FTIR biospectroscopy investigation on cisplatin cytotoxicity in three pairs of sensitive and resistant cell line

Fourier Transformed Infrared Spectroscopy [FTIR] has extensively been used for biological applications. Cisplatin is one the most useful antineoplastic chemotherapy drugs for a variety of different human cancers. One of the clinical problems in its application, which would consequently affect the therapeutic outcome of its application, is the occurrence of resistance to this agent. In this project three different pairs of sensitive and resistant cell lines of human ovarian A2780 and its resistant pair of A2780-CP, human ovarian OV2008 and its resistant pair of C13, and finally human lung carcinoma of HTB56 and its resistant pair of HTB56-CP were grown in the laboratory under the standard procedure. Saline was exposed to control cells, whereas 1, 5 and 10 microg/ml of cisplatin was exposed to experimental cells, for one hour. Cells were then collected and lyophilized from which spectra were taken. According to our results, we could not trigger a well-recognized cells biomolecular band at 1015 cm[-1], being modified after exposure to cisplatin in all cell lines. On the other hand, there was a clear dose-dependent increase in protein beta-sheet structure related peaks shift in resistant cell lines after exposure to cisplatin. This would probably indicate an easier protein interaction site for cisplatin in the resistant cell lines, which would probably inhibit cisplatin from binding to DNA, as the cytotoxic target. As a conclusion, FTIR biospectroscopy has proven its potency to identify the interactions, as well as the false engagement cellular sites for cisplatin in sensitive and resistant cell lines

Immunomodulatory effects of bee venom in human synovial fibroblast cell line

As in Iranian traditional medicine, bee venom [BV] is a promising treatment for the rheumatoid arthritis [RA] which is considered as a problematic human chronic inflammatory disease in the present time. Smoking is considered to be a major risk factor in RA onset and severity. The main aim of this study is to investigate the effects of BV on cigarette smoke-induced inflammatory response in fibroblast-like synoviocytes [FLS]. Cytotoxicity of cigarette smoke condensate [CSC] and bee venom were determined by the tetrazolium [MTT] method in cultured synovial fibroblastes. The expression of interleukin-1 beta and sirtuin1 mRNA were analyzed by SYBR green real-time quantitative PCR. Differences between the mean values of treated and untreated groups were assessed by student t-test. Based on MTT assay, CSC and BV did not exert any significant cytotoxic effects up to 40 micro g/mL and 10 micro g/mL, respectively. Our results showed that interleukin-1 beta mRNA level was significantly up-regulated by CSC treatments in LPS-stimulated synoviocytes in a dose-dependent manner. Conversely, the expressions of IL-1 beta and Sirt1 were up-regulated even in lower concentrations of BV and attenuated at higher concentrations. Also, BV attenuated the CSC-induced and LPS-induced inflammatory responses in synovial fibroblasts. Our results support the epidemiological studies indicating pro-inflammatory effects of CSC and anti-inflammatory effects of BV on FLS cell line

Intracellular GSH alterations and its relationship to level of resistance following exposure to cisplatin in cancer cells

One of the major complications in cancer chemotherapy with cisplatin as one of the important medicines in treatment regimens of different cancers is the development of resistance. One of the most described cellular defense mechanisms involved in resistance is glutathione [GSH], thus in this study, the effects of cisplatin on the total intracellular GSH level [GSHi] in some sensitive and resistant variants of human cell lines [hepatocarcinoma HepG2, sking A375, cisplatin sensitive glioblastoma U373MG and cisplatin resistant glioblastoma U373MGCP, cisplatin sensitive ovary A2780S and cisplatin resistant A2780CP cells] were studied. MTT assay was performed to measure cytotoxicity of cisplatin [33.3 microM for 1 hour]. Following cisplatin exposure, GSHi [per million cells] was evaluated using a photometrical assay up to 90 minutes. Our results indicate that there are significant differences between GSHi content of A2780CP and U373MGCP cells compared to other cell lines. Moreover, IC[50] of cisplatin in different cells seems to have a relation with mean of GSH level in 90 minutes [GSH [mean][90]]. As a conclusion, it seems that resistance to cisplatin in different cell lines is more related with the diverse patterns of GSHi variations following cisplatin exposure than its original level, and/or its cellular increase or decrease. It is also suggested that GSH [mean][90] may be used as a factor for the prediction of cellular resistance to cisplatin

FTIR microspectroscopy reveals chemical changes in mice fetus following phenobarbital administration

