When we try to establish the gene recombinant yeast cell to screen the androgenic endocrine disruptors, the key procedure is the androgen receptor (AR) expression in the yeast cell. For this purpose, we obtained the GPD (glyceraldehyde-3-phosphote dehydrogenase) promoter from the yeast genosome of W303-1A using PCR system and inserting it into Swa I and BamH I sites of pYestrp2. The new constructed vector was named pGPD. The V5 epitope tag DNA with a 5'-BamH I and a 3'-EcoR I sticky end was cloned into the corresponding site of the pGPD vector to yield the vector of pGPDV5. The 2 723 bp full length AR ORF amplified by PCR from pcDNA3.1/AR was fused to V5 epitope tag DNA in pGPDV5 to give the AR yeast expression vector of pGPDV5/AR. This fused vector was transformed into the yeast cell (W303-1A). Western blot was used to detect the V5 fused protein of AR, in the protocol of which the primary monoclonal antibody (IgG(2a)) of mouse anti-V5 and the polyclonal secondary antibody of goat anti-mouse (IgG) linked to horseradish peroxidase (HRP) were used to detect the specific protein in the given sample of the transformed yeast extract. The result showed that the fused protein of AR was expressed successfully in the yeast cell.
Base Sequence ; Endocrine Disruptors ; analysis ; Epitopes ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Glyceraldehyde-3-Phosphate Dehydrogenases ; genetics ; Humans ; Molecular Sequence Data ; Promoter Regions, Genetic ; Receptors, Androgen ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; genetics ; Yeasts ; genetics ; metabolism

To study whether recombinant human erythropoietin (rhEPO) reduces neuronal apoptosis through inhibiting over-expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in nucleus induced by brain ischemia/reperfusion in rats, 48 adult Sprague-Dawley rats were randomly divided into 3 groups: sham, saline and EPO groups. Animal models of brain ischemia/reperfusion were established by middle cerebral artery occlusion in rats. The effects of EPO on the sizes of ischemia tissue were observed by TTC staining. The over-expression of GAPDH in nucleus was detected by Hoechst-33258 and anti-GAPDH antibody double staining. The neuronal apoptosis in penumbral was detected by Nissl's staining and Hoechst-33258 immunofluorescence, respectively. The results showed that rhEPO treatment (3 000 U/kg, three times daily, i.p.) apparently reduced the sizes of infarct brain tissue in ischemia/reperfusion rats. rhEPO inhibited over-expression of GAPDH in nucleus of apoptotic neurons. In the meantime rhEPO decreased the number of apoptotic neurons in ischemia/reperfusion rats. These results suggest that rhEPO may induced reduction of neuronal apoptosis in penumbra may be through inhibiting over-expression of GAPDH in nucleus of apoptotic neurons induced by ischemia/reperfusion. Reduction of GAPDH over-expression in nucleus may play a pivotal role in EPO inhibiting neuronal apoptosis in cerebral ischemia/reperfusion rats, providing experimental evidence for EPO neuro-protecting effects against ischemia/reperfusion.
Animals ; Apoptosis ; Brain ; enzymology ; pathology ; Brain Ischemia ; pathology ; Erythropoietin ; pharmacology ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) ; metabolism ; Humans ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; pharmacology ; Reperfusion Injury ; pathology

OBJECTIVESSurgical repair of Achilles tendon (AT) rupture should immediately be followed by active tendon mobilization. The optimal time as to when the mobilization should begin is important yet controversial. Early kinesitherapy leads to reduced rehabilitation period. However, an insight into the detailed mechanism of this process has not been gained. Proteomic technique can be used to separate and purify the proteins by differential expression profile which is related to the function of different proteins, but research in the area of proteomic analysis of AT 3 days after repair has not been studied so far.

METHODSForty-seven New Zealand white rabbits were randomized into 3 groups. Group A (immobilization group, n equal to 16) received postoperative cast immobilization; Group B (early motion group, n equal to 16) received early active motion treatments immediately following the repair of AT rupture from tenotomy. Another 15 rabbits served as control group (Group C). The AT samples were prepared 3 days following the microsurgery. The proteins were separated employing two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). PDQuest software version 8.0 was used to identify differentially expressed proteins, followed by peptide mass fingerprint (PMF) and tandem mass spectrum analysis, using the National Center for Biotechnology Information (NCBI) protein database retrieval and then for bioinformatics analysis.

