Identification of SRY-box 30 as an age-related essential gatekeeper for male germ-cell meiosis and differentiation.

Although important factors governing the meiosis have been reported in the embryonic ovary, meiosis in postnatal testis remains poorly understood. Herein, we first report that SRY-box 30 (Sox30) is an age-related and essential regulator of meiosis in the postnatal testis. Sox30-null mice exhibited uniquely impaired testis, presenting the abnormal arrest of germ-cell differentiation and irregular Leydig cell proliferation.

Epigenetic Inactivation of SOX30 Is Associated with Male Infertility and Offers a Therapy Target for Non-obstructive Azoospermia.

Non-obstructive azoospermia (NOA) is the most severe form of male infertility. However, the etiology of NOA is largely unknown, resulting in a lack of clinical treatments. Here, we performed a comparative genome-wide profiling of DNA methylation and identified SOX30 as the most notably hyper-methylated gene at promoter in testicular tissues from NOA patients.

Identification and characterization of the CD226 gene promoter.

CD226 is one of the main activating receptors on natural killer cells, and it can induce cytotoxicity to target cells through interaction with its ligands CD155 or CD112. CD226 is also involved in T cell differentiation, activation, and cytotoxicity. The expression of CD226 on natural killer cells and T cells can be regulated by cytokines and chemical stimuli; however, the mechanism of the regulation of the CD226 gene is still unknown.

Dynamic changes of apoptosis-inducing ligands and Th1/Th2 like subpopulations in Hantaan virus-induced hemorrhagic fever with renal syndrome.

The expression of the apoptosis-inducing ligands, TNF-alpha, FasL and TRAIL on peripheral blood mononuclear cells (PBMC) and the levels of their soluble form (TNF-alpha, sFasL and sTRAIL) in plasma from 40 hemorrhagic fever with renal syndrome (HFRS) patients as well as 26 healthy blood donors were determined by flow cytometry (FCM) analysis and sandwich ELISA, respectively. The status of Th1, Th2, Tc1 and Tc2 subsets in PBMC was evaluated by intracellular cytokine staining and FCM. Compared to controls, the expression of membrane bound FasL and TRAIL was up-regulated on surface of PBMC isolated from the HFRS patients, particularly on CD8+ T lymphocytes.

Generation of monoclonal antibodies to cancer/testis (CT) antigen CT10/MAGE-C2.

CT10/MAGE-C2 is a recently identified antigen that, typically of cancer/testis (CT) antigens, can be found in various malignant tumors and in normal adult testis. As with many other CT antigens, our knowledge is based mainly on mRNA expression data. In the present study, we describe the generation of mAbs to CT10/MAGE-C2 for the analysis of its protein expression.

[Preparation, purification and identification of the rabbit antibody against extracellular region of mLAIR-1].

Aim: To express and purify the fusion protein of the extracellular region of murine leukocyte-associated Immunoglobulin-like receptor-1 (mLAIR-1) molecule with Fc fragment of human IgG and to prepare the rabbit antibody against mLAIR-1 extracellular region.

Methods: The eukaryotic expression vector pIg/3c-mLAIR-1 was constructed. Rabbits were immunized with the fusion protein purified from the culture medium of mLAIR-1-Fc vector-transfected CHO cells with protein A column.

[Identification of cell lines expressing the putative ligand(s) for LAIR-1(CD305) and LAIR-2(CD306)].

Aim: To identify cell lines expressing the putative ligand(s) for LAIR-1 and LAIR-2.

Methods: CHO cell lines secreting LAIR-1-Fc or LAIR-2-Fc fusion protein were prepared and the supernatants from the CHO cell lines were collected and purified by protein A affinity chromatography column. Several cell lines were stained with the purified LAIR-1-Fc and LAIR-2-Fc fusion proteins and then analyzed by flow cytometry for detecting the expression of their putative ligand(s).

[Construction and expression of human EpCAM eukaryotic expression vectors and identification of their products].

Aim: To construct EpCAM eukaryotic expression vectors pIg-EpCAM and pEGFP-EpCAM and to express them in COS7 cells.

