Effect of Helicobacter pylori lipopolysaccharide on expression of Gli and Ptch-1 proteins in sonic hedgehog signaling pathway of gastric mucosa GES-1 cells / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the effect of Helicobacter Pylori lipopolysaccharide (Hp-LPS) on expression of Gli and Ptch-1 proteins in sonic hedgehog (Shh) signaling pathway of gastric mucosa GES-1 cells.</p><p><b>METHODS</b>The LPS was extracted from Hp by hot phenol water method, and then the concentration of LPS was detected by the kinetic turbidimetric assay. GES-1 cells were stimulated by different concentrations of Hp-LPS (0, 1, 10, 20, 30 and 40 μg/ml). The inhibition rates of cell growth were measured by MTT assay after treated with Hp-LPS for 24 h. The expression of Gli and Ptch-1 proteins were determined by Western Blot.</p><p><b>RESULTS</b>MTT assay showed that the inhibition rates of GES-1 cell growth after treatment by different concentrations of Hp-LPS (1, 10, 20, 30 and 40μg/ml) were 25.8% ± 2.7%, 34.2% ± 3.1 %, 46.3% 3.4%, 60.8% ± 2.1% and 82.9% ± 2.8% respectively (r=0.985, P<0.001). Western blot showed that the expressions of Gli and Ptch-1 proteins were decreased after Hp-LPS treatment (0, 1, 10, 20, 30 and 40 μg/ml): the relative expression values of Gli were 1.286 ± 0.180, 0.963 ± 0.067, 0.850 ± 0.085, 0.566 ± 0.058, 0.549 ± 0.056 and 0.377 ± 0.047, respectively (r=-0.945, P<0.001); those of Ptch-1 were 1.688 ± 0.088, 1.466 ± 0.061, 1.170 ± 0.065, 1.042 ± 0.064, 0.648 ± 0.057 and 0.482 ± 0.074, respectively (r=-0.985, P<0.001).</p><p><b>CONCLUSION</b>Hp-LPS can decrease the related protein expression of Shh signaling pathway, which indicates that Hp may interfere with the function of Shh signaling pathway in gastric mucosa via the effect of its LPS.</p>

Roles of proto-oncogene c-erbB2 during the initiation growth of rat primordial follicles / 中国应用生理学杂志

<p><b>OBJECTIVE</b>To study the expression and possible roles of proto-oncogene c-erbB2 during the initiation growth of primordial follicles.</p><p><b>METHODS</b>Ovaries were collected from 2-day-old SD rats and cultured in the Waymouth culture system. In-situ hybridization, RT-PCR and immunohistochemistry were performed to assess the expressions of c-erbB2 mRNA and protein during the initiation growth of primordial follicles and after the effect of EGF. Western blot was used to observe the PCNA, p-ERK1/2 contents and correlation analysis was used to study the correlation relationship between contents of p-ERK1/2 and expressions of c-erbB2 mRNA at the same time of the primordial follicles growth.</p><p><b>RESULTS</b>PCNA protein levels appeared to be more intense during the initiation growth of primordial follicles, EGF could promote the proliferation and differentiation of the primordial follicles. c-erbB2 mRNA existed in the oocytes endochylema and ErbB2 existed in the oocytes membrane, the expressions of c-erbB2 mRNA and ErbB2 appeared to be more intense when primordial follicles were cultured for 8 d than cultured for 0 d in the Waymouth culture system and were further increased with 50 ng/ml EGF for 4 d and 8 d. The same results were observed by RT-PCR, too. p-ERK1/2 protein levels were consistent with the changes of c-erbB2 mRNA and protein. Furthermore, Spearman rank correlation analysis showed there was a significant positive correlation relationship between the changes of p-ERK1/2 and the changes of c-erbB2 mRNA during the primordial follicles growth and after the effect of EGF (rs = 0.900, P < 0.05).</p><p><b>CONCLUSION</b>It was suggested that proto-oncogene c-erbB2 may be play an important role during the initiation growth of primordial follicles with EGF, and it is indirectly suggested that c-erbB2 promotes the development of the primordial follicles via ERK-MAPK signal transduction.</p>

Analysis on the molecular characteristics of Japanese encephalitis virus isolated in Northwestern Yunnan province / 中华流行病学杂志

