The influence of matrix metalloproteinase-2, -9, and -12 promoter polymorphisms on Iranian patients with oesophageal squamous cell carcinoma.

AIM OF THE STUDY: Matrix metalloproteinases (MMPs) are a zinc-dependant endopeptidase family that can degrade extracellular matrix components. Their dysregulation has been proven in several diseases, including cancer. Genetic variations in MMP promoter regions can alter their expression. The aim of the present study is to investigate the correlation of MMP-2 (-1306C/T), MMP-9 (-1562C/T), and MMP-12 (-82A/G) single nucleotide polymorphisms (SNPs) with oesophageal squamous cell carcinoma (ESCC) initiation and progression susceptibility in Iranian patients. MATERIAL AND METHODS: MMP-2 (-1306C/T), MMP-9 (-1562C/T), and MMP-12 (-82A/G) SNPs were detected using polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) technique in 70 patients and 60 healthy controls. The genotypes and allele distributions were statistically compared in patients and controls. The correlation of MMP-2 (-1306C/T) and MMP-9 (-1562C/T) polymorphisms with clinicopathological features were investigated in 53 patients. RESULTS: No statistically significant differences were observed in genotype and allele frequencies of MMP-2 (-1306C/T) and MMP-9 (-1562C/T) between patients and controls (p > 0.05). In addition, no relevance was observed in MMP-2 (-1306C/T) and MMP-9 (-1562C/T) SNPs and clinicopathological features. There was no nucleotide variation in MMP-12 (-82) in the case and control groups. CONCLUSIONS: This study indicates that these three SNPs may have no significant association in ESCC risk in Iranian patients.

No evidence of mammary tumor virus env gene-like sequences among Iranian women with breast cancer.

OBJECTIVE: The mouse mammary tumor virus (MMTV) is the well-established etiological agent of mammary tumors in mice. A series of studies have implicated that a human murine mammary tumor virus-like virus occurs in human breast cancer, but it is unclear whether it has any causal role. METHODS: The aim of the present study was to investigate the presence of MMTV env gene-like sequences in a group of Iranian women with or without breast cancer. A total of 65 breast cancer and 65 noncancerous breast specimens from the Department of Pathology of Tabriz University in East Azerbaijan, Iran, were analyzed by nested PCR. RESULTS: All breast cancer and benign breast samples were negative for MMTV env gene-like DNA. CONCLUSION: These results indicate that the MMTV env gene-like virus may not play a significant role in the etiology of breast cancer among Iranian women.

Purification and characterization of a halophilic α-amylase with increased activity in the presence of organic solvents from the moderately halophilic Nesterenkonia sp. strain F.

An extracellular halophilic α-amylase was purified from Nesterenkonia sp. strain F using 80 % ethanol precipitation and Q-Sepharose anion exchange chromatography. The enzyme showed a single band with an apparent molecular weight of 110 kDa by SDS-PAGE. The amylase exhibited maximal activity at pH 7-7.5, being relatively stable at pH 6.5-7.5. Optimal temperature for the amylase activity and stability was 45 °C. The purified enzyme was highly active in the broad range of NaCl concentrations (0-4 M) with optimal activity at 0.25 M NaCl. The amylase was highly stable in the presence of 3-4 M NaCl. Amylase activity was not influenced by Ca²âº, Rb⁺, Li⁺, Cs⁺, Mg²âº and Hg²âº, whereas Fe³âº, Cu²âº, Zn²âº and Al³âº) strongly inhibited the enzyme activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and ß-mercaptoethanol. K(m) value of the amylase for soluble starch was 6.6 mg/ml. Amylolytic activity of the enzyme was enhanced not only by 20 % of water-immiscible organic solvents but also by acetone, ethanol and chloroform. Higher concentration (50 %) of the water-miscible organic solvents had no significant effect on the amylase activity. To the best of our knowledge, this is the first report on increased activity of a microbial α-amylase in the presence of organic solvents.

Molecular characterization analysis of the outer protein layer (VP7) from human rotavirus A genotype G1 isolate identified in Iran: implications for vaccine development.

