Investigation of several biomarkers associated with diabetic nephropathy.

OBJECTIVE: The aim of this study was to facilitate the systematic discovery of diagnostic biomarkers of diabetic nephropathy (DN). METHODS: 3 publicly available independent cohorts were got from Gene Expression Omnibus database. Gene expression array were used to screen for genome-wide relative significance (GWRS) and genome-wide global significance (GWGS). The most significant up- and down-regulated top 100 gene signatures were identified using a fold change based model. Then the protein-protein interaction (PPI) network was constructed, while the hub genes in this PPI network were identified by centrality analysis. Modules detection was performed to explore the functions of the modules. Meanwhile, gene enrichment analysis was performed to illuminate the biological pathways and processes associated with DN. RESULTS: The most significant up- and down-regulated top 100 gene signatures were identified and a PPI network was established. Several hub genes (VEGFA, IL8, MYC, CD14, ALB) were discovered. Several functional modules were revealed. Biological pathways including cytokine-cytokine receptor interaction and p53 signaling pathway, and processes including inflammatory response, response to wounding and enzyme linked receptor protein signaling pathway were identified. CONCLUSION: Our study displayed underlying biomarkers including biological pathways and several hub genes of DN.

Distribution of PDIA3 transcript and protein in rat testis and sperm cells.

Protein disulphide isomerase family A3 (PDIA3) has an activity of thioredoxin, widely expressed in multiple tissues and involved in multiple cellular processes. It was recently found in human and mouse sperm cells and could affect sperm-egg fusion. Therefore, the present investigation aims to identify PDIA3 mRNA and protein in rat testis and sperm cells. Rat PDIA3 cDNA was cloned by RT-PCR. The cRNA riboprobe was transcribed from PDIA3 cDNA and was used to display PDIA3 mRNA location in rat testes by in situ hybridization. PDIA3 protein distribution was also observed in testis and sperm cells by immunohistochemistry and immunocytochemistry, respectively. The rat PDIA3 transcript and protein were localized in the cells from spermatocytes to the spermatozoa phases of rat testes, mostly in the pachytene spermatocytes. PDIA3 protein was also observed on the intact sperm membrane including the tail. The rat PDIA3 gene is transcribed and translated through the whole spermatogenesis process, and the PDIA3 protein is spread all over the sperm cell membrane. The results provide some primary information about PDIA3 in testis and sperm for further study on PDIA3 function in rat spermatogenesis and sperm-egg fusion.

Molecular cloning of sheep and cashmere goat Pdia3 and localization in sheep testis.

Protein disulphide isomerase family A3 (PDIA3) is a member of the protein disulphide isomerase (PDI) family and is multifunctional in many processes. Recently, it has been confirmed as a sperm membrane component and is implicated in sperm-egg fusion under which the molecular mechanism is still obscure. Protein disulphide isomerase family A3 cDNA has been cloned in several mammals; however, goat and sheep counterparts have not been identified. To facilitate the studies on the potential function of PDIA3 protein in sperm-egg fusion in sheep and goat, we cloned the cDNA encoding for Pdia3 of sheep (Ovis aries) and cashmere goat (Capra hircus) and studied its transcript and protein localization in sheep testis. The cloned sheep and cashmere goat Pdia3 cDNA are 1660 bp and 1591 bp, respectively. Bioinformatics analysis showed that sheep and goat Pdia3 cDNA both have a coding region of 1518 bp with the same nucleotides encoding the same 505 amino acids. The predicted peptide, with two typical motifs of Trp-Cys-Gly-His-Cys-Lys (WCGHCK) which is a hallmark of the PDI family, has high homology to that of bovine (99.21%), human (95.05%), rat (89.50%) and mouse (90.89%). Protein disulphide isomerase family A3 protein was observed in cells of various stages of spermatogenesis, from the primary spermatocyte to spermatozoa phases, as well as in the Leydig cells. It was observed in the entire sheep spermatozoa and mostly at the equatorial segment and the forepart of the flagellum. The Pdia3 mRNA was detected over the seminiferous epithelium in all stages of spermatogenesis.

Early meiotic-specific protein expression in post-natal rat ovaries.

Recent studies in mice challenged the basic doctrine that most mammalian females lose neo-oogenesis in post-natal ovaries. In order to provide more information in other species, we examined post-natal rat ovaries by histological sections and detected the germline cell marker protein RVLG (rat vasa-like gene), BrdU (5-bromodeoxyuridine) incorporation in RVLG-expressing cells, for identification of germline cells undergoing mitosis and meiosis in the ovarian surface epithelium (OSE). We also detected the expression of early meiotic-specific proteins disruption of meiotic control 1 (DMC1), stimulated by retinoic acid gene 8 (STRA8) and synaptonemal complex protein 3 (SCP3) by immunohistochemical analysis and Western blotting, and the transcript of SCP1, SCP3 and Sporulation-specific protein 11 (SPO11) by RT-PCR in the post-natal ovarian cortex. However we failed in detecting large ovoid cells in the OSE, which may represent the putative germline stem cells (GSCs) that are supposed to sustain neo-oogenesis, and the transcription of the meiotic-specific genes SCP1, SCP3 and SPO11 by RT-PCR as well as the translation of DMC1, STRA8 and SCP3 by Western blotting. Our data support the postulation that there is no neo-oogenesis occurring in the OSE of rat post-natal ovary through meiosis of GSCs.

Molecular cloning and characterization of CD9 cDNA from sheep and cashmere goat.

CD9 is a glycoprotein of the transmembrane 4 superfamily (TM4SF) and is involved in various cellular processes. Some CD9 cDNA have been cloned in mammals and certain fish genera in recent years, but goat and sheep counterparts of cattle, human and mouse have not been identified. To facilitate the studies, we cloned the cDNA encoding for CD9 of cashmere goat (Capra hircus) and sheep (Ovis aries), and expressed sheep CD9 in Escherichia coli cells. Structural analysis indicated for both goat and sheep that a 1123 bp cDNA spanned an open reading frame of 681 bp which predicted a protein of 226 amino acids with a typical TM4SF structure, including four highly conserved transmembrane domains, two extracellular domains and a CCG motif, which is a hallmark of the TM4SF. The predicted amino acid sequences were highly homologous to those of cattle, mouse and human CD9. Molecular phylogenetic analysis based on CD9 cDNA sequences indicated that goat and sheep CD9 were closely related to CD9 of cattle, which is in agreement with their morphological taxonomy.

A comparison of SCE test in human lymphocytes and Vicia faba: a hopeful technique using plants to detect mutagens and carcinogens.

A comparison of the Vicia faba SCE with the human lymphocyte SCE test was made with regard to their capacity to detect mutagens. Twelve chemical agents that can induce sister-chromatid exchanges (SCEs) in both Vicia faba root tip cells and human lymphocytes were used. The results indicate that the plant SCE technique is a sensitive test for screening various chemical agents including food preservatives, pesticides and essences which may have mutagenic activity. A new simplified procedure was introduced for differential staining of sister chromatids in Vicia faba.