Sigma-1 receptor is involved in diminished ovarian reserve possibly by influencing endoplasmic reticulum stress-mediated granulosa cells apoptosis.

Sigma non-opioid intracellular receptor 1 (sigma-1 receptor), a non-opioid transmembrane protein, is located on cellular mitochondrial membranes and endoplasmic reticulum. Current research has demonstrated that sigma-1 receptor is related to human degenerative diseases. This study is focused on the effects of sigma-1 receptor on the pathophysiological process of diminished ovarian reserve (DOR) and granulosa cells (GCs) apoptosis. Sigma-1 receptor concentration in follicular fluid (FF) and serum were negatively correlated with basal follicle-stimulating hormone (FSH) and positively correlated with anti-mullerian hormone (AMH), antral follicle count (AFC). Sigma-1 receptor reduction in GCs was accompanied by endoplasmic reticulum stress (ERS)-mediated apoptosis in women with DOR. Plasmid transfection was used to establish SIGMAR1-overexpressed and SIGMAR1-knockdown human granulosa-like tumor (KGN) cell and thapsigargin (TG) was used to induce ERS KGN cells. We found that KGN cells treated with endogenous sigma-1 receptor ligand dehydroepiandrosterone (DHEA) and sigma-1 receptor agonist PRE-084 showed similar biological effects to SIGMAR1-overexpressed KGN cells and opposite effects to SIGMAR1-knockdown KGN cells. DHEA may improve DOR patients' pregnancy outcomes by upregulating sigma-1 receptor and downregulating ERS-mediated apoptotic genes in GCs. Thus, sigma-1 receptor may be a potential ovarian reserve biomarker, and ligand-mediated sigma-1 receptor activation could be a future approach for DOR therapy.

Inhibitory effect of dihydroartemisinin on chondrogenic and hypertrophic differentiation of mesenchymal stem cells.

Chondrocytes located in hyaline cartilage may maintain phenotype while the chondrocytes situated in calcified cartilage differentiate into hypertrophy. Chondrogenic and hypertrophic differentiation of mesenchymal stem cells (MSCs) are two subsequent processes during endochondral ossification. However, it is necessary for chondrocytes to hold homeostasis and to inhibit hypertrophic differentiation in stem cell-based regenerated cartilage. Dihydroartemisinin (DHA) is derived from artemisia apiacea which has many biological functions such as anti-malarial and anti-tumor. Whereas the effects of DHA on chondrogenic and hypertrophic differentiation are poorly understand. In this study, the cytotoxicity of DHA was determined by CCK8 assay and the cell apoptosis was analyzed by flow cytometry. Additionally, the effects of DHA on chondrogenic and hypertrophic differentiation of MSCs are explored by RT-PCR, western blotting and immunohistochemistry. The results showed that DHA inhibited expression of chondrogenic markers including Sox9 and Col2a1 by activating Nrf2 and Notch signaling. After induced to chondrogenesis, cells were treated with hypertrophic induced medium with DHA. The results revealed that hypertrophic markers including Runx2 and Col10a1 were down-regulated following DHA treatment through Pax6/HOXA2 and Gli transcription factors. These findings indicate that DHA is negative to chondrogenesis and is protective against chondrocyte hypertrophy to improve chondrocytes stability. Therefore, DHA might be not suited for chondogenesis but be potential as a new therapeutic candidate to maintain the biological function of regenerated cartilage.

[Prevention against and treatment of doxorubicin-induced acute cardiotoxicity by dexrazoxane and schisandrin B].

In this study, it is to compare the effectiveness of prevention against and treatment of doxorubicin (DOX) induced cardiotoxicity by dexrazoxane and schisandrin B (Sch B) in rats. Sprague-Dawley (SD) rats were randomly divided into the following 6 groups: normal saline group, DOX group, DOX+DEX group, DOX+Sch B (80 mg x kg(-1)) group, DOX+Sch B (40 mg x kg(-1)) group and DOX+Sch B (20 mg x kg(-1)) group. The results showed that Sch B could combat the increase of myocardial enzymes in peripheral blood, decrease of the enzyme activity of myocardial tissue antioxidant enzymes and disorders of systolic and diastolic function of heart in rats intravenously injected with doxorubicin (15 mg x kg(-1)). Sch B was better than DEX in protecting rat against DOX-induced the symptoms. Sch B could protect rat against DOX-induced acute cardiomyopathy and has clinical potential applications.

