To investigate whether there is association between the-2548G/A functional polymorphism in the promoter region of leptin gene and weight gain following antipsychotic agents (APS) acute treatment in schizophrenic patients. Eight-four Chinese Han untreated schizophrenia patients in 70 nuclear families were recruited. The polymorphism of leptin gene was determined with PCR-RFLP technique. Body weight was measured in the patients on admission the and after 10 weeks treatment with risperidone or chlorpromazine. There was an average (8.00+/-6.13)% increases in baseline weight after the 10 week treatment. There were significant differences in the distribution of allele frequencies (chi2=4.031, P=0.045) between the patients with weight changed >or=7% and <7% subgroups. Family-based association analysis further confirmed the above significant finding by transmission disequilibrium test but not by quantitative trait transmission disequilibrium test. The finding confirms that the-2548G/A polymorphism in promoter region of leptin gene is associated with APS-induced weight gain.
To isolate and identify the potential binding partners of LRRK2, a gene linked to both dominant familial form and sporadic form of Parkinson's disease, thus to further our knowledge of its function. We used a sequence containing full-length of COR domain and part of ROC and MAPKKK domain as bait. The bait amplified by polymerase chain reaction (PCR) was then cloned into a yeast expression plasmid pGBKT7. After being sequenced and analyzed, pGBKT7-bait was transformed into the yeast strain AH109. Western blot was performed to confirm the expression of pGBKT7-bait in AH109 yeast strain. Then human fetal brain cDNA library was transformed into that yeast strain, which could express pGBKT7-bait fusion protein. The yeast strain which contained pGBKT7-bait and human fetal brain cDNA library was plated on quadruple dropout medium (SD/-Trp/-Leu/-His/-Ade) containing X-alpha-gal. We retested these positive colonies using 2 independent yeast strains AH109 contained pGBKT7-bait or pGBKT7, respectively. At last, these plasmids isolated from these true positive colonies were analyzed by bioinformatics. We obtained 9 true positive colonies, these colonies were sequenced, and we performed sequence Blast in GenBank. Three colonies of the 9 positive colonies were not in open reading-frames. Among other 6 colonies, there were known proteins including spermatid perinuclear RNA-binding protein (STRBP) and BCL2-associated athanogene 5 isoform b (BAG5), as well as unknown proteins including tyrosine phosphatase non-receptor type (PTPN23), l(3)mbt-like 3 isoform b (L3MBTL3), RALY RNA binding protein-like isoform 1 (RALYL), and Homo sapiens mRNA for KIAA1783 protein, partial cds (KIAA1783). True positive colonies of LRRK2 are successfully obtained by the yeast 2-hybrid. Our screened proteins may provide a new research clue for revealing biological functions of LRRK2, pathogenesis of Parkinson's disease, and other neurodegerations.
To explore the effect of all-trans-retinoic acid (ATRA) on the growth inhibition and cellular differentiation of C6 glioma cells. Human glioma C6 cells were treated with 5 mg/L ATRA,and the inhibition of cell growth was assessed by methyl thiazolyl tetrazolium assay. The differentiation of C6 cells was determined by flow cytometry, microscopy,transmission electron microscope, and immunohistochemical technique. Treatment of ATRA could result in the growth inhibition of C6 cells, and the cell density significantly decreased(P0.05).Whereas, early apoptosis was observed under the transmission electron microscope, the vacuoles increased, the mitochondria and endoplasmic reticulum were abundant in the cytoplasm, and the cellular structures tended to be normal.The expression of glial fibrillaryacidic protein in C6 cells increased in the treatment group. ATRA can inhibit the proliferation, and induce the differentiation of C6 glioma cells.
To investigate the effect of alanyl-glutamine (Ala-Gln) on acute lung injury (ALI) in rats induced by sepsis and its mechanisms. Forty healthy adult Wistar rats were randomly divided into a control group, a lipopolysaccharide (LPS)-induced shock group, an Ala-Gln treated group, and a glutamine (Gln) treated group. The control group received an intravenous infusion of 28 mL/kg lactated Ringeros solution(LR). The LPS-induced shock group received an intravenous administration of 25 mL/kg LR, and then 3 mL/kg (6 mg/kg) LPS (L-2880, Sigma, America). The Ala-Gln treated group received 4.5% Dipeptiven (25 mL/kg, equaling 0.75 g/kg Gln) immediately before 3 mL/kg (6 mg/kg) LPS.The Gln treated group received 3% glutamine ( 25 mL/kg, 0.75 g/kg) immediately before 3 mL/kg (6 mg/kg) LPS. Serum (1 mL) was drawn via the femoral vein or cardiac puncture before LPS injection (T(0)) and 6 h after the administration of LPS (T(1)), respectively. All rats were killed 6 h after LPS infusion. The samples of pulmonary tissue and lung lavage fluid were collected after experiments. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-8 (IL-8) in the serum at T(0) and T(1) were detected by ELISA. Apoptosis in the lung epithelial cells was detected with TUNEL assays. The lung wet/dry(W/D)weight ratio and total protein in the bronchoalveolar lavage fluid (BALF) were measured. Six hours after the infusion of LPS (T(1)), the plasma concentrations of TNF-alpha, IL-1beta, and IL-8 were much lower in the Ala-Gln treated group and the Gln treated group than those in the LPS-induced shock group (P<0.05). Compared with the LPS-induced shock group, Ala-Gln and Gln significantly reduced the increase in the lung wet/dry weight ratio (P<0.05) and attenuated the morphological lung damage. Intravenous administration of Ala-Gln can effectively protect the lung from sepsis induced injury.
