Inhibitory Effect of CCK-8 on Methamphetamine-Induced Apoptosis.

OBJECTIVES: To investigate the inhibitory effect of cholecystokinin octapeptide (CCK-8) binding to cholecystokinin 2 receptor (CCK2R) on methamphetamine (METH)-induced neuronal apoptosis, and to explore the signal transduction mechanism of ß-arrestin 2 in CCK-8 inhibiting METH-induced neuronal apoptosis. METHODS: SH-SY5Y cell line was cultured, and HEK293-CCK1R and HEK293-CCK2R cell line were constructed by lentivirus transfection. Small interfering RNA (siRNA) was used to knockdown the expression of ß-arrestin 2. Annexin Ⅴ-FITC/PI staining and flow cytometry were used to detect the apoptotic rate of cells, and Western blotting was used to detect the expression of apoptosis-related proteins. RESULTS: The apoptosis of SH-SY5Y cells was induced by 1 mmol/L and 2 mmol/L METH treatment, the number of nuclear fragmentation and pyknotic cells was significantly increased, and the expression of apoptosis-related proteins Bax and cleaved caspase-3 were increased. CCK-8 pre-treatment at the dose of 0.1 mmol/L and 1 mmol/L significantly reversed METH-induced apoptosis in SH-SY5Y cells, and inhibited cell nuclear fragmentation, pyknosis and the changes of apoptosis-related proteins induced by METH. In lentivirus transfected HEK293-CCK1R and HEK293-CCK2R cells, the results revealed that CCK-8 had no significant effect on METH-induced changes of apoptosis-related proteins in HEK293-CCK1R cells, but it could inhibit the expression level of apoptosis-related proteins in HEK293-CCK2R cells induced by METH. The inhibitory effect of CCK-8 on METH-induced apoptosis was blocked by the knockdown of ß-arrestin 2 expression in SH-SY5Y cells. CONCLUSIONS: CCK-8 can bind to CCK2R and exert an inhibitory effect on METH-induced apoptosis by activating the ß-arrestin 2 signal.

Association of ATM and BMI-1 genetic variation with breast cancer risk in Han Chinese.

We tested the hypothesis that genetic variation in ATM and BMI-1 genes can alter the risk of breast cancer through genotyping 6 variants among 524 breast cancer cases and 518 cancer-free controls of Han nationality. This was an observational, hospital-based, case-control association study. Analyses of single variant, linkage, haplotype, interaction and nomogram were performed. Risk was expressed as odds ratio (OR) and 95% confidence interval (CI). All studied variants were in the Hardy-Weinberg equilibrium and were not linked. The mutant allele frequencies of rs1890637, rs3092856 and rs1801516 in ATM gene were significantly higher in cases than in controls (P = .005, <.001 and .001, respectively). Two variants, rs1042059 and rs201024480, in BMI-1 gene were low penetrant, with no detectable significance. After adjustment, rs189037 and rs1801516 were significantly associated with breast cancer under the additive model (OR: 1.37 and 1.52, 95% CI: 1.10-1.71 and 1.14-2.04, P: .005 and .005, respectively). In haplotype analysis, haplotypes A-C-G-G (in order of rs189037, rs3092856, rs1801516 and rs373759) and A-C-A-A in ATM gene were significantly associated with 1.98-fold and 6.04-fold increased risk of breast cancer (95% CI: 1.36-2.90 and 1.65-22.08, respectively). Nomogram analysis estimated that the cumulative proportion of 3 significant variants in ATM gene was about 12.5%. Our findings collectively indicated that ATM gene was a candidate gene in susceptibility to breast cancer in Han Chinese.

Structural characterization of polysaccharides from Cordyceps militaris and their hypolipidemic effects in high fat diet fed mice.

Cordyceps militaris is a crude dietary therapeutic mushroom with high nutritional and medicinal values. Mushroom-derived polysaccharides have been found to possess antihyperglycemic and antihyperlipidemic activities. This study aimed to partially clarify the structural characterization and comparatively evaluate hypolipidemic potentials of intracellular- (IPCM) and extracellular polysaccharides of C. militaris (EPCM) in high fat diet fed mice. Results indicated that IPCM-2 is α-pyran polysaccharide with an average molecular weight of 32.5 kDa, was mainly composed of mannose, glucose and galactose with mass percentages of 51.94%, 10.54%, and 37.25%, respectively. EPCM-2 is an α-pyran polysaccharide with an average molecular weight of 20 kDa that is mainly composed of mannose, glucose and galactose with mass percentages of 44.51%, 18.33%, and 35.38%, respectively. In in vivo study, EPCM-1 treatment (100 mg kg-1 d-1) showed potential effects on improving serum lipid profiles of hyperlipidemic mice, reflected by decreasing serum total cholesterol (TC), triglyceride (TG) and low density lipoprotein-cholesterol (LDL-C) levels by 20.05%, 45.45% and 52.63%, respectively, while IPCM-1 treatment (100 mg kg-1 d-1) remarkably decreased TC, TG and LDL-C levels by 20.74%, 47.93%, and 38.25%, respectively. In addition, EPCM-1 ameliorated hyperlipidemia possibly through upregulating the expression of serum lipoprotein lipase (LPL) and down-regulating the expression of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), as determined by enzyme-linked immunosorbent assay (ELISA) method, while IPCM-1 remarkably upregulated the expression of serum LPL. This study confirms polysaccharides from C. militaris could be explored as functional foods or natural medicines for preventing hyperlipidemia.