[The expression of nicotinamide phosphoribosyl transferase and vascular endothelial growth factor-A in gastric carcinoma and their clinical significance].

OBJECTIVES: To study the expression of nicotinamide phosphoribosyl transferase (Nampt) and vascular endothelial growth factor-A (VEGF-A) in gastric carcinoma and investigate their correlations to clinicopathologic features and prognostic significance. METHODS: The proteins of Nampt and VEGF-A in 68 specimens of gastric carcinoma and 59 specimens normal gastric tissue were detected by immunohistochemistry during January 2000 to December 2004, and the 68 patients were followed up. RESULTS: Nampt protein was detected in the cytoplasm of both tissues, and Nampt in gastric carcinoma (13 ± 5) were significantly higher than that in normal gastric tissue (6 ± 3) (t = 7.46, P < 0.01). The expression of Nampt was correlated to invasive depth (F = 4.693, P = 0.034), lymph node metastasis (F = 4.027, P = 0.049), clinical TNM stage (F = 9.979, P = 0.002), but not to gender, age, tumor location, tumor size, differentiation (P > 0.05). The expression of Nampt is correlated with survival of patients that underwent surgical resection for gastric cancer. The survival rate of patients in negative of Nampt was very higher than that of the positive patients, and its co-expression with VEGF-A showed a trend towards poorer survival. The positive correlation was found between the expression of Nampt and VEGF-A in gastric carcinoma (r = 0.293, P = 0.015). CONCLUSIONS: The expression of Nampt is positively correlated to that of VEGF-A in gastric carcinoma. The correlation between the expression of Nampt and VEGF-A in gastric carcinoma plays an important role cooperatively in carcinogenesis, development and metastasis of gastric carcinoma.

[Functional difference of malate-aspartate shuttle system in liver between plateau zokor (Myospalax baileyi) and plateau pika (Ochotona curzoniae)].

To explore the adaptive mechanisms of plateau zokor (Myospalax baileyi) to the enduring digging activity in the hypoxic environment and of plateau pika (Ochotona curzoniae) to the sprint running activity, the functional differences of malate-aspartate shuttle system (MA) in liver of plateau zokor and plateau pika were studied. The ratio of liver weight to body weight, the parameters of mitochondria in hepatocyte and the contents of lactic acid in serum were measured; the open reading frame of cytoplasmic malate dehydrogenase (MDH1), mitochondrial malate dehydrogenase (MDH2), and the partial sequence of aspartate glutamate carrier (AGC) and oxoglutarate malate carrier (OMC) genes were cloned and sequenced; MDH1, MDH2, AGC and OMC mRNA levels were determined by real-time PCR; the specific activities of MDH1 and MDH2 in liver of plateau zokor and plateau pika were measured using enzymatic methods. The results showed that, (1) the ratio of liver weight to body weight, the number and the specific surface of mitochondria in hepatocyte of plateau zokor were markedly higher than those of plateau pika (P < 0.01 or P < 0.05), but the content of lactic acid in serum of plateau pika was significantly higher than that of plateau zokor (P < 0.01); (2) MDH1 and MDH2 mRNA levels as well as their enzymatic activities in liver of plateau zokor were significantly higher than those of plateau pika (P < 0.01 or 0.05), AGC mRNA level of the zokor was significantly higher than that of the pika (P < 0.01), while no difference was found at OMC mRNA level between them (P > 0.05); (3) mRNA level and enzymatic activity of MDH1 was significantly lower than those of MDH2 in the pika liver (P < 0.01), MDH1 mRNA level of plateau zokor was markedly higher than that of MDH2 (P < 0.01), but the activities had no difference between MDH1 and MDH2 in liver of the zokor (P > 0.05). These results indicate that the plateau zokor obtains ATP in the enduring digging activity by enhancing the function of MA, while plateau pika gets glycogen for their sprint running activity by increasing the process of gluconeogenesis. As a result, plateau pika converts the lactic acid quickly produced in their skeletal muscle by anaerobic glycolysis and reduces dependence on the oxygen.