To investigate the distribution of pulmonary surfactant protein A (SP-A) like molecules and the bridge of frontier host defense and adaptive immune response cell of CD68 positive macrophages in inflammatory bowel disease (IBD). Surgical specimens derived from involved areas and normal area of the colon with Crohn disease (CD) and ulcerative colitis (UC) were obtained from Department of Pathology, Rhode Island Hospital, Brown University Medical Center. The distribution of SP-A like molecule in intestine of IBD was detected by immunohistochemistry. SP-A like molecule located in epithelia of intestine, the surface of intestine villi, blood vessels of connective tissue, and some inflammatory cells. The number of macrophages with both SP-A like molecule and CD68 positive was dramatically increased in the inflammatory area than the normal area. Some CD68 positive macrophages expressed SP-A like immunoreactivity by immunofluorescence double labeling. SP-A is an important host defense molecule in lung, and SP-A expression in large intestine may reflect a close relation between 2 organs in immune response towards inflammation.
To investigate the protective effect of isoflurane delayed preconditioning on myocardial ischemia reperfusion injury and the potential mechanism in rabbits. Thirty New Zealand male white rabbits were randomly assigned to 3 groups: Control group; I/R group; and 2.0% isoflurane group. Isoflurane group was exposed to 2.0% isoflurane-100% oxygen for 2 hours. Control group and I/R group were exposed to 100% oxygen for 2 hours and served as untreated controls. Twenty-four hours later I/R group and isoflurane group underwent 40 minutes of coronary occlusion followed by 2 hours of reperfusion. Blood samples were taken from the arterial line at 20 minutes before the occlusion(T1), 20 minutes after the occlusion(T2), 40 minutes after the occlusion(T3), 1 hours after the reperfusion(T4), and 2 hours after the reperfusion(T5) to determine the plasma level of TNF-alpha. At the end of the reperfusion, infarct size and area at risk were defined by Evans and TTC staining. The heart was harvested and levels of the p38MAPK activity were determined by Western blot, and ultrastructures were observed under the electron microscope. The p38MAPK activity of isoflurane group was significantly lower than that of I/R group (P<0.05). Isoflurane significantly (P<0.05) reduced the infarct size(19.7%+/-2.8% in isoflurane group) of the left ventricular area at risk as compared with the controls (37.8%+/-1.7% in I/R group).The injury of I/R group was worse than that of isoflurane group under the light microscope. Isoflurane group had a lower level of TNF-alpha than I/R group. Isoflurane can inhibit p38MAPK activity during myocardial ischemia reperfusion and modulate the cytokine expression, which may be one of the molecular mechanisms of isoflurane delayed preconditioning on cardioprotection.
To construct a technological platform of 2-dimensional tumor microvascular architecture phenotype (2D-TAMP) expression. Thirty samples of non-small cell lung cancer (NSCLC) were collected after surgery. The corresponding sections of tumor tissue specimens to the slice of CT perfusion imaging were selected. Immunohistochemical staining,Gomori methenamine silver stain, and electron microscope observation were performed to build a technological platform of 2D-TMAP expression by detecting the morphology and the integrity of basement membrane of microvasculature, microvascular density, various microvascular subtype, the degree of the maturity and lumenization of microvasculature, and the characteristics of immunogenetics of microvasculature. The technological platform of 2D-TMAP expression was constructed successfully. There was heterogeneity in 2D-TMAP expression of non-small cell lung cancer. The microvascular of NSCLC had certain characteristics. 2D-TMAP is a key technology that can be used to observe the overall state of micro-environment in tumor growth.
