MAP3K1 regulates female reproductive tract development.

Mitogen-activated protein 3 kinase 1 (MAP3K1) has a plethora of cell type-specific functions not yet fully understood. Herein, we describe a role for MAP3K1 in female reproductive tract (FRT) development. MAP3K1 kinase domain-deficient female mice exhibited an imperforate vagina, labor failure and infertility. These defects corresponded with shunted Müllerian ducts (MDs), the embryonic precursors of FRT, that manifested as a contorted caudal vagina and abrogated vaginal-urogenital sinus fusion in neonates. The MAP3K1 kinase domain is required for optimal activation of the Jun-N-terminal kinase (JNK) and cell polarity in the MD epithelium, and for upregulation of WNT signaling in the mesenchyme surrounding the caudal MD. The MAP3K1-deficient epithelial cells and MD epithelium had reduced expression of WNT7B ligands. Correspondingly, conditioned media derived from MAP3K1-competent, but not -deficient, epithelial cells activated a TCF/Lef-luciferase reporter in fibroblasts. These observations indicate that MAP3K1 regulates MD caudal elongation and FRT development, in part through the induction of paracrine factors in the epithelium that trans-activate WNT signaling in the mesenchyme.

Development of sexual dimorphism of skeletal muscles through the adrenal cortex, caused by androgen-induced global gene suppression.

The zona fasciculata (zF) in the adrenal cortex contributes to multiple physiological actions through glucocorticoid synthesis. The size, proliferation, and glucocorticoid synthesis characteristics are all female biased, and sexual dimorphism is established by androgen. In this study, transcriptomes were obtained to unveil the sex differentiation mechanism. Interestingly, both the amount of mRNA and the expressions of nearly all genes were higher in females. The expression of Nr5a1, which is essential for steroidogenic cell differentiation, was also female biased. Whole-genome studies demonstrated that NR5A1 regulates nearly all gene expression directly or indirectly. This suggests that androgen-induced global gene suppression is potentially mediated by NR5A1. Using Nr5a1 heterozygous mice, whose adrenal cortex is smaller than the wild type, we demonstrated that the size of skeletal muscles is possibly regulated by glucocorticoid synthesized by zF. Taken together, considering the ubiquitous presence of glucocorticoid receptors, our findings provide a pathway for sex differentiation through glucocorticoid synthesis.

Sex differences in metabolic pathways are regulated by Pfkfb3 and Pdk4 expression in rodent muscle.

Skeletal muscles display sexually dimorphic features. Biochemically, glycolysis and fatty acid ß-oxidation occur preferentially in the muscles of males and females, respectively. However, the mechanisms of the selective utilization of these fuels remains elusive. Here, we obtain transcriptomes from quadriceps type IIB fibers of untreated, gonadectomized, and sex steroid-treated mice of both sexes. Analyses of the transcriptomes unveil two genes, Pfkfb3 (phosphofructokinase-2) and Pdk4 (pyruvate dehydrogenase kinase 4), that may function as switches between the two sexually dimorphic metabolic pathways. Interestingly, Pfkfb3 and Pdk4 show male-enriched and estradiol-enhanced expression, respectively. Moreover, the contribution of these genes to sexually dimorphic metabolism is demonstrated by knockdown studies with cultured type IIB muscle fibers. Considering that skeletal muscles as a whole are the largest energy-consuming organs, our results provide insights into energy metabolism in the two sexes, during the estrus cycle in women, and under pathological conditions involving skeletal muscles.

Cinnamomum burmanini Blume increases bone turnover marker and induces tibia's granule formation in ovariectomized rats.

BACKGROUND: Bone fragility and an increase in susceptibility to fracture osteoporosis is characterized by a reduction in bone mass and the micro-architectural deterioration of bone tissue. There is no previous study regarding the effect of Cinnamomum burmanini Blume on osteoporosis. OBJECTIVE: This study was aimed to investigate the effect of C. burmanini Blume on bone turnover marker, mineral elements, and mesostructure of ovariectomized rats. MATERIALS AND METHODS: Thirty female Wistar rats were randomly divided into five groups, which included a control group (sham surgery), ovariectomy group (OVX), and ovariectomy groups in the presence of ethanolic extract of C. burmanini Blume (EECB) at doses of 12.5; 25; 50 mg/kg body weight (BW). Analysis of serum C-telopeptide collagen type I (CTX) and osteocalcin (OC) were done by enzyme-linked immunosorbent assay (ELISA). Tibia mineral elements and mesostructure were analyzed by X-ray Fluorescence and Scanning Electron Microscopy, respectively. In silico study was performed by modeling protein structure using SWISS-MODEL server and Ramachandran plot analysis. RESULTS: The increase in OC and CTX were significantly attenuated by treatments of EECB. Ovariectomy significantly decreased Cu/Zn ratio compared to sham-operated rats (p < 0.05). Mesostructure of ovariectomized rats was significantly different compared with the control group. CONCLUSION: Cinnamon was able to normalize bone turnover markers, but, the mesostructure of hydroxyapatite crystal growth was achieved at the highest dose extract. In silico study showed that the active compound of EECB could not only support osteoclastogenesis process by decreasing the binding energy between RANKL and RANK, but also by inhibiting the interaction between OPG and RANK.

THE COMPARING OF ANTIMICROBIAL ACTIVITY OF CSN1S2 PROTEIN OF FRESH MILK AND YOGHURT GOAT BREED ETHAWAH INHIBITED THE PATHOGENIC BACTERIA.