The full open reading frame of the outer protein layer VP7 from an isolate of human rotavirus identified in 2010 in an Iranian child admitted to hospital with gastroenteritis was amplified from a clinical stool specimen and subjected to molecular characterization. Genetic and phylogenetic analyses indicated that the analyzed gene falls into the G1 genotype forming a sub-cluster with sequences recently identified in Iran and geographically distant countries. Such results were confirmed by protein sequence alignment, showing a highly conserved "G1-like?? amino acid sequence pattern within the known three main immunodominant regions. These results are extremely relevant in a perspective of vaccine development. Indeed, the present study confirms that the A group G1 genotype is the most prevalent Rotavirus circulating in Iran and supports the development of G1 genotype-based rotavirus vaccine for this country.

Crosstalk of EGF-directed MAPK signalling pathways and its potential role on EGF-induced cell proliferation and COX-2 expression in human mesenchymal stem cells.

Epidermal growth factor (EGF) promotes proliferation in human mesenchymal stem cells (hMSCs) during in vitro propagation. In this study, we investigated the effects of PI3K/AKT, ERK1/2, P38 and JNK on EGF signalling in hMSCs. The effects of EGF on MAPKs and PI3K/AKT crosstalk were investigated by immunoblotting; cyclooxygenase-2 (COX-2) expression was studied by real-time RT-PCR; and cell proliferation was evaluated by methylthiazolyl tetrazolium bromide assay. Our results showed that EGF immediately activated all four pathways, induced proliferation and increased COX-2 expression. Interestingly, inhibition of PI3K/AKT-enhanced EGF-stimulated ERK1/2 activity, and inhibition of ERK1/2 and JNK reduced AKT phosphorylation. Furthermore, EGF-induced proliferation as well as EGF-augmented COX2 expression was hindered by ERK1/2 and p38 inhibitors. The results of this study provide evidences to be used in extended proliferation of hMSCs for cell therapy.

Purification and characterization of an organic-solvent-tolerant halophilic α-amylase from the moderately halophilic Nesterenkonia sp. strain F.

A halophilic α-amylase produced by Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange, and Sephacryl S-200 gel filtration chromatography. The purified amylase exhibited specific activity of 357 unit/mg protein that corresponds to twofold purification. The molecular mass of the amylase was determined to be 57 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography. The optimal pH and temperature for enzyme activity were 6.5 and 45°C, respectively. The amylase was active over a wide range of salt concentrations (0-4 M) with maximum activity at 0.75-1 M NaCl. The α-amylase activity was stimulated by Ca(2+) and inhibited by ethylenediamine tetraacetic acid (EDTA), suggesting that this enzyme is a metalloenzyme. The purified enzyme showed remarkable stability towards surfactants (Tween 20, Tween 80, and Triton X-100), and its activity was increased by ß-mercaptoethanol. The halophilic α-amylase was stable in the presence of various organic solvents such as benzene, chloroform, toluene, and cyclohexane. These properties indicate wide potential applications of this α-amylase in starch-processing industries.

Paramagnetic nanoparticle-based detection of hepatitis B virus using cathodic stripping voltammetry.

A nanoparticle-based electrochemical method for detection of hepatitis B virus DNA sequences has been developed. This method relies on the adsorption of amplified hepatitis B virus DNA strands on to probe-coated paramagnetic particles and electrochemical detection of hybridized strands using a hanging mercury drop electrode. For hepatitis B virus detection, a combination of dynamic DNA hybridization on transportable reactive surfaces and label-free detection of DNA based on the electrochemical determination of adenines was used. Separation of the hybridization area from the detection electrode eliminates non-specific adsorption of long DNAs, and combining this method with dynamic synthesis of probe may result in new flexible approaches for detection of other infectious agents by lab-on-a-chip technology.

Production, optimization and purification of a novel extracellular protease from the moderately halophilic bacterium Halobacillus karajensis.