Objective To analyze the molecular characteristics of Japanese encephalitis virus (JEV) isolated in Northwestern Yunnan province, and to clarify the differences between the strains isolated in Northwestern and other parts of Yunnan province. Methods PrM, E and 3' untranslated region nueleotide acid sequences of the isolates were amplified by RT-PCR and then sequenced. Sequence alignment and phylogenetic analysis were performed by using Clustal 1.8X, DNASTAR, GENEDOC and Mega 3.1 programs. Results 12 of the 13 isolates of JEV obtained in Northwestern Yunnan were identified as genotype Ⅰ, only one strain was genotype Ⅲ of JEV. The 12 strains of genotype Ⅰ were clustered in different branches with other isolates obtained in other parts of Yunnan province. Data from sequence analysis on E gene found that the nucleotide identity was 0.2%-13.9% between the Northwestern isolates and other Yunnan strains. There were two kinds of nucleotides deletion patterns at 3' untranslated region with three and one deletions was found after termination eodon in genotype Ⅰ and Ⅲ isolates, respectively. Conclusion There were two genotypes of Ⅰ and Ⅲ in 13 strains of JEV in this study and genotype Ⅰ isolates were predominant (12/13). There were no apparent differences in E gene sequence between isolates obtained in the Northwestern and other parts of Yunnan. Three deletions were found in 3' untranslated region in genotypes Ⅰ isolates and one deletion was in genotypes Ⅲ.

Molecular characterization of full-length genome of Japanese encephalitis virus isolated in Liaoning Province in 2008 / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To sequence and analyze the whole genome of Japanese encephalitis virus (JEV) isolated from mosquitoes in Liaoning province in 2008.</p><p><b>METHODS</b>Using RT-PCR to amplify fragments with genome sequencing primer. The full-length genome was obtained by sequencing and splicing. The differentiation analysis for nucleotides, deduced amino acid sequence and phylogenetic tree was performed by the software of Clustal X (1.83), ATGC (V4), DNAStar, GENEDOC (3.2) and Mega (4.0).</p><p><b>RESULTS</b>The whole genome of strain LN0828 possesses 10 965 nucleotides. An open reading frame from 97 to 10 392 including 10 296 nucleotides is capable of coding for a 3432 amino acid polyprotein. Comparison of strain LN0828 genomic sequence with those of 32 JEV isolates in GenBank showed that nucleotide sequence divergence ranges from 1.6% to 16.4%, which resulted in amino acid sequence divergence from 0.3% to 5.1%. In comparison with live attenuated vaccine stain SA14-14-2 in open reading frame, strain LN0828 has a total of 1186 nucleotide substitutions, 86 amino acid divergences. Based on phylogenetic analysis, the strain LN0828 belongs to the genotype I JEV.</p><p><b>CONCLUSION</b>The whole genome of strain LN0828 is close to those of isolates from Liaoning in 2002 and 2007, which were grouped into genotype I JEV.</p>

The genotype-specific primers for amplying and sequencing the genotype I & II Japanese encephalitis virus / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>For constructing the high-throughput platform of sequencing the JEV whole genome, the two systems of multiplex primers for genotype I and III should be designed and used for detected the whole genome of genotype I and III JEV in the research.</p><p><b>METHODS</b>The two systems of JEV genotype-specific primers was designed based on the reference sequence of all the available genotype I and III JEV genome sequence on GenBank, then, they were used to amply and sequence the 121 JEV strains isolated in China contains 63 GIII JEV and 58 GI JEV.</p><p><b>RESULTS</b>The self-designed genotype-specific primers for genotype I and genotype III JEV were 16 pairs and 17 pairs respectively, which were used for detecting the whole genome of 121 JEV. The average quality value for GI JEV is 40.037. The average quality value for GIII JEV is 40.857.</p><p><b>CONCLUSION</b>The two systems of JEV genotype-specific primers could sequenced the genotype I and III JEV qualified and specific. It is the basis of the high throughput platform of sequencing the JEV whole genome.</p>

Molecular characterization of full-length genome of Japanese encephalitis virus strain patient's cerebrospinal fluid in China / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To sequence and analyze the whole genome of Japanese encephalitis virus (JEV) strain named 47 which was isolated from patient's cerebrospinal fluid sample in Heilongjiang province in 1950.</p><p><b>METHODS</b>RNA was extracted from the recovery strain 47 and amplified with self-designed JEV genome sequencing primers. The differentiation analysis for nucleotides and coding amino acids and phylogenetic analysis were performed by the software of DNAStar, Modeltest, and Phylip.</p><p><b>RESULTS</b>The whole genome of strain 47 has 10,977 nucleotides. An open reading frame from 95 to 10,391 including 10,296 nucleotides is capable of coding a 3432 amino acid polyprotein. The nucleotide difference between strain 47 and 5 vaccine strains is 2.4%-4.4%, the amino acid difference between strain 47 and 5 vaccine strains is 0.3%-1.1%. The best evolution model for the whole genome is GTR + I + G. Based on the phylogenetic analysis, strain 47 belongs to the genotype III JEV.</p><p><b>CONCLUSION</b>Strain 47 is highly conserved on whole genome nucleotide and amino acid sequence. And it is belongs to the genotype III JEV.</p>