[Impact of CACNA1C polymorphisms on antihypertensive efficacy of calcium channel blocker].

OBJECTIVE: To explore the relationship between genetic polymorphisms of CACNA1C that encoded the a1c subunit of the L-type calcium channel and the efficacy of calcium channel blocker (CCB, Nifedipine extended release tablet/20 mg/d) in essential hypertension (EH) patients of Han Chinese in Wenzhou. METHODS: For the enrolled 103 EH patients, Multiplex Polymerase Chain Reaction (Multi-PCR) and matrix assisted laser desorption ionization time of flight MS (MLDI-TOF MS) were performed to detect their genotypes (rs216008, rs1051375, rs2299661, rs10848683, rs215976), blood pressure (BP) after CCB monotherapy was compared among patients with different genotypes. RESULTS: (1) Blood pressure was significantly reduced in all patients post CCB (P < 0.05 vs. pre-CCB). (2) Diastolic blood pressure reduction was more significant in subjects with rs2299661 C/C genotype (wild genotype) than in subjects with rs2299661C/G and rs2299661G/G genotype (mutational genotype) [(12.46 ± 7.91) mm Hg (1 mm Hg = 0.133 kPa) vs. (7.22 ± 8.01) mm Hg and (5.93 ± 9.77) mm Hg, P < 0.05]. (3) Systolic blood pressure reduction was more significant in subjects with rs216008 C/C genotype (wild genotype) than in subjects with rs216008 C/T genotype (mutational genotype) [(20.60 ± 12.35) mm Hg vs. (13.62 ± 10.21) mm Hg, P < 0.05]. (4) Blood pressure reduction was similar between subjects with genotype of rs1051375, rs10848683 and rs215976. CONCLUSION: EH patients with wild genotype of rs2299661 and rs216008 in CACNA1C are more likely to be responders of CCB monotherapy.

[Functional analysis of promoter fragments of salt-tolerance related genes in Spirulina].

In this work, the functions of promoter fragments of two potential salt-tolerance related genes of Spirulina (Spirulina platensis Geitl.) were studied using green fluorescent protein gene (gfp) as a reporter. The promoter structures of two salt-tolerance related genes of Spirulina were predicted using online promoter prediction software. pMD18-T and pUC18 vectors were used to clone the promoter sequences as well as the gfp gene and kanamycine resistance (kan) gene. The fragments containing pro-gfp-kanr were further cloned into pKW1188 vector and the resulting recombinant plasmids were then transformed into a host strain Synechocystis sp. (Synechocystis pevalekii Ercegovic) PCC6803. The resulting bacterial strains were grown under various concentrations of salinity for defining time intervals. The bacterial fluorescence was observed using laser confocal microscope. Our results showed that the transgenic bacteria grown at different concentrations of salinity for various periods produced varying fluorescence intensities. The bacteria treated with NaCl at the concentrations of 0.4mol/L to 0.6mol/L for 6 to 8 h showed the strongest fluorescent intensity. From the result of high salt induced expression of gfp, we predicted that the genes under control of these two promoters are likely to play important roles in the salt tolerance of Spirulina. Accordingly, we believed that a research platform for the studying functions of the promoters of the salt-tolerance related genes in Spirulina has been developed with the gfp as a reporter, the kanr gene as the selection marker, and Synechocystis. sp. PCC6803 as the expression host.

Genetic distance of SARS coronavirus from the recent natural case.

Phylogenetic analysis of SARS coronavirus isolates based on the spike gene and protein sequence using Neighbor-Joining, maximum likelihood and Bayesian inference methods indicated that a recent human SARS-CoV isolate was closer to some human SARS-CoV isolates from earlier epidemic phase than to the SARS-CoV-like viruses isolated from wild animals during previous epidemic phase. A reasonable judgment based on phylogenetic relationship and sequence variations it is likely that the recent human SARS-CoV isolate is closer to an unknown SARS-CoV predecessor.