The production of a protease was investigated under conditions of high salinity by the moderately halophilic bacterium Halobacillus karajensis strain MA-2 in a basal medium containing peptone, beef extract, maltose and NaCl when the culture reached the stationary growth phase. Effect of various temperatures, initial pH, salt and different nutrient sources on protease production revealed that the maximum secretion occurred at 34 degrees C, pH 8.0-8.5, and in the presence of gelatin. Replacement of NaCl by various concentrations of sodium nitrate in the basal medium also increased the protease production. The secreted protease was purified 24-fold with 68% recovery by a simple approach including a combination of acetone precipitation and Q-Sepharose ion exchange chromatography. The enzyme revealed a monomeric structure with a relative molecular mass of 36 kDa by running on SDS-PAGE. Maximum caseinolytic activity of the enzyme was observed at 50 degrees C, pH 9.0 and 0.5 M NaCl, although at higher salinities (up to 3 M) activity still remained. The maximum enzyme activity was obtained at a broad pH range of 8.0-10.0, with 55 and 50% activity remaining at pH 6 and 11, respectively. Moreover, the enzyme activity was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), Pefabloc SC and EDTA; indicating that it probably belongs to the subclass of serine metalloproteases. These findings suggest that the protease secreted by Halobacillus karajensis has a potential for biotechnological applications from its haloalkaline properties point of view.

Increased cytotoxicity of cisplatin in SK-MEL 28 melanoma cells upon down-regulation of melanoma inhibitor of apoptosis protein.

BACKGROUND: Malignant melanoma is a highly metastatic cutaneous cancer and typically refractory to chemotherapy. Deregulated apoptosis has been identified as a major cause of cancer drug resistance, and upregulated expression of the inhibitor of apoptosis protein melanom, an inhibitor of apoptosis (ML-IAP) is frequent in melanoma. METHODS: Based on the conclusion that ML-IAP expression contributes to a malignant phenotype, we down-regulated the ML-IAP mRNA using sequence optimized antisense oligonucleotides. RESULTS: As measured by real-time PCR, oligonucleotides M706 and M711 inhibited ML-IAP mRNA expression by 47% and 52%, respectively in the highly metastatic and drug resistant SK-MEL28 cell line. Oligonucleotide M706, which was previously evaluated in G361 cells as the most efficient inhibitor of ML-IAP expression, was chosen to compare cell viability and drug sensitivity of these two melanoma cell lines with different p53 functionality. Protein expression was reduced by oligonucleotide M706 to 49% of the normal level and resulted in a dose-dependent specific reduction of cell viability with a maximum of 39% at 600 nM. Typical morphological changes showed that loss of viability was mainly due to cell death. In combination experiments, the use of oligonucleotide M706 resulted in a two-fold increase of cisplatin cytotoxicity at different concentrations of oligonucleotide and cisplatin (P<0.05). This is in line with our previous findings in G361 melanoma cell line, in which oligonucleotide M706 caused a 3-fold increase in cisplatin cytotoxicity. CONCLUSION: Our data suggest the use of ML-IAP antisense oligonucleotides to overcome drug resistance in metastatic melanoma, in spite of its p53 status.

Molecular cloning and sequence analysis of a novel zinc-metalloprotease gene from the Salinivibrio sp. strain AF-2004 and its extracellular expression in E. coli.

In this work the first protease gene encoding a novel zinc-metalloprotease from the moderately halophilic bacterium Salinivibrio sp. strain AF-2004 has been cloned, sequenced and reported to the GenBank. We have generated a library containing about 10,000 transformants whose screening yielded one clone harboring plasmid pBluescript with 3.6 kb inserted fragment (pBlueSVP2) with positive caseinolytic activity. Nucleotide sequence analysis of the selected clone revealed a single open reading frame (ORF) of 1833 bp encoding 611 amino acids. The deduced amino acid sequence includes a zinc-metalloprotease HEXXH-E consensus motif which is highly conserved in the M4 family of proteases. The primary amino acid sequence alignment search in the database revealed a moderate homology between the deduced amino acid sequence and the known zinc-metalloproteases including vibriolysin from Vibrio vulnificus and Pseudomonas aeruginosa elastase. The full length of SVP2 gene was subcloned into pQE-80L (pQEVP1) and transformed into Escherichia coli BL21 (DE3) for recombinant overexpression of the protease. Following induction by IPTG, active enzyme was found within cells and in the extracellular medium, where it slowly accumulated to high levels. Mass spectrometric fingerprinting of trypsin digested rSVP2 analysis identified the processed mature protease which starts at Ala-200 of a SVP2 full length protein. Although this result suggested a mature protein of 412 amino acids (44.8 kDa), electrospray-ionisation mass spectrometry revealed that the molecular mass of purified rSVP2 was only 34.2 kDa, which indicates a further cleavage site at the C-terminal.

