Loss of heterozygosity on chromosome 5 in Iranian esophageal cancer patients.

There is a high incidence of esophageal squamous cell carcinoma (ESCC) in Iran. Non-functionality of some tumor suppressor genes has been reported in esophageal cancer. Loss of heterozygosity on chromosome 5 has also been reported in esophageal carcinomas. We assessed loss of heterozygosity along a region of the long arm of chromosome 5 (5q), from 5q23.1 to 5q23.2, by PCR amplifying DNA fragments of tumor tissues from patients with ESCC and their corresponding normal samples. The PCR products were electrophoresed on 6% non-denaturing polyacrylamide gels, and band intensity was shown by silver staining. Of 40 patients with ESCC, 27, 25 and 36% of informative cases showed allelic losses at microsatellite markers D5S1384, D5S1478 and D5S1505, respectively. Two of the 40 patients studied had microsatellite instability at marker D5S1384. Based on the fact that loss of heterozygosity with more than 22% incidence for a specific marker cannot be regarded as a random event, we add support to previous reports concerning the presence of tumor suppressor genes in this chromosome region and that they affect esophageal cancer development. According to the data in NCBI UniSTS, the PCR product size of human DNA with primers of the D5S1505 marker ranges from 243 to 275 bp, containing about 20 repeats of the TAGA tetranucleotide, while the amplicon size of one allele of one of our cases was 207 bp, with about 10 repeats of the TAGA tetranucleotide, which would be the shortest sequence reported so far.

Nanoparticle-based electrochemical detection of hepatitis B virus using stripping chronopotentiometry.

A selective and sensitive gold nanoparticle-based electrochemical method for detection of hepatitis B virus DNA sequences was used. This method relies on the hybridization of amplified hepatitis B virus DNA strands with probes that are extended on paramagnetic beads. After separation of noncomplementary sequences, hybridized magnetic beads were treated with streptavidin-modified gold followed by silver enhancement. High selectivity and high sensitivity were obtained using electrochemical stripping detection of silver ions that were deposited on gold nanoparticles. With a signal/noise ratio of approximately 4.6, the detection limit was estimated to be 0.7ng/ml.

A natural variant of Bacillus licheniformis alpha-amylase isolated from flour mill wastewaters sheds light on the origin of high thermostability.

AIMS: Understanding the origin of high thermostability exhibited by the alpha-amylase produced by a natural strain of Bacillus licheniformis. METHODS AND RESULTS: The MSH320 alpha-amylase gene has been cloned from a native strain of B. licheniformis isolated from flour mill wastewaters in Kashan, central Iran, and its nucleotide sequence was determined (GenBank Accession Number AF438149). Whereas previously cloned B. licheniformisalpha-amylase (BLA) genes are nearly identical, the MSH320 gene coding sequence presents only 93% identity with the reference 'wild-type' BLA gene, most of the nucleotide changes leading to silent mutations. Amino acid substitutions occurred at 19 of the 483 residues of the matured protein, distributed all along the protein sequence. Nevertheless, the natural BLA variant presents thermoinactivation kinetics similar to that of the reference BLA. Protein modelling and structural predictions at the substitution sites suggest that half of the mutations may have a significant stabilizing or destabilizing effect on the protein structure. Compensatory mutations thus occurred in the natural variant in order to maintain thermostability to the level of the reference enzyme. CONCLUSIONS: The exceptional high thermostability of BLA, although produced by a nonthermophilic organism, is not fortuitous but subject to a selective pressure still at work in natural environments. SIGNIFICANCE AND IMPACT OF THE STUDY: BLA thermal performances are not naturally maximized and can be substantially improved by protein engineering.

Expression and secretion of an alpha-amylase gene from a native strain of Bacillus licheniformis in Escherichia coli by T7 promoter and putative signal peptide of the gene.

The gene encoding a hyperthermostable alpha-amylase from a Bacillus licheniformis native strain was cloned in pET24d transcription vector containing T7 promoter, and expressed in Escherichia coli BL21(DE3) cells. Having confirmed the alpha-amylase activity through activity staining method on SDS-PAGE gel, the yields of production were determined in two separated intra and inter-cellular phases and compared using enzymatic assay methods. Extracellular production of the active recombinant enzyme implies the recognition of the putative signal peptide of this Bacillus sp. by E. coli secretory system. This may be because of the amino acid sequence of this signal peptide which covers all the structural parameters of a standard signal peptide processed by Lep B, the major signal peptidase in E. coli secretory system. This study recommends the use of this signal peptide for extracellular production of other foreign proteins in E. coli.

Neurite outgrowth of dorsal root ganglia is delayed and arrested by aspirin.

In this study, the effect of increasing doses of aspirin on the neurite outgrowth of Dorsal Root Ganglia (DRG) was investigated. DRG were cultured in complete medium (DMEM + 10% FCS +100 ng/ml NGF + collagen Type1 in substratum in 96 multiwell plate) in the presence of concentration of 1.25, 2.5, 5 and 10 mM aspirin. The neurite outgrowth of DRG was followed in comparison with controls that lack aspirin. 10 mM aspirin treated DRG showed delayed neurite outgrowth and after 7 days it reached the same DRG neurite outgrowth control wells after 18 hrs. This growth has delayed approximately one week and showed no further development and in such stage the cells became apoptos. However at concentrations of 1.25, 2.5, 5 mM of aspirin, outgrowth was observed after 18-24 hrs. Although the rate of growth was lower than control, it was not significant. In the other experiment, when DRG cultured for one week in complete medium then treated with aspirin, at 10 mM, DRG neurite outgrowth was stopped, while it was continued in the control. It seem that the aspirin affected DRG became apoptosis.