Adaptive evolution of cry genes in Bacillus thuringiensis: implications for their specificity determination.

The cry gene family, produced during the late exponential phase of growth in Bacillus thuringiensis, is a large, still-growing family of homologous genes, in which each gene encodes a protein with strong specific activity against only one or a few insect species. Extensive studies are mostly focusing on the structural and functional relationships of Cry proteins, and have revealed several residues or domains that are important for the target recognition and receptor attachment. In this study, we have employed a maximum likelihood method to detect evidence of adaptive evolution in Cry proteins, and have identified 24 positively selected residues, which are all located in Domain II or III. Combined with known data from mutagenesis studies, the majority of these residues, at the molecular level, contribute much to the insect specificity determination. We postulate that the potential pressures driving the diversification of Cry proteins may be in an attempt to adapt for the "arm race" between delta-endotoxins and the targeted insects, or to enlarge their target spectra, hence result in the functional divergence. The sites identified to be under positive selection would provide targets for further structural and functional analyses on Cry proteins.

Polymorphism of SARS-CoV genomes.

In this work, severe acute respiratory syndrome associated coronavirus (SARS-CoV) genome BJ202 (AY864806) was completely sequenced. The genome was directly accessed from the stool sample of a patient in Beijing. Comparative genomics methods were used to analyze the sequence variations of 116 SARS-CoV genomes (including BJ202) available in the NCBI GenBank. With the genome sequence of GZ02 as the reference, there were 41 polymorphic sites identified in BJ202 and a total of 278 polymorphic sites present in at least two of the 116 genomes. The distribution of the polymorphic sites was biased over the whole genome. Nearly half of the variations (50.4%, 140/278) clustered in the one third of the whole genome at the 3' end (19.0 kb-29.7 kb). Regions encoding Orf10-11, Orf3/4, E, M and S protein had the highest mutation rates. A total of 15 PCR products (about 6.0 kb of the genome) including 11 fragments containing 12 known polymorphic sites and 4 fragments without identified polymorphic sites were cloned and sequenced. Results showed that 3 unique polymorphic sites of BJ202 (positions 13 804, 15 031 and 20 792) along with 3 other polymorphic sites (26 428, 26 477 and 27 243) all contained 2 kinds of nucleotides. It is interesting to find that position 18379 which has not been identified to be polymorphic in any of the other 115 published SARS-CoV genomes is actually a polymorphic site. The nucleotide composition of this site is A (8) to G (6). Among 116 SARS-CoV genomes, 18 types of deletions and 2 insertions were identified. Most of them were related to a 300 bp region (27,700-28,000) which encodes parts of the putative ORF9 and ORF10-11. A phylogenetic tree illustrating the divergence of whole BJ202 genome from 115 other completely sequenced SARS-CoVs was also constructed. BJ202 was phylogeneticly closer to BJ01 and LLJ-2004.

Molecular evolution and multilocus sequence typing of 145 strains of SARS-CoV.

In this study, we have identified 876 polymorphism sites in 145 complete or partial genomes of SARS-CoV available in the NCBI GenBank. One hundred and seventy-four of these sites existed in two or more SARS-CoV genome sequences. According to the sequence polymorphism, all SARS-CoVs can be divided into three groups: (I) group 1, animal-origin viruses (such as SARS-CoV SZ1, SZ3, SZ13 and SZ16); (II) group 2, all viruses with clinical origin during first epidemic; and (III) group 3, SARS-CoV GD03T0013. According to 10 special loci, group 2 again can be divided into genotypes C and T, which can be further divided into sub-genotypes C1-C4 and T1-T4. Positive Darwinian selections were identified between any pair of these three groups. Genotype C gives neutral selection. Genotype T, however, shows negative selection. By comparing the death rates of SARS patients in the different regions, it was found that the death rate caused by the viruses of the genotype C was lower than that of the genotype T. SARS-CoVs might originate from an unknown ancestor.