Kinetic study of cytokines production by human peripheral blood mononuclear cells in response to Brucella DNA.

In spite of reports on cytokines induction by the Brucella DNA in murine model, there is no comparison between pathogenic and appropriate vaccine strains in human. We investigated the cytokines profile of human peripheral blood mononuclear cells (PBMCs) induced by DNA extracted from pathogenic isolates of Brucella melitensis and B. abortus as well as Rev1 and S19; the appropriate vaccine strains. It was observed that despite differential induction of Interleukin(IL)-12 and IL-10 production, identical IL-12/IL-10 concentration ratio was obtained by all Brucella strains DNAs that was 2 after 24 h and 4 after 5 days of incubation. In addition, IL-2 and Interferon(IFN)-gamma production were profoundly increased compared to the medium at day 3 and 5 respectively but IFN-alpha was not induced. Therefore, Brucella strains DNAs are Th1 inducing component with similar pattern in human PBMCs.

Relationship between lung cancer and human papillomavirus in north of Iran, Mazandaran province.

Lung cancer is a major health problem and the leading cause of cancer deaths in the world. The pathogenesis of lung cancer is complex, and is believed to be due to the interaction between environmental and genetic factors. Various evidences show that HPV might be involved in bronchial carcinogenesis. In this study, 141 lung cancer patients and 92 non-cancer control subjects were enrolled to examine whether HPV DNA existed in lung tumor and normal tissues in Mazandaran, north part of Iran by nested PCR. Our data showed that 33 of 129 lung tumors had HPV DNA compared with 8 of 90 non-cancer control subjects (25.6% vs. 9.0%, P=0.002). The infection of HPV had an OR of 3.48 (95% CI 1.522-7.958; P=0.002). Meanwhile infection of high risk HPV types (16 and 18) had a significantly high OR of lung cancer incidence as 8.00 (95% CI 1.425-44.920; P=0.021) compared with 4.423 (95% CI 2.407-8.126; P0.0001) of smoking status. This result suggests that HPV infection is associated with lung cancer development in Mazandaran, Iran.

An increased lung cancer risk associated with codon 72 polymorphism in the TP53 gene and human papillomavirus infection in Mazandaran province, Iran.

The TP53 gene has a polymorphism in exon 4 at codon 72 that presents the arginine or proline genotype. The association of TP53 codon 72 polymorphism with lung cancer risk has been studied by several groups, although with inconsistent results. Our previous study showed that the human papillomavirus (HPV) infection is associated with the development of lung cancer in Mazandaran, north part of Iran (cases=25.6% versus controls=9.0%, P=0.002). The frequency of TP53 codon 72 polymorphism was studied in a north part Iranian group of 92 healthy controls and 141 lung cancer patients. The allelic distribution of the three genotypes (ArgArg, ArgPro, ProPro) in healthy normal controls was 46.1, 32.6 and 21.3%, respectively, which differs from that of lung cancer patients showing genotype frequency as 42.6, 49.6 and 7.8%. A relation between the presence of the Arg allele and lung cancer risk was observed. Our study reveals that Arg allele, active smoking and HPV infection are the important risk factors in lung cancer development in the north part of Iran, Mazandaran province.

Identification of squamous cell carcinoma associated proteins by proteomics and loss of beta tropomyosin expression in esophageal cancer.

AIM: To assess the proteome of normal versus tumor tissue in squamous cell carcinoma of the esophagus (SCCE) in Iranian patients and compare our results with former reports by using proteomics. METHODS: Protein was extracted from normal and tumor tissues. Two dimensional electrophoresis was carried out and spots with differential expression were identified with mass spectrometry. RNA extraction and RT-PCR along with immunodetection were performed. RESULTS: Fourteen proteins were found whose expression levels differed in tumor compared to normal tissues. Mass spectrometric analysis resulted in the identification of beta-tropomyosin (TMbeta), myosin light chain 2 (and its isoform), myosin regulatory light chain 2, peroxyredoxin 2, annexin I and an unknown polypeptide as the down regulated polypeptides in tumor tissue. Heat shock protein 70 (HSP70), TPM4-ALK fusion oncoprotein 2, myosin light polypeptide 6, keratin I, GH16431p and calreticulin were the up-regulated polypeptides found in tumor tissue. Several of these proteins, such as TMbeta, HSP70, annexin I, calreticulin, TPM4-ALK and isoforms of myosins, have been well recognized in tumorigenesis of esophageal or other types of cancers. CONCLUSION: Our study not only supports the involvement of some of the formerly reported proteins in SCCE but also introduces additional proteins found to be lost in SCCE, including TMbeta.