BACKGROUND: Goat milk is reported to have antimicrobial activity of several pathogen bacteria that contained on food materials. The research related with antimicrobial activity of Alpha-S2 casein from goat milk is relatively less than other casein components. Herein, we reported the antimicrobial activity of caprine Alpha-S2 Casein (CSN1S2) protein from Ethawah breed goat milk and yoghurt in Gram positive (Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus) and negative pathogen bacteria (Escherichia coli, Salmonella typhi and Shigella flexneri). Those bacteria were known as pathogens that caused gastrointestinal infection. METHODS: Serial dilution and agar diffusion analysis with three different concentrations of caprine CSN1S2, 1.25 mg/ml, 2.5 mg/ml, and 5 mg/ml were used to test the inhibition effect of protein on the viability of bacteria cells. The inhibitory activity of caprine CSN1S2 was based on dose dependent manner. Agar diffusion analysis was showed the larger diameter of clear zone at B. cereus and S. flexneri. RESULTS: The serial dilution analysis was shown the inhibition of almost in all groups of bacteria with concentration 5 mg/ml higher by CSN1S2 protein of goat fresh milk than yogurt. The inhibitory activity caprine CSN1S2 protein of fresh milk was shown a vary inhibition clear zone with optimal concentration 5 mg/ml, however CSN1S2 protein of goat yogurt intermediate effectively was only in gram negative bacteria. The weakness bacteria against inhibition activity caprine CSN1S2 protein was B. cereus (Gram positive) and S. flexneri (Gram negative). Meanwhile the strongest bacteria against inhibition activity caprine CSN1S2 protein was S. typhi (Gram negative), may cause in this bacteria has lipopolysaccharide prevent to interact with that protein as proper. CONCLUSION: This study result concluded that the caprine CSN1S2 protein has inhibition activity in opposition to pathogenic bacteria by optimal concentration 5 mg/ml in all bacteria and indicated caprine CSN1S2 protein as anti-microbial agent.

A novel CDKL5 mutation in a Japanese patient with atypical Rett syndrome.

Rett syndrome (RTT) is a severe X-linked dominant inheritance disorder with a wide spectrum of clinical manifestations. Mutations in Methyl CpG binding protein 2 (MECP2), Cyclin dependent kinase-like 5 (CDKL5) and Forkhead box G1 (FOXG1) have been associated with classic and/or variant RTT. This study was conducted to identify the responsible gene(s) in atypical RTT patient, and to examine the effect of the mutation on protein function. DNA sequence analysis showed a novel heterozygous mutation in CDKL5 identified as c.530A>G which resulted in an amino acid substitution at position 177, from tyrosine to cysteine. Genotyping analysis indicated that the mutation was not merely a single nucleotide polymorphism (SNP). We also revealed that patient's blood lymphocytes had random X-chromosome inactivation (XCI) pattern. Further examination by bioinformatics analysis demonstrated the mutation caused damage or deleterious in its protein. In addition, we demonstrated in vitro kinase assay of mutant protein showed impairment of its activity. Taken together, the results suggested the mutant CDKL5 was responsible for the disease.

Generation of rat induced pluripotent stem cells using a plasmid vector and possible application of a keratan sulfate glycan recognizing antibody in discriminating teratoma formation phenotypes.

Induced pluripotent stem cells (iPSCs) offer an invaluable tool for biological research and regenerative medicine. We report establishment of rat iPSCs (riPSCs) using a plasmid vector encoding four transcription factors, Oct3/4, Sox2, c-Myc and Klf4. Although all riPSC clones were generated and cultured under the same conditions, expressed hallmark pluripotency markers and differentiated successfully in vitro, the expression of a keratan sulfate glycan epitope with unique properties defined by R-10G antibody varied in the riPSC clones. In contrast, tumor rejection antigen (TRA)-1-81 epitope expression was comparable. A clone highly reactive to R-10G antibody formed teratomas in vivo consisting of cells from all three germ layers. However, clones expressing a lower level of the epitope defined by R-10G resulted in tumors with rapid growth consisting of undifferentiated cells. Additionally, riPSCs could be successfully differentiated into a neuronal lineage including glutamate neurons that responded to agonist stimulation. These observations demonstrate a glycophenotypic difference that may potentially serve as a useful probe for riPSC evaluation and to study the role of glycans in pluripotency and carcinogenesis in these cells.

Allele-specific real-time polymerase chain reaction as a tool for urate transporter 1 mutation detection.

Allele-specific polymerase chain reaction (ASPCR) method has long been applied for the detection of nucleotide variations and genotyping, which are detected by the presence or absence of DNA amplification PCR products. Recently, Real-Time PCR genotyping has fast developed and offered a rapid method of detecting mutations without the need of gel electrophoresis as with ASPCR. Here, we describe an easy and rapid touchdown real-time PCR method for the detection of nucleotide variations. Using our method we successfully detect two main mutations in human urate transporter 1 (SLC22A12), W258X and R90H, and validate the results. The method can potentially be applied to genotype of various other nucleotide variations.

Idursulfase enzyme replacement therapy in an adult patient with severe Hunter syndrome having a novel mutation of iduronate-2-sulfatase gene.

Mucopolysaccharidosis II (Hunter syndrome), a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS), has variable clinical phenotypes. Total by nearly 400 different mutations have been identified in IDS gene from patients with Hunter syndrome. Herein, we reported a patient who has a novel mutation in IDS gene with a severe clinical phenotype. Genetic analysis of the IDS gene revealed a novel 1-bp deletion in position c.1053T in exon 8 and resulting in a frameshift with a premature stop codon. Enzyme replacement therapy (ERT) using idursulfase (Elaprase®) was conducted to the patient and it improved hepatosplenomegaly, white blood cells and platelets number, and decreased the level of urinary glycosaminoglycan. ERT was proved to be effective at least in part in even an adult patient with severe type of Hunter syndrome.