[Factors that influence direct sequencing of PCR products].

Factors influenced direct sequencing of PCR (polymerase chain reaction) products were investigated in this paper. It showed that the specialization of PCR products played a key role in their sequencing reactions and only which could be sequenced directly. It also showed that the PCR reaction residues (including dNTP, primers,and metal ion) affected badly on the sequencing quality, so the purification of PCR products was necessary before sequencing. In addition,the optimum templates amount in sequencing reaction rose with the increasing of their DNA size in a certain range.

Prevalence of plasmid-mediated quinolone resistance in Escherichia coli isolates in Wenzhou, Southern China, 2002-2008.

A total of 514 consecutive clinical Escherichia coli isolates, irrespective of resistance background, were collected in the period 2002-2008 in Wenzhou, southern China, to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR). The dominant PMQR gene was aac(6')-Ib-cr, followed by qnr, whereas qepA was absent. A total of 253 (49.2%) of these isolates were aac(6')-Ib-positive. Subsequently, 134 of these isolates were sequenced and 42 (31.3%) found to harbor aac(6')-Ib-cr, 18 to harbor new aac(6')-Ib mutants, and 74 to harbor wild-type aac(6')-Ib. The genes qnrA, qnrB, and qnrS were found in 2 (0.4%), 6 (1.2%), and 14 (2.7%) of 514 isolates, respectively, with 2 isolates co-harboring qnrB and qnrS genes. Sequencing allowed us to identify qnrA1, qnrB4, qnrB6, and qnrS1 in 20 qnr-positive isolates, with qnrS1 being the most prevalent allele. The genes qnrC and qnrD were not found in any isolates. Interestingly, 35% of qnr-positive isolates and 16.7% of aac(6')-Ib-cr-positive isolates were susceptible to ciprofloxacin. PMQR genes are therefore present in both quinolone-resistant and -susceptible isolates and can also be transferred by conjugation experiments, thus suggesting a likely future increase in quinolone resistance.

[Association of CD14 gene polymorphism with atopic diseases in Chinese Han ethnic group children].

OBJECTIVE: To investigate the distribution characteristics of the single nucleotide polymorphisms (SNPs) of the human CD14 gene in Chinese Han ethnic group children in Wenzhou, and their association with atopic diseases. METHODS: Totally 113 cases were recruited in atopic disease group who met the following criteria: 2 - 12 years old, clinically diagnosed as asthma or allergic rhinitis or atopic dermatitis, elevation of serum total IgE levels and serum specific IgE. Sixty-seven healthy children were enrolled in control group. The related regions of CD14 gene were sequenced to identify and characterize the SNPs, and plasma TIgE and SIgE were detected by immunoassay system and uniCAP system, respectively. The frequency of genotypes and alleles between two groups, as well as the levels of IgE in different genotypes, were compared. RESULTS: CD14/-159 SNP was present in Han ethnic group population of Wenzhou. The frequency of each genotype was 57.0% (TT), 28.0% (TC), 15.0% (CC) in normal children, and 46.9% (TT), 35.4% (TC), 17.7% (CC) in atopic children. No significant difference was found in the distribution of CD14/-159 polymorphism between atopic children and healthy control (chi(2) = 1.918, P > 0.05) according to Hardy-Weinberg principle statistics. There were no significant difference in frequency of each genotype between boys and girls. No significant difference was found in the total plasma IgE levels among groups of TT genotypes [(2520 +/- 460) IU/L], TC genotypes [(2400 +/- 460) IU/L] and CC genotype [(2500 +/- 460) IU/L] (F = 0.807, P > 0.05). CONCLUSION: CD14/-159 SNP is present in Han ethnic group children in Wenzhou, and other SNP in CD14 gene was not found. TT genotype was the primary genotype in CD14/-159 SNP in the children studied. No relationship between CD14/-159 SNP and atopic disease or serum total IgE level was found.