Microsatellite analysis of genetic diversity in the Tupaia belangeri yaoshanensis.

The Chinese tree shrew (Tupaia belangeri yaoshanensis) has long been proposed to serve as an animal model for studying human diseases. However, its overall genetic diversity and population structure remain largely unknown. In the present study, we investigated the genetic diversity of population microsatellite DNA in wild Tupaia belangeri yaoshanensis. Sixteen microsatellite loci were assessed in 76 wild Tupaia belangeri yaoshanensis. The target microsatellite DNA fragments were amplified from the peripheral blood DNA of the animals by polymerase chain reaction (PCR), and the PCR-amplified products were verified by DNA sequencing and used for the analysis of allele, effective allele, genetic heterozygosity, polymorphism and population structure. Our results showed that of the 16 microsatellite loci examined, 5 microsatellite loci were monomorphic and 11 microsatellite loci were polymorphic. We detected 61 alleles in the polymorphic loci and found 2-10 (with an average of 5.5455) alleles per locus. Our data also showed that the observed and expected heterozygosities ranged from 0.087 to 0.8947 and 0.1368 to 0.7892 with an average of 0.3968 and 0.4796, respectively. Taken together, the results revealed a considerably high heterozygosity and high genetic diversity at the molecular level in the population of wild Tupaia belangeri yaoshanensis. The identified markers from the present study may be useful for individual identification and parentage testing, as well as for the quantification of population heterogeneity in the Chinese tree shrew.

High expression and frequently humoral immune response of melanoma-associated antigen D4 in glioma.

MAGE-D4 is a novel member of MAGE super-family. It has preliminarily been demonstrated that MAGE-D4 mRNA is not expressed in majority of normal tissues except for brain and ovary in which only trace amount of MAGE-D4 mRNA can be detected, but predominantly expressed in glioma. MAGE-D4 protein expression and its immunogenicity in glioma have not been elucidated well. This study was designed to analyze MAGE-D4 expression both at mRNA and protein level, characteristic of humoral immune response, and their relationships with glioma patients' clinicopathological parameters. Recombinant MAGE-D4 protein and antiserum were generated. Quantitative RT-PCR analysis revealed that MAGE-D4 mRNA expression was overall up-regulated in 41 glioma specimens compared with that in 14 normal brain tissues. Immunohistochemistry analysis showed that 78% (21/27) glioma tissues expressed MAGE-D4 protein, which was predominantly located in the cytoplasm of tumor cells, but absent in any neuroglia cell of normal brain tissues. ELISA analysis demonstrated that humoral response against MAGE-D4 was detected in 17% (7/41) of glioma patients' sera but not in 77 healthy donors. No apparent correlation was observed between the expression and immunogenicity of MAGE-D4 with clinicopathological parameters of glioma. In summary, these results indicate that MAGE-D4 is highly expressed in glioma and can develop specifically humoral response in glioma patients, which supports that it may be a promising biomarker for glioma diagnosis and immunotherapy.

[Fingerprints identification of Gynostemma pentaphyllum by RAPD and cloning and analysis of its specific DNA fragment].

OBJECTIVE: To identify the resources of Gynostemma pentaphyllum and its spurious breed plant Cayratia japonica at level of DNA. METHODS: Two random primers ( WGS001, WGS004) screened were applied to do random amplification with genomic DNA extracted from Gynostemma pentaphyllum and Cayratia japonica which were collected from different habitats. After amplificated with WGS004, one characteristic fragment about 500 bp which was common to all Gynostemma pentaphyllum samples studied but not to Cayratia japonica was cloned and sequenced. Then these sequences obtained were analyzed for identity and compared by Blastn program in GenBank. RESULTS: There were obvious different bands amplified by above two primers in their fingerprints of genomic DNA. On the basis of these different bands of DNA fingerprints, they could distinguish Gynostemma pentaphyllum and Cayratia japonica obviously. Sequence alignment of seven cloned bands showed that their identities ranged from 45.7% - 94.5%. There was no similar genome sequences searched in GenBank. This indicated that these seven DNA fragments had not been reported before and they should be new sequences. CONCLUSION: RAPD technique can be used for the accurate identification of Gynostemma pentaphyllum and its counterfeit goods Cayratia japonica. Besides, these specific DNA sequences for Gynostemmna pentaphyllum in this study are useful for the further research on identification of species and assisted selection breeding in Gynostemma pentaphyllum.

[Apoptosis of hepatocarcinoma cell line HLE induced by the combination of wild type P53 gene and 1,2:5,6-dianhydro-3,4-diacetylgalactitol].

BACKGROUND & OBJECTIVE: P53 gene is a tumor suppressor gene,and its mutation in human tumor cells is frequently observed. Previous studies revealed that wild type p53 (wt-p53)gene can suppress proliferation ,and induce apoptosis of tumor cells. However,the enhancive effect of wt-p53 on apoptosis of tumor cells is not so obvious when it is used alone. Therefore,it is a new field for tumor research that wt-p53 gene combined with drug is used to enhance apoptosis rate of tumor cells. This study was to investigate the enhancement effect of the combination of wt-p53 and 1,2:5,6-dianhydro-3,4-diacetylgalactitol (DADAG)on apoptosis of human hepatocarcinoma cell line HLE. METHODS: HLE cells were transfected with pUHD10-3-p53 plasmid contained wt-p53 gene,and treated with DADAG. After 96-hour treatment,apoptosis was evaluated by flow cytometry and DNA electrophoresis. RESULTS: The apoptosis rates were: 1.4% in untreated group, 3.5% in pUHD10-3-transfection group, 32.6% in DADAG group, 43.4% in pUHD10-3-p53-transfection group, and 74.6% in pUHD10-3-p53-transfection plus DADAG-treatment group. DNA ladder was observed in pUHD10-3-p53-transfection plus DADAG-treatment group. CONCLUSION: Apoptosis of HLE cells could be induced by both wt-p53 gene and DADAG,and the effect was more obvious when HLE cells were treated by the combination of wt-p53 gene and DADAG.