Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica最新文献

In order to elucidate the role of EBV-LMP1 in the nasopharyngeal carcinogenesis, the expression vector was constructed with subjecting the N-LMP1 gene to double regulation of two specific regulators: EDL-2 and PLUNC-p. The N-LMP1 related transgenic mice model has been constructed successfully by pronucleus microinjection. 58 founder mice were born, 4 of which were founded to be positive by PCR and Southern blot. Immunohistochemistry assay showed that N-LMP1 protein was expressed in the nasopharynx, tongue and forestomach of transgenic mice.

Glial cell is an ideal vehicle for gene therapy of brain diseases. However, there are many limits in using primary glial cells. Therefore, an immortalized rat glial cell line (RGLT) was established by SV40 large T-antigen (LTag) gene from the primary rat fetal glial cells. The RGLT cell was shown to be non-tumorigenic after transplantation to nude mice (up to 4 weeks) and rat striatum (up to 18 months). Rat tyrosine hydroxylase (TH) gene was transfected into RGLT cell to obtain RGLT-TH cell. The TH immunohistochemical staining and HPLC-ECD analysis demonstrated the TH expression and dopamine (DA) production in RGLT-TH cells in vitro. When implanting RGLT-TH cells into the striatum of 6-hydroxydopamine (6-OHDA) lesioned hemiparkinsonism model rats, TH immunohistochemical staining showed the TH presence in striatum and HPLC-ECD analysis held at 6 months after cell implantation showed an increase of DA content in striatum. The asymmetric rotation of rats receiving RGLT-TH cells was reduced by 50%-60% and this reduction persisted stably at least for 18 months. These results suggest that the immortalized glial cell line could serve as an ideal vehicle for therapeutic gene delivery system to achieve a long-term gene therapy of neurodegenerative diseases.

To study the biological basis of selenium in resisting senescence through its effects on cellular telomerase activity and telomere length. In the experiments, the cell line of hepatocytes L-02 was divided into three groups supplemented with sodium selenite at final concentrations of 0, 0.5 and 2.5 micromol/L, respectively. Cellular telomerase activity was measured by telomeric repeat amplification protocol and enzymatic luminometric inorganic pyrophosphate detection assay. RT-PCR was used to semi-quantitatively detect human telomerase reverse transcriptase (hTERT) gene expression. The change of telomere length was assayed through flow cytometry and fluorescence in situ hybridization. Results showed that L-02 cells had low telomerase activity and hTERT gene expression level when cultured in the normal way. The cells grew well after 3-week-cultivation in the media supplemented with 0.5 or 2.5 micromol/L sodium selenite. Besides, sodium selenite significantly increased cellular telomerase activity and hTERT gene expression level. The telomere length of L-02 cells was also extended after 4-week-cultivation with sodium selenite. Thus, sodium selenite at nutritional doses could prolong the life span of hepatocytes L-02 through increasing telomerase activity and telomere length. This result provides a possible mechanism for explaining the anti-senescence function of selenium.

The GATA-1 of Xenopus (xGATA-1), which has two subtypes xGATA-1a and xGATA-1b, is a necessary factor for erythroid differentiation and maturation as similar as that of other GATA-1s. Although both xGATa-1a and xGATA-1b are able to stimulate erythropoiesis, only xGATA-1b is capable of inhibiting neurogenesis in Xenopus embryos. Compared between their structures, xGATA-1a and xGATA-1b are very similar in nucleotide and amino acids composition, but not identical. Therefore, it is responsible for studying the role of the diverse codons between the two genes, so the desired mutations: S(168), H(169) double deletion and point mutation of T(304)-->A, T(359)-->A, were introduced into xGATA-1b gene through site-directed mutagenesis. Then, mRNA from each mutant as well as wtxGATA-1b was co-injected with DN-BR mRNA or separately injected into Xenopus stage 2 embryos, and the role of mutants in erythropoiesis and neurogenesis was analyzed by using animal cap culture system. The results showed that the neural-inhibiting activity of xGATA-1b, but not hematopoiesis-inducing activity, was aborted because of deletion of Ser(168) and His(169) or point mutation of T(359)-->A. So it is demonstrated for the first time that Ser(168) and His(169) or Thr(359)in xGATA-1b may be one of the structural basis for explanting the different function between xGATA-1b and xGATA-1a.

