[In vitro killing activity of the fractional proteins from microtus fortis serum to Schistosoma japonicum juveniles].

OBJECTIVE: To explore the killing effect of different fractional proteins from Microtus fortis (Mf) serum to S. japonicum juveniles, and to find possible association of the proteins with the natural resistance to schistosome infection. METHODS: The proteins in Mf serum were separated by means of ion-exchange column chromatography and molecular sieve column chromatography. After desalted by dialysis and lyophilized, the proteins were dissolved in DMEM medium which contained 300 U/ml penicillin and 300 microg/ml streptomycin, and the two-day old schistosomula were added in for in vitro cultivation (100 +/- 20/well). The killing activity of the fractional proteins to the juvenile worms was defined by mortality rate. RESULTS: 58 fractional proteins were separated from Mf serum, in which six proteins were confirmed to have a significant killing activity to schistosomula. The mortality of schistosomula all reached 37% and above, and the highest mortality (87.5%) was observed in the fraction 18.1, while the negative control was 25.09% (P < 0.01). CONCLUSION: Some fractional proteins in Microtus fortis serum show an effect in the natural resistance to schistosome infection.

[Comparison of specific genomic DNA fragment between Microtus fortis calamorum and Microtus fortis fortis].

Microtus fortis(Taxonomy ID: 100897), also named as reed vole, is classified as Microtus, Micotinae, Cricetidae, Rodentia, Mammalia on taxonomy. Microtus fortis mainly distributes in China. Some areas of Russia, North Korea and Mongolia close to Northeast borderland of China also have a small number of Microtus fortis in distribution. Microtus fortis in China has principally 4 subspecies, and most of them live is the drainage area of Yangtse River. Schistosoma japonicum (one of commonly parasites in China) can infect about 40 kinds of mammalian animals, including the human being, but could not infect Microtus foris. It is known as the only animal in Dongting Lake region of China which has the ability of natural resistance to Schistosoma japonicum. The Microtus fortis domesticated in laboratory has the same biological characteristics as the wild one and these characteristics could be inherited to its progeny steadily. We got a specific DNA fragment from genomic library of Microtus fortis. This DNA fragment in genomic DNA of human beings, Kunming mice, Balb/c mice and C57BL/6J mice could not be detected by dot blot hybridization and PCR, apart from genomic DNA of Microtus fortis. In this report, the differences of genomic DNA in 34 Microtus fortis were compared between Microtus fortis calamorum(Dongting Lake region of southern China) and Microtus fortis fortis (Ningxia province of northern China). The residing localion of these two subspecies is far away about 1,200 kilometers from each other. The genomic DNA of Microtus fortis calamorum and Microtus fortis fortis were extracted and amplified by PCR according to the specific genomic DNAs sequence of Microtus fortis reported previously (Accession number in GenBank: AF277394). The amplified DNA fragments were inserted into pGEM-T easy vector and sequenced. The DNA fragment sequencing results from the two subspecies were compared to detect whether there was any difference. 19 alleles were found from Microtus fortis (20 of Microtus fortis calamorum and 14 of Microtus fortis fortis). The results of multiple sequence alignment showed that 25 single nucleotide polymorphism (SNP) sites were existed in the different Microtus fortis individuals, including transition (G-->A,A-->G,T-->C,C-->T), transversion(G-->T,A-->T,T-->A,C-->A), insertion (CA) and deletion (TGTTTT). The difference of genomic DNA from two subspecies were obvious, especially on the sites of 146, 192, 223, 224 and 235. The insertion of CA on the sites of 223 and 224 as well as A-->G transition on the site of 235 was only occurred in Microtus fortis fortis. They are not found in Microtus fortis calamorum so far. They could be divided into two groups according to phylogenetic tree analysis results, one was the genomic DNA of Microtus fortis calamorum and the other one was that of Microtus fortis fortis. However, the homologues reached up to 98% between two subspecies. These results are very important for us to further understand the genetic background, biological characteristics, evolutionary rule and the anti-schistosoma japonicum mechanism of Microtus fortis at the molecular levels. The specific base changes of the DNA fragment between the two subspecies are probably correlative with the animal immigration, survival conditions, and species evolution.

Identification of Novel Regulatory Elements of Human Stem Cell Factor Gene in MCF Cell.

Human stem cell factor(hSCF)is a pluripotent growth factor that regulates proliferation, differentiation and migration of certain mammalian stem cells, such as primordial germ cells etc. It is shown that hSCF and its receptor are commonly co-expressed in human breast cancer cells. Up to now, the definite regulatory mechanism of hSCF gene in breast cancer cells is unclear, except that its 5'flanking sequence contains essential elements for regulating transcription. To localize the regulatory elements responsible for the regulation of the hSCF gene, we performed transient transfection study in MCF cells, with a series of luciferase reporter gene constructs, containing different 5x end deletions of hSCF gene. This study indicates that the region of -1190 -853 significantly enhanced the luc gene expression, while the region of -339 -162 inhibited the expression. Eletrophoretic mobility shift assay confirmed that MCF nuclear extract proteins bound to both -1190 -853 and -339 -273 regions, forming specific DNA-protein complexes, indicating that there were nuclear protein binding sites in these regions. The results suggest that both -1190 -853 and -339 -273 DNA fragments of the hSCF 5'flanking sequence may be novel regulatory elements, and may play a role in the regulation of hSCF gene expression in MCF cells.