Methods: EpCAM cDNA was amplified by PCR and then inserted into the pIg and pEGFP vectors respectively to construct recombinant vectors pIg-EpCAM and pEGFP-EpCAM. The two recombinant vectors were transfected into COS7 cells under the mediation of liposome.

[Changes and significance of TNF, sIL-2R, IL-6, IL-4 and IFN-gamma levels in plasma from the patients with hemorrhagic fever with renal syndrome].

Aim: To explore the changes of TNF, sIL-2R, IL-6, IL-4 and IFN-gamma levels in plasma from patients with hemorrhagic fever with renal syndrome(HFRS) and analyze the correlation between these cytokine levels and alanine aminotransferase(ALT) level in sera from the patients.

Methods: The cytokine levels were measured by double mAb sandwich ELISA. An automatic biochemical detector(RA-1000) was employed to determine the ALT level.

[Cloning genes sensitive to mechanical stretch in osteoblasts through subtractive hybridization technique].

Objective: In this experiment, genes sensitive to mechanical stretch in osteoblast like cells were cloned through subtractive hybridization technique.

Methods: Two dimensional mechanical stretch with deformation of 12% and frequency of 6 cycles was loaded on human osteoblastic like cell line Saos-2. Complementary deoxyribonucleic acid (cDNA) library of cells was constructed 12 h after loading, acting as tester.

[The inhibition of TNF-induced NF-kappaB nuclear translocation in ECV304 cells by mAbs against human TNF].

Aim: To identify the inhibition of TNF-induced NF-kappaB nuclear translocation by three anti-human TNF mAbs, D2, E6 and F6.

Methods: TNF solutions were pretreated with mAbs D2, E6 and F6 as well as control mAb at 37 degrees Celsius for 1 h, respectively, and then they were added to ECV304 cell cultures. After 1 hour, the cells were harvested and nuclear proteins were extracted.

[Characterization of neutralizing activity of murine monoclonal antibodies for construction of anti-human TNF chimeric antibody].

Aim: To screen high quality murine monoclonal antibodies (mAb) for construction of anti-human TNF chimeric antibody.

Methods: 3 hybridoma cells D2, E6 and F6 were selected from one group of hybridoma cells secreting anti-human TNF mAbs and then ascitic fluids were prepared according to routine protocol. After salted out with saturated ammonium sulphate, the mAb (IgG2b) secreted by D2 cells was purified through protein A affinity chromatography column.

[Effects of perindopril on plasma soluble TRAIL and receptor soluble DR5 in patients with congestive heart failure].

Aim: To explore the levels of soluble TRAIL and DR5 in plasma of patients with congestive heart failure (CHF) and the effects of perindopril.

Methods: 58 CHF patients were randomly divided into two groups, namely perindopril treatment group (30 cases) and routine treatment group (28 cases). The levels of sTRAIL and sDR5 in plasma of 30 CHF patients treated with perindopril, 28 CHF patients treated with routine method before and after treatment and 20 healthy persons were detected by ELISA.

[Eukaryotic expression and application of human CD226 Ig fusion protein containing 3C protease-restricted site].

Aim: To construct, express and characterize the eukaryotic expression vector of encoding human CD226 (PTA1) extracellular region Ig fusion protein gene containing 3C protease-restricted site.

Methods: The gene fragment encoding extracellular region of human CD226 was cloned into the eukaryotic expression vector p-3C-Ig containing 3C protease-restricted site and human Ig Fc fragment gene. After sequencing, the vector was transfected into COS7 cells, and the expressed molecule was purified by affinity chromatography.

[Cloning, prokaryotic expression of LAIR and identification of their immunological reactivities].

Aim: To clone and express the leukocyte-associated immunoglobulin-like receptor (LAIR) and to identify the immune reactivity of LAIR-2 to anti-LAIR-1 specific monoclonal antibodies(mAb).

Methods: Genes encoding LAIR-1 and LAIR-2 were cloned by RT-PCR from human peripheral blood mononuclear cells(PBMC) and two leukemia cell lines Jurkat and HL-60. The extracellular region gene of LAIR1 and LAIR-2 were inserted into vector pGEX-4T-3 expressing GST fusion protein, expressed on IPTG induction and purified through glutathione-sepharose 4B column.