Infection of hepatitis B virus (HBV) continues to be a significant health problem. alpha interferon (IFN-alpha) and gamma interferon (IFN-gamma) have been proven to be effective in inhibiting HBV replication. To study the global effect of HBV persistent existence on IFN induced cellular gene expression, cDNA microarrays dotted with 14 112 human genes were used to examine the transcriptional changes between an HBV DNA transfected cell line (HepG2.2.15) and its parental cell line (HepG2) after the treatment of IFN-alpha or IFN-gamma for 6 h. The results showed that many genes related to cell cycle, proliferation, apoptosis and new ESTs were regulated by IFN-alpha and/or IFN-gamma. Many genes involved in kinase and signal transduction, transcription regulation, antigen presentation and processing were differentially regulated between these two cell lines post IFN-alpha or IFN-gamma treatment. Interestingly, several IFN-differentially regulated genes, such as MyD88 and Diubiquitin, were found to inhibit HBV gene expression, and MyD88 was proved to inhibit HBV replication. Taken together, our results revealed the global effects of HBV persistent existence on IFN-induced cellular gene expression. The novel antiviral genes identified by microarray could be potentially developed as new anti-HBV drugs or for novel therapies.

Based on the phenomenon of the natural anti-schistosomiasis in Microtus fortis, the sera from normal Microtus fortis were employed to immunoscreen the cDNA library of adult Schistosoma japonicum (Chinese strain), two positive clones were obtained, RACE technique was further applied to amplify one of the clones, and a cDNA fragment with an ORF was identified. Sequencing revealed that it was a novel gene of Schistosoma japonicum, and it was named SjMF4 (Schistosoma japonicum Microtus fortis 4). Then the structure and functional motifs of SjMF4 were analysed. The gene was subcloned into pET-28a(+) vector; the recombinant protein showed good antigenicity in Western blotting. The gene was further subcloned into eukaryotic expression vector pcDNA3 to construct the DNA vaccine containing SjMF4. Immune experiments in mice showed significant protection that the recombinant plasmid did induce 28.64%+/-3.82% worm reduction and 21.73%+/-3.98% egg reduction than controls against the Schistosoma japonicum cercaria challenge.

A novel big defensin was isolated and characterized from the plasma of Ruditapes philippinesis. It was purified to homogeneity by means of precipitation with (NH(4))(2)SO(4), gel-exclusion chromatography, two kinds of cation-exchange chromatography and named RPD-1. Its relative molecular mass was 24.8 kD by means of SDS-PAGE. By means of ABI437 amino acid sequence analyser, its 11 NH(2)-terminal amino acid sequence is AVPDVAFNAYG. Two databank systems (NCBI and EBI/EMBL) was indexed, no sequence with homology to RPD-1 was found. Furthermore, it exhibited strong inhibition on the growth of Gram-negative and -positive bacteria. Minimal inhibitory concentration (MIC) of RPD-1 against Staphylococcus aureus, Bacillus subtilis, Micrococcus tetragenus, Escherichia coli, Vibrio parahaemolyticus, Vibrio anguillarum was 9.6 mg/L, 76.8 mg/L, 38.4 mg/L, 76.8 mg/L, 19.2 mg/L, 19.2 mg/L respectively.

The gene duplication, fusion and horizontal transfer are the frequent events during evolution of many proteins, including the aminoacyl-tRNA synthetases (AARSs). However, in this work, it was shown that the main event during evolution of phenylalanyl-tRNA synthetase (PheRS) is a domain loss, and the function/activity of PheRS is not affected by domain losing. Generally, the size of genome and number of genes are increased during evolution from bacteria to eukaryote, but the interesting thing is that the type and number of PheRS domains in eukaryote are obviously less than those in bacteria. The evolution of PheRS by domain losing seems to be related to the functional evolution of some AARSs from the multiple specificities to the single specificity.

The cDNA of human B lymphocyte stimulator C-terminal peptide (C-BLyS) was amplified by nested PCR from cDNA library of human fetal brain. The expression plasmid pT7450-C-BLyS was constructed and transformed into E. coli BL21 CodonPlus (DE3) RIL which can recognize many rare codons. The C-BLyS protein was expressed as inclusion body in E. coli BL21 CodonPlus (DE3) RIL and the inclusion body of C-BLyS was denatured and then refolded by dialysis and purified by ion exchange chromatography. The refolded and purified C-BLyS can specifically bind with BCMA-Fc, a fusion protein of BLyS receptor and human IgG1 Fc. Furthermore, C-BLyS is proved to be an effective stimulator in mouse splenocytes proliferation in vitro and effective immunostimulant in vivo.

Domain is a protein architecture in a subunit. It might be defined as a basic unit for structure, function, folding, evolution and design. Different combinations of domains lead to the formation of various tertiary structures with various functions for proteins. The delineation of domains for a protein is important both conceptually and practically, which remains up to date a challenging and unsolved problem. Based on the above definition, a method was previously proposed based on refolding free energy to define continuous domains in proteins. By constructing a residue-residue contact matrix, using correspondence analysis, and then selecting optimal partition function of a protein according to refolding free energy and some empirical scoring functions, a new computer program, PDOM, was developed, which was applicable to both continuous and discontinuous domains. When compared with the manual partition results reported by crystallographers, PDOM has achieved an accuracy of 76% on a test data set including 55 protein structures frequently used. The differences in 13 proteins between PDOM, literature as well as SCOP have been discussed extensively.