RESULTSA mean of 446.33, 436.33 and 462.67 protein spots on Achilles tendon samples of 13 rabbits in Group A, 14 rabbits in Group B and 13 rabbits in Group C were successfully detected in the 2D-PAGE. There were 40, 36 and 79 unique proteins in Groups A, B and C respectively. Some differentially expressed proteins were enzyme with the gel, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We successfully identified 9 and 11 different proteins in Groups A and B, such as GAPDH, phosphoglycerate kinase 1, pro-alpha-1 type 1 collagen, peroxiredoxin 1, alpha-1-antiproteinase E a-1 and MAD2L1 binding protein, etc. And some with the molecular chaperone, oxidative stress, energy metabolism, signal transduction, coupled with the tendon cell expression and protein synthesis, proliferate, differentiate and are closely related to the AT healing. The GAPDH protein was further validated through Western blotting. It was indicated that some differentially expressed proteins were involved in various metabolism pathways and may play an important role in initial healing of AT rupture.

CONCLUSIONDifferentially expressed proteins in rabbit healing AT model may contribute to 3 days healing of AT rupture through a new mechanobiological mechanism due to the application of postoperative early kinesitherapy.

Achilles Tendon ; injuries ; Animals ; Blotting, Western ; Computational Biology ; Electrophoresis, Gel, Two-Dimensional ; Exercise Therapy ; Glyceraldehyde-3-Phosphate Dehydrogenases ; analysis ; Male ; Proteins ; analysis ; Rabbits ; Rupture ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Tendon Injuries ; metabolism ; rehabilitation ; surgery ; Wound Healing ; physiology

OBJECTIVETo investigate the differentiation-related proteins in human gastric carcinoma cell lines by comparative proteomics.

METHODSThe holoproteins of human gastric carcinoma cell lines MKN28, SGC7901 and BGC823 were measured by two-dimensional gel electrophoresis and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Some proteins identified by proteomics were tested by Western blot in the cell strains and tissues of gastric carcinoma.

RESULTS14 differential protein spots were found in the 3 gastric carcinoma cell lines, among them 8 spots were identified by MALDI-TOF-MS. These proteins were probably thioredoxin peroxidase, glyceraldehyde-3-phosphate dehydrogenase (GAPD), beta-tubulin polypeptide, hypothetical protein, zinc finger protein (ZNF) 139, protein-tyrosine kinase, calreticulin precursor, and tropomyosin, proteins related with biological behavior of gastric carcinoma cells such as signal transduction, cellular homeostasis, glycolysis, antioxidation action, multidrug resistance(MDR), etc. The expressions of those proteins in gastric cancer cells and tissues identified by Western blot were consistent with the results obtained by proteomics.

CONCLUSIONDifferential proteins are found in 3 human gastric carcinoma cell lines, mainly, proteins related with cell signaling, maintenance of homeostasis, glycolysis, metabolism of anti-cancer drug and anti-oxidative injury, etc.

Adenocarcinoma ; metabolism ; pathology ; Adult ; Aged ; Blotting, Western ; Cell Line, Tumor ; Electrophoresis, Gel, Two-Dimensional ; Female ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; Glyceraldehyde-3-Phosphate Dehydrogenases ; metabolism ; Humans ; Male ; Middle Aged ; Protein-Tyrosine Kinases ; metabolism ; Proteome ; analysis ; Proteomics ; methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Stomach Neoplasms ; metabolism ; pathology

In order to characterize the Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, immunogenicity and immunoprotection of the Staphylococcus aureus (S. aureus) surface protein GapC, gapC gene of S. aureus was amplified from strain BMSA/855/23-1 by PCR, and was inserted into pQE-30 vector subsequently. The recombinant plasmid, designated as pQE/gapC, was transformed into E. coli strain M15 (pREP4). The recombinant GapC fusion proein was successfully expressed in E. coli M15 induced with IPTG and its GAPDH activity was confirmed by GAPDH activity assay. Then, the recombinant GapC protein, inactivated S. aureus whole cell and placebo (PBS) were administrated to healthy rabbits respectively. The IgG antibody titers, concentration of IFN-gamma and IL-4 cytokines in immunized rabbit sera were measured with Enzyme-Linked Immunosorbnent Assay (ELISA). Finally, immunized rabbits were challenged with S. aureus strain Wood46 to evaluate the immunoprotection. The IgG antibody titers against GapC and whole cell in rabbit sera reached their peaks at day 28 after boost immunization (1:64,000). The concentration of IL-4 and IFN-gamma in GapC groups rabbit sera increased significantly (P<0.05) at day 14 after boost immunization, while the concentration of those in whole cell group did not increase (P>0.05) compared with the placebo group. 4 rabbits in 5 of the protein immunized group were protected against challenge with 1 x 10(8) CFU S. aureus. The results above indicate that the expressed recombinant GapC protein have high GAPDH activity and immunogenicity, can also protect against S. aureus challenge to some extent. S. aureus GapC protein could be an attractive target for further genetic engineering vaccine.
Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; genetics ; metabolism ; Bacterial Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Glyceraldehyde-3-Phosphate Dehydrogenases ; biosynthesis ; genetics ; immunology ; Immunization ; Male ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Staphylococcal Vaccines ; immunology ; Staphylococcus aureus ; enzymology ; genetics ; immunology ; Vaccines, Synthetic ; immunology

AIMTo develop a high-throughput multiplex, fast and simple assay to scan azoospermia factor (AZF) region microdeletions on the Y chromosome and establish the prevalence of Y chromosomal microdeletions in Chinese infertile males with azoospermia or oligozoospermia.