To compare the behavioral improvement to find the best transplantation approach for treating brain injury through transplanting amniotic-derived mesenchymal stem cells into brain injured rats in different ways. Eighty brain injured Wista rats were randomly divided into a control group with brain injury alone (n=20) and a treatment group(n=60) which were further evenly divided into Group A (transplanted through the vena caudalis), Group B (transplanted through the ventriculus cerebri lateralis), and Group C (transplanted through the injured brain area). Each group was transplanted with amniotic-derived esenchymal stem cells, and their therapeutic efficacy would be evaluated through the neurological severity score (NSS). Compared with other groups, the behaviors of Group C had markedly improved. There was statistically significant difference in the 2 groups (P0.05). Transplanting the amniotic-derived mesenchymal stem cells into the injured brain area may be effective for brain injury in rats.
To determine the effect of a selective COX-2 inhibitor celecoxib on cell proliferation and apoptosis of gastric cancer cell line BGC-823 to seek an effective and safe drug for gastric cancer chemoprevention. Gastric cancer cell line BGC-823 was cultured to 80% fusion. MTT assay and flow cytometry were used to quantify the influence of celecoxib in the proliferation, cell period, and apoptosis of gastric cancer cell line BGC-823. The expression of p21 and Fas by RT-PCR were investigated on gastric cancer cell line BGC-823 by the effect of different celecoxib concentrations. Growth of BGC-823 cells was inhibited by celecoxib in a dose-and time-dependent manner (P<0.05).Flow cytometry showed that celecoxib increased the proportion of cells in G1 phase, whereas decreased the proportion of cells in S phase and increased the apoptotic rates of cells in a concentration-dependent manner from 0 to 100 micromol/L in gastric cancer cell line BGC-823 (P<0.05). RT-PCR detection showed that the treated BGC-823 cells had increased the expression of p21 and Fas, which was also in a dose-dependent manner (P<0.05). Celecoxib inhibited cell proliferation and apoptosis of human gastric cancer cell line BGC-823, which may be related to blocking the cell cycle progress by increasing the expression of p21 and inducing the apoptosis of gastric cancer cells by increasing the expression of Fas.
To explore the frequency of vitamin D receptor (VDR) Fok I polymorphism in healthy Chinese and colorectal tumor patients,and to study the correlation between VDR Fok I polymorphism and the pathogenesis of colorectal tumor. After the preparation of gDNA by common method,VDR genotypes were determined by Fok I restriction endonuclease digestion of PCR-amplified DNA in 69 colorectal cancer patients and 218 healthy persons. The F allele frequencies of VDR in healthy persons and in colorectal tumors patients were 61.5% and 49.3%, respectively, with statistical difference (P< 0.05). The genotype frequencies of FF, Ff and ff in healthy persons and in colorectal tumors patient were (39.5%,44%,16.5%) and (29.1%,40.5%,30.4%), respectively,with statistical differences (P< 0.05). The odds ratio of ff and Ff genotypes were 2.51 (95% confidence interval,1.21 approximately 5.18) and 2.00 (95% confidence intervals,1.01approximately 3.96), respectively (P< 0.01). Fok I polymorphism is a common single nucleotide polymorphism (SNP) of VDR in Chinese population,and the VDR Fok I polymorphism may lower the risk of colorectal tumor.
To observe the internalization of beta-amyloid protein (Abeta) in primary cultured neurons and effect of astrocyte on it. The purified cortical neurons of mouse were cultured for 14 d, and were divided into a control group and an Abeta group. Each group was further divided into 3 subgroups. The neurons and 3 different concentration fluorescein or Abeta1-42-fluo were co-incubated for 24 h. The internalization of Abeta and the location of Abeta in subcellular structure were examined by the laser scanning confocal microscope combined with the image analysis method directly or after immunofluorescence staining. Neurons and astrocytes were co-cultured for 14 d. The cultured neurons and astrocytes were divided into a control group and a Abeta group. The cultures were treated with 200 nmol/L fluorescein or 200 nmol/L Abeta1-42-fluo for 24 h respectively. The effect of astrocyte on the internalization was analyzed by the above method. There were no fluorescent granules within neurons in every fluorescein group. The purified cortical neurons could internalize 100 nmol/L, or 200 nmol/L Abeta1-42-fluo in 24 h. The fluorescent granules of Abeta1-42 distributed within perikaryon and processes. The internalization was related to the concentration of Abeta. The part of Abeta was located in the lysosome of neurons indicated by immunofluorescence staining. Compared with the purified neurons, the neurons co-cultured with the astrocytes internalized Abeta increased in the internalization of Abeta. There was significant difference between the purified neurons and the co-cultured neurons with astrocytes (P<0.05). Neurons could internalize the proper concentration of Abeta. Astrocyte might facilitate the internalization of Abeta in neurons.