Ghrelin and ghrelin receptor (GHSR) in Chinese alligator, alligator sinensis: Molecular characterization, tissue distribution and mRNA expression changes during the active and hibernating periods.

The Chinese alligator (Alligator sinensis) is a freshwater crocodilian endemic to China. So far, the endocrine regulation of feeding and growth in Chinese alligator is poorly understood. In this study, the molecular structure and tissue expression profiles of ghrelin and its receptor GHSR in the Chinese alligator were characterized for the first time. The full-length cDNA of ghrelin was 1770 bp, including a 37 bp 5 '-UTR (untranslated region), a 435 bp ORF (open reading frame) and a 1298 bp 3 '-UTR. The ORF encodes a ghrelin precursor, which consists of 145 amino acid residues, including a signal peptide with 52 amino acid residues at the N-terminus, a mature peptide with 28 amino acid residues, and a possibly obestain at the C-terminus. The full-length cDNA of GHSR was 3961 bp, including a 5'-UTR of 375-bp, an ORF of 1059-bp and a 3' -UTR of 2527-bp. The ORF encodes a protein of 352 amino acid residues containing seven transmembrane domains, with multiple N glycosylation modification sites and conserved cysteine residue sites. The active core "GSSF" of Chinese alligator ghrelin was identical to that of mammals and birds, and the ghrelin binding site of GHSR was similar to that of mammals. The amino acid sequences of both ghrelin and GHSR share high identity with American alligator (Alligator mississippiensis) and birds. Ghrelin was highly expressed in cerebrum, mesencephalon, hypothalamus and multiple peripheral tissues, including lung, stomach and intestine, suggesting that it could play functions in paracrine and/or autocrine manners in addition to endocrine manner. GHSR expression level was higher in hypothalamus, epencephalon and medulla oblongata, and moderate in multiple peripheral tissues including lung, kindey, stomach and oviduct, implicating that ghrelin/GHSR system may participate in the regulation of energy balance, food intake, water and mineral balance, gastrointestinal motility, gastric acid secretion and reproduction. During hibernation, the expression of ghrelin and GHSR in the brain was significantly increased, while ghrelin was significantly decreased in heart, liver, lung, stomach, pancreas and ovary, and GHSR was significantly decreased in heart, liver, spleen, lung, kindey, stomach, ovary and oviduct. These temporal changes in ghrelin and GHSR expression could facilitate the physiological adaption to the hibernation of Chinese alligator. Our study could provide basic data for further studies on the regulation of feeding, physiological metabolism and reproduction of Chinese alligator, which could also be useful for the improvement of artificial breeding of this endangered species.

Molecular cloning, characterization, and gene expression behavior of glucocorticoid and mineralocorticoid receptors from the Chinese alligator (Alligator sinensis).

The Chinese alligator is an endemic crocodilian species in China. We isolated and obtained the glucocorticoid and mineralocorticoid receptor genes coding from the kidney of Alligator sinensis by nested polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE). The glucocorticoid receptor (GR) gene has 2343 base pairs encoding 780 amino acids, while the mineralocorticoid receptor (MR) gene is 2958 bp in length encoding 985 amino acids. Quantitative real-time PCR was used to detect the distribution of messenger RNA (mRNA) levels. The maximum mRNA expressions were observed in the ovary and kidney, suggesting that these receptors may be involved in basic cellular functions or stress response of alligators. Besides this, RT-qPCR was performed to analyze the abundance of GR and MR mRNA transcripts in early embryonic development of the Chinese alligator in the kidney, liver, and heart. The mRNA levels of GR and MR at earlier stages in kidney, liver, and heart indicates that they might involve in the transcriptional regulation of early embryos and activate many precise developmental effects in fetal tissues. We also measured the protein expression in the liver embryonic developmental stages and found that the GR and MR proteins were restricted to both the nuclei and cytoplasm. The protein expression levels in the liver at different embryonic developmental stages have extremely prominent differences. Taken together, our results showed the full coding regions of GR and MR, their characteristics, and embryonic developmental mRNA and protein expressions of both genes in A. sinensis. This study could provide the necessary information for further investigating the diverse functions of GR and MR in A. sinensis.

Cloning, characterisation and expression profile of kisspeptin1 and the kisspeptin1 receptor in the hypothalamic-pituitary-ovarian axis of Chinese alligator Alligator sinensis during the reproductive cycle.

Kisspeptin1 (Kiss1), a product of the Kiss1 gene, plays an important role in the regulation of reproduction in vertebrates by activating the Kiss1 receptor (Kiss1R) and its coexpression with gonadotrophin-releasing hormone (GnRH) in GnRH neurons. The purpose of this study was to clone the Kiss1 and Kiss1R genes found in the brain of Alligator sinensis and to explore their relationship with reproduction. The full-length cDNA of Kiss1 is 816bp, the open reading frame (ORF) is 417bp and the gene encodes a 138-amino acid precursor protein. The full-length cDNA of Kiss1R is 2348bp, the ORF is 1086bp and the gene encodes a 361-amino acid protein. Quantitative polymerase chain reaction showed that, except for Kiss1R expression in the hypothalamus, the expression of Kiss1 and Kiss1Rduring the reproductive period of A. sinensis was higher than that in the hypothalamus, pituitary gland and ovary during the hibernation period. The changes in GnRH2 mRNA in the hypothalamus were similar to those of GnRH1 and peaked during the reproductive period. This study confirms the existence of Kiss1 and Kiss1R in A. sinensis and the findings strongly suggest that Kiss1 and Kiss1R may participate in the regulation of GnRH secretion in the hypothalamus of alligators during the reproductive period. Furthermore, this is the first report of the full-length cDNA sequences of Kiss1 and Kiss1R in reptiles.