Phenobarbital is a phenobarbiturate used as a sedative, anticonvulsant or hypnotic with the doses prescribed and can cause teratogenic effects. The goal of this study was to examine an alternative method for the recognition of the mechanism or the bimolecular potential changes in mice fetus caused by Phenobarbital using FTIR micro spectroscopy. The mice were injected with Phenobarbital [120 mg/Kg] on gestation day 9. Fetuses were dissected on day 15 of gestation and morphological and histological studies on the fetus were carried out. Sections [10 microm] of normal and Phenobarbital treated fetus brains and livers were used for FTIR measurement in the wave number region of 400- 4000 cm. The results were shown by 2 derivatization of spectra and also subtracting from control spectra. In liver, the intensity at 1054 cm, 1155 cm, 1353 cm, 1453cm,1645 cm, 1622 cm, 2944 cm, 2913 cm and 2845 cm were shifted and increased. In the brain, the intensity at 879 cm, 911 cm, 955 cm, 1223 cm, 1256 cm, 1304 cm, 1360 cm, 1453 cm, 1529 cm, 1636 cm, 2845 cm, 2915 cm and 2950 cm were increased and shifted. The most important changes of the fetus brain tissue are on the beta structure of proteins due to the amide I bands at 1636 cm, while extensive effects on the DNA structure were obvious for the Phenobarbital treated liver tissues. As a conclusion, FTIR spectroscopy might well be assumed as a potentially powerful teratogenic measurement instrument with a unique ability to identify the modified bimolecular structures

FTIR-microspectroscopy detection of metronidazole teratogenic effects on mice fetus

Metronidazole is used to treat trichomoniasis, bacterial vaginosis, and other diseases. There are controversy aspects about its teratogenicity. A teratogenic agent can alter morphology or subsequent function of the fetus. The aim of this study was to examine an alternative method for the recognition of the mechanism or the bimolecular potential changes in mice fetus caused by Metronidazole using FTIR micro spectroscopy. The mice were injected with metronidazole [60 mg/Kg] on gestation day 9. Fetuses were dissected on day 15 of gestation and morphological and histological studies on the fetus were carried out. Serial sectioning [10 micro m] of normal and metronidazole-treated brains and livers were used for FTIR measurement in the wave number region of 600- 3600 cm[-1].The results showed that there were some variations between the fetus of normal and treated brain and liver. The band intensities in fetus brain and liver of test animals were reduced and shifted at 707 cm[-1], 1155 cm[-1], 1054 cm[-1], 1256 cm[-1] and 1219 cm[-1], 1453 cm[-1] and 1525 cm[-1], 1622 cm[-1], 1645 cm[-1] and 2944 cm[-1],while the band intensities were increased and shifted at 879 cm[-1], 810 cm[-1], 1223 cm[-1], 1256 cm[-1] 1360 cm[-1], 1723 cm[-1]. It was concluded that most of variations in brain and liver of Metronidazole treated fetuses are in amid bands, nucleic acid and carbohydrate related bands. Based on these findings FTIR spectroscopy can be a useful tool for bio diagnostic

Toxicity effect of silver nanoparticles on mice liver primary cell culture and HepG2 cell line

Nano-silver [AgNP] has biological properties which are significant for consumer products, food technology, textiles and medical applications [e.g. wound care products, implantable medical devices, in diagnosis, drug delivery, and imaging]. For their antibacterial activity, silver nanoparticles are largely used in various commercially available products. Thus, the use of nano-silver is becoming more and more widespread in medicine. In this study we investigated the cytotoxic effects of AgNPs on liver primary cells of mice, as well as the human liver HepG[2] cell. Cell viability was examined with MTT assay after HepG[2] cells exposure to AgNPs at 1, 2, 3, 4, 5, 7.5, 10 ppm compared to mice primary liver cells at 1, 10, 50, 100, 150, 200, 400 ppm for 24h. AgNPs caused a concentration-dependent decrease of cell viability in both cells. IC50 value of 2.764 ppm [Micro g/ml] was calculated in HepG[2] cell line and IC[50] value of 121.7 ppm [Micro g/ml] was calculated in primary liver cells of mice. The results of this experiment indicated that silver nanoparticles had cytotoxic effects on HepG[2] cell line and primary liver cells of mice. The results illustrated that nano-silver had 44 times stronger inhibitory effect on the growth of cancerous cells [HepG[2] cell line] compared to the normal cells [primary liver cells of mice]. which might further justify AgNPs as a cytotoxic agents and a potential anticancer candidate which needs further studies in this regard

effects of extending of co-planarity in a series of structurally relative polypyridyl palladium[II] complexes on DNA-binding and cytotoxicity properties