[Gene coding and mRNA expression of vascular endothelial growth factor as well as microvessel density in brain of plateau zokor: comparison with other rodents].

Vascular endothelial growth factor (VEGF) plays an important role in tissues angiogenesis. The adaptation of animals to hypoxic environment is relative to the microvessel density (MVD) in tissues. To further explore the adaptation mechanisms of plateau zokor (Myospalax baileyi) to the hypoxic-hypercapnic burrows, the VEGF mRNA and the MVD in cerebral tissues of the plateau zokor were studied. Total RNA was isolated from liver, and VEGF cDNA was obtained by RT-PCR, then the VEGF cDNA was cloned and sequenced. The coding sequence of plateau pika (Ochotona curzniae), rat (Rattus norvegicus) and mouse (Mus musculus) VEGF cDNA are obtained from GenBank, and the nucleotide and amino acid sequence homology of plateau zokor VEGF cDNA coding sequence with that of plateau pika, rat and mouse were analyzed and compared by using of bioinformatics software. The VEGF mRNA was detected by real-time PCR, and the MVDs in cerebral tissues of the plateau zokor, plateau pika and Sprague-Dawley (SD) rat were measured by immunohistochemical staining. The results showed that the open reading frame of the plateau zokor VEGF was 645 bp, and the coding sequence of the plateau zokor VEGF cDNA shared 92.1%, 93.6% and 93.8% nucleotide sequence homology to that of the plateau pika, rat and mouse, respectively. The deduced amino acid sequence of the plateau zokor VEGF cDNA was composed of 188 amino acids and the amino acids from 1 to 26 were signal peptide sequence. The plateau zokor VEGF188 was 90.2%, 94.9% and 94.4% homologous to that of plateau pika, rat and mouse. The level of VEGF mRNA in brain of the plateau zokor was significantly lower than that of SD rat, but there was no obvious difference in VEGF mRNA level between plateau zokor and plateau pika. The MVD in brain of the plateau zokor was markedly higher than that of plateau pika and SD rat. In conclusion, plateau zokor enhances its adaptation to the hypoxic environment by increasing the MVD. The level of VEGF mRNA in the brain of plateau zokor is lower than that of SD rat, which may be as a result of inhibition by the higher concentration of carbon dioxide in the burrow.

Effects of early excision and grafting on cytokines and insulin resistance in burned rats.

UNLABELLED: Burn wound excision and grafting is a common clinical practice that decreases patient morbidity and mortality. It is not known, however, if the salutary effects of this procedure are related to effects on interleukin 6 (IL-6) and tumor necrosis factor (TNF-) α, and to reducing insulin resistance after burn. Sprague-Dawley rats were randomly divided into three groups: control, burn, burn ± excision groups. Rats in burn group were given a third-degree scald burn covering 30% total body surface area (TBSA) and no wound excision. Rats in burn ± excision group were subjected to a 30% third-degree burn followed by complete excision and allografting of the injury site within 15 min after burn. The rats in control group were treated in the same manner as the burn group, except that they were immersed in a room-temperature water. Glucose tolerance tests (GTT) were observed at 3 days after burn, euglycemic-hyperinsulinemic glucose clamps were performed at 4 days after burn and interleukin 6 (IL-6) and tumor necrosis factor (TNF-) α were determined after euglycemic-hyperinsulinemic glucose clamps. The levels of IL-6 and TNF-α increased after burn. Significant differences in GTT were observed between control and burn groups, and the rate of glucose infused measured in burned rats was significantly decreased compared with that in control at 4 days after burn. Early excision and grafting significantly decreased levels of IL-6 and TNF-α, and further reduced insulin resistance following thermal injury compared with burn group. CONCLUSION: Early excision and grafting appeared to have an effect on inflammatory mediators and further reduced insulin resistance induced by major burns.