[Data mining for SSRs in ESTs and development of EST-SSR marker in oilseed rape].

Totally 2803 SSRs distributed in 2443 ESTs were mined out and accounted for 13.58% of 17987 non-redundant ESTs from oilseeed rape, with the average distance of distribution about 4.26 kb. Dinucleotide and trinucleotide repeats are the dominant type, with similar frequency and accounting for 89.05% together in all SSRs. AG/CT and AAG/CTT are the most frequent motifs, accounting for 84.31% and 37.71% in dinucleotide and trinucleotide repeats respectively. Further, 23 primer pairs for EST-SSRs were designed and the suitable annealing temperature for each primer pair was determined by gradient PCR. The amplification and polymorphism displayed by these primers in 10 varieties of oilseed rape were detected by using silver staining of nondenaturing polyacrylamide gel. 21 primer pairs showed the amplification, accounting for 91.30% of total primers, and 12 primer sets showed polymorphisms, accounting for 57.14% of primers available. These results indicate that it is an effective and feasible approach to develop SSR markers based on ESTs in oilseed rape.

[Development of EST (Expressed Sequence Tags) Marker in Chinese Cabbage and its Transferability to Repeseed.].

28 pairs of primers were designed according to the expressed sequence tags in Chinese cabbage. After testing on the annealing temperature and the concentration of primer, dNTP and MgCl2, a suitable PCR system was established. Under the condition of reaction system developed, primers designed specific to ESTs were screened against genomic DNA of inbreed line A from which the cDNA library was constructed. Among them, 18 pairs of primers showed the amplification. Then all the primers available in line A were subjected to PCR for DNAs from 17 cabbage varieties. Polymorphism was detected by electrophoresis with agarose gel, and 10 of 18 primer sets could reveal polymorphisms among cabbage varieties, which accounted for 55.6% of primers selected. To examine the transferability of EST markers developed in cabbage, all primers were further used for PCR-mediated amplification of genomic DNA from different varieties of rapeseeds. Of 28 pairs of primers, 24 were able to produce amplified product(s) and 18 showed polymorphisms, accounting for 85.7% and 64.3% of total primers respectively. All of 18 primer sets that amplified in cabbage also showed amplified products in rapeseed and 13 of them were polymorphic. Even amongst the 10 primer sets that were unable to amplify in cabbage, 6 pairs produced amplification and 5 could reveal the polymorphism in rapeseeds. Results obtained in the present paper proved that developing polymorphic markers based on EST could be feasible and this kind of marker would be transferable to closed related species.

[Differences of gene expression in bud stage of backcross hybrid between Ogura-type male-sterile Brassica napus L and B campestris L versus parents].

Crosses between female parent of Ogura male sterility Brassica napus L. and male parents of B. campestris ssp. chinensis Makino were made and F(1), BC(1) and BC(2) generations produced. Gene expression of two Chinese cabbage backcross hybrid BC(1), BC(2) and their parents at bud stage was analyzed by means of cDNA-AFLP technique. The results indicated that the patterns of gene expression differ significantly between BC(1) and BC(2) generations and their parents. There were many patterns of gene expression, including gene overexpression and gene silencing. Five patterns (seven kinds) of gene expression were observed, which include: (1) bands occurring in only one parent (two kinds); (2) bands observed in hybrids and one parent (two kinds); (3) bands occurring in only parents (one kind); (4) bands visualized in only hybrids (one kind); (5) bands observed in parents and hybrids (one kind). In accompany with the addition of backcross, the increase trend in backcross hybrids and their parents were described in the aspects of differential gene expression, bands expressed only in one parent and bands expressed only in both parents. The declined trend in backcross hybrids and their parents were observed in the aspects of bands expressed in both hybrids and one parent (two kinds), bands visualized in only hybrids and bands observed in parents and hybrid. Fifteen patterns of gene expression were observed in F(1)bBC(1)bBC(2) and backcross parents. The percent of bands expressed in F(1)bBC(1)bBC(2) and backcross was highest.