METHODSIn total, 178 infertile patients with azoospermia (non-obstructed), 134 infertile patients with oligozoospermia as well as 40 fertile man controls were included in the present study. The samples were screened for AZF microdeletion using optimized multi-analyte suspension array (MASA) technology.

RESULTSOf the 312 patients, 36 (11.5%) were found to have deletions in the AZF region. The microdeletion frequency was 14% (25/178) in the azoospermia group and 8.2% (11/134) in the oligospermia group. Among 36 patients with microdeletions, 19 had deletions in the AZFc region, seven had deletions in AZFa and six had deletions in AZFb. In addition, four patients had both AZFb and AZFc deletions. No deletion in the AZF region was found in the 40 fertile controls.

CONCLUSIONThere is a high prevalence of Y chromosomal microdeletions in Chinese infertile males with azoospermia or oligozoospermia. The MASA technology, which has been established in the present study, provides a sensitive and high-throughput method for detecting the deletion of the Y chromosome. And the results suggest that genetic screening should be advised to infertile men before starting assisted reproductive treatments.

Adult ; Azoospermia ; epidemiology ; genetics ; China ; epidemiology ; Chromosomes, Human, Y ; genetics ; ultrastructure ; DNA ; genetics ; isolation & purification ; Female ; Gene Deletion ; Genetic Loci ; Glyceraldehyde-3-Phosphate Dehydrogenases ; genetics ; Humans ; In Situ Hybridization ; Infertility, Male ; epidemiology ; genetics ; Male ; Oligonucleotide Probes ; Oligospermia ; epidemiology ; genetics ; metabolism ; Protein Array Analysis ; Reproducibility of Results ; Reverse Transcriptase Polymerase Chain Reaction ; Seminal Plasma Proteins ; genetics

Mesenchymal stem cells (MSCs) secrete a variety of neuroregulatory molecules, such as nerve growth factor, brain-derived neurotrophic factor, and glial cell-derived neurotrophic factor, which upregulate tyrosine hydroxylase (TH) gene expression in PC12 cells. Enhancing TH gene expression is a critical step for treatment of Parkinson's disease (PD). The objective of this study was to assess the effects of co-culturing PC12 cells with MSCs from feline bone marrow on TH protein expression. We divided the study into three groups: an MSC group, a PC12 cell group, and the combined MSC + PC12 cell group (the co-culture group). All cells were cultured in DMEM-HG medium supplemented with 10% fetal bovine serum for three days. Thereafter, the cells were examined using western blot analysis and immunocytochemistry. In western blots, the co-culture group demonstrated a stronger signal at 60 kDa than the PC12 cell group (p < 0.001). TH was not expressed in the MSC group, either in western blot or immunocytochemistry. Thus, the MSCs of feline bone marrow can up-regulate TH expression in PC12 cells. This implies a new role for MSCs in the neurodegenerative disease process.
Animals ; Antigens, Surface/metabolism ; Blotting, Western ; Cats/*physiology ; Cell Culture Techniques ; Cells, Cultured ; *Gene Expression Regulation, Enzymologic ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating)/metabolism ; Immunohistochemistry ; Mesenchymal Stem Cells/*cytology/metabolism ; Microscopy, Phase-Contrast ; PC12 Cells/cytology/*enzymology ; Rats ; Tyrosine 3-Monooxygenase/*metabolism

OBJECTIVETo observe the different effects of Puerarin and Daidzein on the expression of proliferating vascular smooth muscle cells, and to discuss the mechanism.

METHODMT was used to detect the state of VSMC (vascular smooth muscle cell) activity. The expression levels of Survivin, Bcl-xl, Bax and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA (mRNA) were analyzed quantitatively by reverse transcriptase polymerase chain reaction (Rt-PCR).

RESULTCompared with Puerarin groups, VSMC activity in daidzein groups was lower, and the ratio of Bax/Gapdh/Bcl-xl/Gapdh was higher.

CONCLUSIONThe inhibition effect of daidzein on VSMC proliferation is stronger than that of puerarin.

Cells, Cultured ; Gene Expression Regulation ; drug effects ; Glyceraldehyde-3-Phosphate Dehydrogenases ; biosynthesis ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; Isoflavones ; isolation & purification ; pharmacology ; Microtubule-Associated Proteins ; biosynthesis ; genetics ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Neoplasm Proteins ; Plants, Medicinal ; chemistry ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Pueraria ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Vasodilator Agents ; pharmacology ; bcl-2-Associated X Protein ; bcl-X Protein