comparing to single nucleotide polymorphism database. However, in‑silico analysis of the missense mutation revealed to be a pathogenic mutation.

Apigenin inhibits cell migration through MAPK pathways in human bladder smooth muscle cells

Apigenin, a nonmutagenic flavonoid, has been shown to possess free radical scavenging activities, anticarcinogenic properties, antioxidant and anti-inflammatory effects. Recently, apigenin was reported to cause gastric relaxation in murine. To assess possible effects of apigenin on migration of bladder smooth muscle (SM) cell, we isolated SM cells from peri-cancer tissue of human bladder and established a cell model that was capable to overexpress transiently MEKK1 (MEK kinase 1). Results showed that overexpression of active human MEKK1 by adenoviruses infection induced migration of human bladder smooth muscle (hBSM) cells and phosphorylation of MAPKs, ERK, JNK and p38, which are the downstream molecules of MEKK1. Then, hBSM cell overexpressing MEKK1 were exposed to apigenin (50 microM). Our data indicated that apigenin inhibited significantly activation/phosphorylation of MAPKs and migration of hBSM cells induced by MEKK1 overexpression. Besides, apigenin inhibited actin polymerization, which underlines muscle contraction and cell migration. The results suggest that apigenin inhibits activation of MAPKs and thereby the cell migration. The mechanism might be that apigenin blocks signal transmission from MEKK1 to MAPKs.

Salvianolic acid B alleviate the disruption of blood-brain barrier in rats after cerebral ischemia-reperfusion by inhibiting MAPK pathway / 药学学报

The aim of the study is to investigate the effect of salvianolic acid B (SalB) on blood-brain barrier (BBB) in rats after cerebral ischemia-reperfusion, and to illustrate its possible mechanisms. Cerebral ischemia-reperfusion was induced by middle cerebral artery occlusion in rats. The break-down of BBB was indicated by extravasations of immunoglobulin (IgG) monitored with immunohistochemistry. The expression of MMP-9 and NOS2 in the brain was determined by immunohistochemistry, and the expression of p-p38 and p-ERK1/2 was detected by Western blotting. It was shown that on day 2 after ischemia-reperfusion the IgG accumulated around the vascular boundary zone, suggesting the break-down of BBB, and the expression of MMP-9 and NOS2 up-regulated at the same time. The result of Western blotting suggested that the expression of p-p38 and p-ERK1/2 increased. On day 7 after ischemia-reperfusion the. expression of MMP-9 and NOS2 was about the same level as day 2, the expression of p-p38 was higher than that on day 2 and the expression of p-ERK1/2 was slightly lower than that on day 2. SalB (1 and 10 mg x kg(-1)) significantly alleviated the extravasations of immunoglobulin induced by cerebral ischemia-reperfusion (P < 0.05). On day 2 and day 7 SalB attenuated the expression of MMP-9 and NOS2 (P < 0.05). SalB (10 mg x kg(-1)) reduced the expression of p-p38 and p-ERK1/2 apparently on day 2 and 7 after ischemia-reperfusion (P < 0.05). SalB (1 mg x kg(-1)) inhibited the expression of p-p38 on day 7 after ischemia-reperfusion (P < 0.05). The results indicate that SalB protects blood-brain barrier in rats after cerebral ischemia-reperfusion by inhibiting the MAPK pathway.