Optimization of extraction of phenolics from leaves of Ficus virens.

In this research, the conditions for extraction of phenolics from leaves of Ficus virens were optimized using response surface methodology (RSM). The extraction abilities of phenolics (EAP) and flavonoids (EAF), the 2,2-diphenyl-1-pierylhydrazyl (DPPH) free-radical scavenging potential, and the ferric reducing/antioxidant power (FRAP) were used as quality indicators. The results of single-factor experiments showed that temperature, ethanol concentration, extraction time, and the number of extraction cycles were the main influencing variables, and these provided key information for the central composite design. The results of RSM fitted well to a second degree polynomial model and more than 98% of the variability was explained. The ideal extraction conditions for EAP, EAF, DPPH free-radical scavenging potential, and FRAP were obtained. Considering the four quality indicators overall, the ideal extraction conditions were 58% ethanol at 57 °C for 37 min with three extraction cycles. At the ideal extraction conditions, the values of EAP, EAF, DPPH free-radical scavenging potential, and FRAP were 5.72%, 3.09%, 58.88 mg ascorbic acid equivalent (AAE)/g dry weight (DW), and 15.86 mg AAE/g DW, respectively. In addition, linear correlations were observed between EAP, EAF, and antioxidant potential.

Gay saunas and the risks of HIV and syphilis transmissions in China--results of a meta-analysis.

INTRODUCTION: Previous studies suggest the risk of human immunodeficiency virus (HIV) transmission among men who have sex with men (MSM) is associated with characteristics of venues. However, very few studies have systematically compared HIV/sexually transmitted disease (STD) prevalence among MSM according to key venue type for sourcing sex partners. AIMS: The aim of this study was to investigate the associations between HIV/syphilis prevalence and the types of venues, namely saunas, parks, gay bars, and the Internet, which are mostly used for sourcing male sex partners by MSM in China. METHODS: Meta-analyses using fixed-effect and random-effect methods were conducted. Secondary data were obtained from 10 concurrent surveys conducted in 10 cities basing on a common protocol. Pairwise comparisons (e.g., "saunas" vs. "parks") were made. MAIN OUTCOMES MEASURES: Odds ratios (OR) for HIV/syphilis infection in relations to venue type for partner sourcing. RESULTS: The distribution of the most commonly used source to recruit male sex partner was 59.32% (Internet), 18.47% (gay bars), 14.18% (gay saunas), and 8.02% (parks). The median prevalence of HIV/syphilis was 10.49% and 19.38% (gay saunas), 2.12% and 16.25% (parks), 6.06% and 15.45% (Internet), and 4.41% and 11.90% (gay bars). We found significant between-group differences when comparing "sauna" against "Internet" (HIV: OR= 2.27, 95% confidence interval [CI] = 1.65-3.12; syphilis: OR = 1.61, 95% CI= 1.07-2.41), "sauna" against "bars" (HIV: OR= 1.65, 95% CI= 1.14-2.39; syphilis: OR= 1.35, 95% CI= 1.02-1.78), and "parks" against "Internet" (syphilis: OR= 1.55, 95% CI= 1.12-2.15) as the main source to recruit male sex partners. Other pairwise comparisons were not statistically significant. CONCLUSIONS: The results of this study suggest that those sourcing partners mainly from gay saunas have higher prevalence of HIV/syphilis when compared with those doing so via the Internet or gay bars. Venue based (sauna-based) interventions using socio-ecological approaches are greatly warranted in order to reduce HIV and syphilis prevalence among MSM in China.

Analysis of unculturable bacterial communities in tea orchard soils based on nested PCR-DGGE.

The bacterial communities in the soils from tea orchards and their adjacent wasteland in Anhui Province, China were analysed by nested PCR-DGGE technique combined with sequencing. DGGE profiles revealed that the DGGE patterns of different soils were similar to each other and the most intensely bands appeared in all lanes. The bacterial genetic diversity index of tea orchard soils was lower than that of wasteland. For the tea orchard soils, Shannon's diversity index decreased in the order: 45-year-old tea orchard >25-year-old tea orchard >7-year-old tea orchard >70-year-old tea orchard. The analysis of 16S rRNA gene sequences indicated that the fragments belong to Proteobacteria, Acidobacteria, TM7, Cyanobacteria and Firmicutes. A comprehensive analysis of the bacterial community structure in the tea orchard soils indicated the bacterial community was dominantly composed of Acidobacteria, followed by Proteobacteria (Gamma and Alpha), Firmicutes, Cyanobacteria and candidate division TM7. The RDA combined with UPGMA clustering analysis showed that the more similar the environmental variables were, the more similar the bacterial community structures in tea orchard soils were.