Differential gene expression between squamous cell carcinoma of esophageus and its normal epithelium; altered pattern of mal, akr1c2, and rab11a expression.

AIM: To identify the altered gene expression patterns in squamous cell carcinoma of esophagus (ESCC) in relation to adjacent normal esophageal epithelium. METHODS: Total RNA was extracted using SV total RNA isolation kit from snap frozen tissues of ESCC samples and normal esophageal epithelium far from the tumor. Radio-labeled cDNA were synthesized from equal quantities of total RNAs of tumor and normal tissues using combinations of 24 arbitrary 13-mer primers and three different anchoring oligo-dT primers and separated on sequencing gels. cDNA with considerable different amounts of signals in tumor and normal tissue were reamplified and cloned. Using southern blot, the clones of each band were controlled for false positive results caused by probable heterogeneity of cDNA population with the same size. Clones that confirmed differential expression by slot blot selected for sequencing and northern analysis. Corresponding full-length gene sequences was predicted using human genome project data, related transcripts were translated and used for various protein/motif searches to speculate their probable functions. RESULTS: The 97 genes showed different levels of cDNA in tumor and normal tissues of esophagus. The expression of mal gene was remarkably down regulated in all 10 surveyed tumor tissues. Akr1c2, a member of the aldo-keto reductase 1C family, which is involved in metabolism of sex hormones and xenobiotics, was up-regulated in 8 out of 10 inspected ESCC samples. Rab11a, RPL7, and RPL28 showed moderate levels of differential expression. Many other cDNAs remained to further studies. CONCLUSION: The mal gene which is switched-off in all ESCC samples can be considered as a tumor suppressor gene that more studies in its regulation may lead to valuable explanations in ESCC development. Akr1c2 which is up-regulated in ESCC probably plays an important role in tumor development of esophagus and may be proposed as a potential molecular target in ESCC treatments. Differential display technique in spite of many disadvantages is still a valuable technique in gene function exploration studies to find new candidates for improved ones like gene chips.

No detection of 'high-risk' human papillomaviruses in a group of Iranian women with breast cancer.

The presence of viral DNA in breast cancer cells is controversial. However, some studies have revealed a possible role for the human papillomavirus in the pathogenesis of breast cancer. The aim of the present study was to investigate the presence of HPV-DNA in breast tissue in a group of Iranian women with and without breast cancer and identification of the detected HPV types. Paraffin-embedded specimens from 65 malignant breast cancer cases and 65 cases with benign breast lesions were investigated for presence of HPV-DNA by nested polymerase chain reaction. We found HPV-DNA in 22 (33.8%) of the breast cancer specimens. All non-cancerous specimens were negative. Low and high-risk HPV types, including HPV-6 (26.2%), HPV-16 (1.5%), HPV-35 (1.5%), HPV-52 (1.5%), and HPV-11 (1.5%) were detected in our study. HPV-6 was the most prevalent type in the breast cancer specimens. Although high-risk HPV types have been shown to have a major role in cervix cancer, there have been no data that support the same relevance for other types of malignancies. Furthermore, presence of low-risk HPV types in malignancies still is a matter of debate. The data presented in this study indicates a strong need for epidemiological studies correlating different HPV types in human breast cancer.

Effects of paraoxonase 1 activity and gene polymorphisms on long-term pulmonary complications of sulfur mustard-exposed veterans.