[Damage analyse of X-ray radiated cellobiohydrolase II molecule from Trichoderma viride].

After X-ray treatment at 1.82 keV and 40 mA and 4 hours, the cellobiohydrolase II (CBH II) aqueous solution of Trichoderma viride was analysed, and the damage state of the irradiated molecule was detected using some cysteine residue relative parameters in Raman spectroscopic methods. The results show that S-H stretch modes of CBH II exhibited some shift, which means that the hydrogen proton donor state of sulfhydryl groups was stronger and weaker, respectively. The 2 554 cm(-1) peak of irradiated sample was wide. The -S-S- construction of disulfide bonds was not broken, and the geometrical conformation types did not change either, but its bond length was somewhat shortened. Before irradiation, C-S isomer mode content of cysteine residue was Pc type slightly more than both P(N) and P(H) types, while P(N) and P(H) types increased after irradiation. Besides, CH2 rocking mode of cysteine residue was weakened remarkably after the treatement, and the protein molecule structure did not show important damage in sulfhydryl and disulfide bonds, but showed some change because of the X-ray irradiation condition.

Improved stability and yield of Fv targeted superantigen by introducing both linker and disulfide bond into the targeting moiety.

Bacterial superantigens (SAg) are the most potent activators of human T lymphocytes and recombinant immunotoxin using bacterial SAg shows promising clinical values. To engineer superantigen for immunotherapy of hepatocellular carcinoma, we genetically fused the superantigen staphylococcus enterotoxin A (SEA(D(227)A)) to the single-chain disulfide-stabilized Fv (scdsFv) of anti-hepatoma monoclonal antibody HAb25 through a short peptide GGGSGGS. We expressed this recombinant protein in Escherichia coli and extract it from inclusion bodies. We found purified scdsFv-targeted SAg contains equivalent binding affinity with disulfide-stabilized Fv (dsFv) targeted SAg and single-chain Fvs (scFv) targeted SAg, but more stable and more suitable for large scale production. The MTS(3-(4,5-dimethylthiazole-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliu m, inner salt) assay shows that the scdsFv-targeted SAg also shares the ability to activate a large number of T lymphocytes and has cytotoxic activity on human hepatoma cell line SMMC-7721. Therefore, this novel generation of recombinant immunotoxins using scdsFv has a high potential in hepato cancer treatment and the same strategy may also be applied to other cancer treatments.

[Expression, purification, and characterization of single-chain disulfide-bond Fv (ScdsFv) antibody fused with targeted superantigen SEA (D227A)].

AIM: To express, purify, and characterize scdsFv antibody fused with superantigen SEA(D227A). METHODS: The expression plasmid of scdsFv-SEA(D227A) was constructed by standard molecular cloning procedures. The recombinant protein was induced to express in E. coli BL21plusS by IPTG and purified by Q Sepharose HP column and Hiprep 26/60 Sephacryl S-200 HR column. Formation of the intramolecular disulfide bond of the purified protein was analysed by AMS alkylation and PAGE electrophoresis. The binding activity, stability and killing activity of the purified protein were assayed by ELISA and MTS, respectively. RESULTS: The recombinant protein was expressed as inclusion body, accounting for more than 30% of total bacterial protein. After purification by Q Sepharose HP and Hiprep 26/60 Sephacryl S-200 HR, the yield of the purified protein was 60 mg per liter of induced culture. AMS alkylation and PAGE electrophoresis analysis showed that intramolecular disulfide bond formed correctly in the recombinant protein. The purified protein had similar binding affinity as dsFv fused SEA and scFv fused SEA have and similar killing activity as native SEA has to human hepatoma cell line, but more stable, in vitro, as compared with dsFv fused SEA and scFv fused with SEA. CONCLUSION: The scdsFv fused with SEA, as a novel form of immunotoxin, might be used in cancer treatment.