Sulfur mustard (SM) is an alkylating agent with prolonged adverse effects. The antioxidant paraoxonase 1 (PON1), an endogenous free radical scavenger, plays a protective role against oxidative stress. The possible roles of oxidative stress in the pathogenesis of SM, together with the antioxidant activity of PON1, are enough to warrant the analysis of PON1 polymorphisms and allelic variants in incapacitated veterans. PON1 55 L/M and 192 Q/R polymorphisms were assayed in 289 male veterans with severe pulmonary conditions, who were exposed to SM 20-25 years ago, and 66 gender-, age- and ethnic-matched healthy controls. As we showed previously the PON1 activity decreased significantly in veterans. However, PON1 55 L/M and 192 Q/R genotype distributions were not significantly different between the veterans and the controls. R and L allele carriers have also significantly higher basal and salt-stimulated PON1 activity than Q and M allele carriers. Paraoxonase and arylesterase activities in individuals with the QQ+(MM or LM) genotype were significantly lower than those with the (RR or QR)+LL genotype. Furthermore, basal and salt-stimulated paraoxonase activity in veterans with the (RR or QR)+LL genotype was significantly lower than that in the controls. A positive correlation has been determined between serum PON1 activity and pulmonary function test in QR/LL genotypes. Some of the veterans with RR+QR genotypes have also shown a novel missense change of Asn227Ser in exon 6 of the enzyme. This substitution is close to the binding domain of PON1 and so modifies enzyme activity.

Molecular epidemiology of human herpesvirus 8 variants in Kaposi's sarcoma from Iranian patients.

Kaposi's sarcoma (KS) is a rare cancer in Iran and there is no epidemiological and molecular information about HHV-8 variants circulating among the Iranian population. In this study HHV-8 sequences have been analyzed in 43 cutaneous KS biopsies from Iranian patients mainly affected by classic KS. DNA samples were subjected to PCR amplification of HHV-8 ORF26, T0.7 and K1 followed by direct nucleotide sequencing and phylogenetic analysis. The analysis of ORF26 showed that 30 (69.8%) and 13 (30.2%) samples belonged to subtypes A/C and K, respectively. In general, the clustering of HHV-8 T0.7 variants paralleled that of ORF26. Genotyping of K1 sequences showed that the majority of samples (39 out of 41) fall into the large C clade with only 2 belonging to the A clade. In conclusion, HHV-8 variants identified among classic Iranian KS are largely related to Eurasian genotypes previously identified in KS from Mediterranean, Middle East, and East Asian regions.

Biodesulfurization of dibenzothiophene by a newly isolated Rhodococcus erythropolis strain.

A new dibenzothiophene (DBT) desulfurizing bacterium was isolated from oil-contaminated soils in Iran. HPLC analysis and PCR-based detection of the presence of the DBT desulfurization genes (dszA, dszB and dszC) indicate that this strain converts DBT to 2-hydroxybiphenyl (2-HBP) via the 4S pathway. The strain, identified as Rhodococcus erythropolis SHT87, can utilize DBT, dibenzothiophene sulfone, thiophene, 2-methylthiophene and dimethylsulfoxide as a sole sulfur source for growth at 30 degrees C. The maximum specific desulfurization activity of strain SHT87 resting cells in aqueous and biphasic organic-aqueous systems at 30 degrees C was determined to be 0.36 and 0.47 micromol 2-HBP min(-1) (gdrycell)(-1), respectively. Three mM DBT was completely metabolized by SHT87 resting cells in the aqueous and biphasic systems within 10h. The rate and the extent of the desulfurization reaction by strain SHT87 suggest that this strain can be used for the biodesulfurization of diesel oils.

Mutations of RAS gene family in specimens of bladder cancer.

INTRODUCTION: Studies have shown different types of RAS mutations in human bladder tumors with a wide range of mutation frequencies in different patient populations. This study aimed to assess the frequency of specific-point mutations in the RAS gene family of a group of Iranian patients with bladder cancer. MATERIALS AND METHODS: We examined the tumor specimens of 35 consecutive patients with transitional cell carcinoma. The DNA samples were evaluated for the occurrence of HRAS, KRAS, and NRAS activation using a polymerase chain reaction-restriction fragment length polymorphism technique. RESULTS: None of the patients had mutations in the RAS gene family "hot spots" including codons 12, 13, and 61. CONCLUSION: We failed to find RAS mutations in our bladder tumor samples. These observations may reflect the involvement of different etiological factors in the induction of bladder tumor of which RAS mutation might not be present in all populations.