In depth interaction studies between calf thymus deoxyribonucleic acid [CT-DNA] and a series of four structurally relative palladium[II] complexes [Pd[en][HB]][NO[3]][2] [a-d], where en is ethylenediamine and heterocyclic base [HB] is 2, 2›-bipyridine [bpy, a]; 1, 10-phenanthroline [phen, b]; dipyridoquinoxaline [dpq, c] and dipyridophenazine [dppz, d] [Figure 1], were performed. These studies have been investigated by utilizing the electronic absorption spectroscopy, fluorescence spectra and ethidium bromide [EBr] displacement and gel filtration techniques. a-d complexes cooperatively bind and denature the DNA at low concentrations. Their concentration at midpoint of transition, L1/2, follows the order a >> b > c > d. Also the g, the number of binding sites per 1000 nucleotides, follows the order a >> b c > d. EBr and Scatchard experiments for a-d complexes suggest efficient intercalative binding affinity to CT-DNA giving the order: d > c > b > a. Several binding and thermodynamic parameters are also described. The biological activity of these cationic and water soluble palladium complexes were tested against chronic myelogenous leukemia cell line, K562. b, c and d complexes show cytotoxic concentration [Cc[50]] values much lower than cisplatin

Anticancer Activity of Cobra Venom Polypeptide, Cytotoxin-II, against Human Breast Adenocarcinoma Cell Line (MCF-7) via the Induction of Apoptosis / 한국유방암학회지

PURPOSE: Breast cancer is a significant health problem worldwide, accounting for a quarter of all cancer diagnoses in women. Current strategies for breast cancer treatment are not fully effective, and there is substantial interest in the identification of novel anticancer agents especially from natural products including toxins. Cytotoxins are polypeptides found in the venom of cobras and have various physiological effects. In the present study, the anticancer potential of cytotoxin-II against the human breast adenocarcinoma cell line (MCF-7) was investigated. METHODS: The cytotoxic effects of cytotoxin-II were determined by morphological analysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The mode and mechanism of cell death were investigated via acridine orange/ethidium bromide (AO/EtBr) double staining, flow cytometric analysis of cell death, detection of mitochondrial membrane potential, measurement of intracellular reactive oxygen species (ROS), annexin V/propidium iodide staining, and caspase-9 activity assays. RESULTS: The half maximal inhibitory concentration (IC50) of cytotoxin-II in MCF-7 cells was 4.18+/-1.23 microg/mL, while the value for cisplatin was approximately 28.02+/-1.87 microg/mL. Morphological analysis and AO/EtBr double staining showed typical manifestations of apoptotic cell death (in doses lower than 8 microg/mL). Dose- and time-dependent ROS generation, loss of mitochondrial membrane potential, caspase-9 activation, and cell cycle arrest were observed in their respective tests. CONCLUSION: In conclusion, cytotoxin-II has potent anticancer effects in the MCF-7 cell line, which are induced via the intrinsic pathways of apoptosis. Based on these findings, cytotoxin-II is a suitable choice for breast cancer treatment.

[Frequency of sialic acid binding adhesin gene in Helicobacter pylori isolated from patient with gastroduodenal diseases]

Sialic acid binding adhesin gene is one of the most important factors contributing to adhesin of Helicobacter pylori [H. pylori] to epithelial cell layer of stomach. The prevalence rates of sialic acid binding adhesin gene vary in different geographic areas. The aim of this study was to determine the frequency of sialic acid binding adhesin coding gene in the patients with different gastroduodenal diseases. This is a cross-sectional study. One hundred twenty patients with GI symptoms were enrolled in this study. Two gastric biopsy specimens were taken from each of the patients for rapid urease test [RUT] and DNA extraction. Presence of H. pylori was investigated by RUT and urease A gene [ureA] PCR. sialic acid binding adhesin gene was detected by using gene specific primers. Among 120 samples, presence of H. pylori was confirmed in 82 cases, of which 64 strains [78%] were positive for sialic acid binding adhesin gene. The frequency of this gene was 84.6%, 86.7%, 77.8% and 72.2% for gartric cancer, gastric ulcer, duodenal ulcer and gastritis [%66.7] respectively. The frequency of sialic acid binding adhesin gene in different samples was almost the same. Discrepancies in the frequency of this gene in different studies may be related to geographical diversity or use of different primers for detection of this gene

frequency of extended spectrum beta lactamase and CTX M-I of Escherichia coli isolated from the urine tract infection of patients by phenotypic and PCR methods in the city of Khoy in Iran

The production of Extended Spectrum Beta Lactamases [ESBLs] by Escherichia coli is the main cause of resistance to Cephalosporins. In the past decade, CTX-M enzymes have become the most prevalent ESBLs in Europe, Canada, and Asia. In this study, the frequency of ESBL- producing E.coli and molecular detection of the CTX-M-I group was investigated. A total of 400 urine samples were collected from both hospitalized and out-patients in Khoy's hospitals between November 2009 and April 2010. Out of these samples, 188 were identified as E.coli by standard biochemical tests. The antibiotic Susceptibility tests to 10 antibiotics were performed by the-disk-agar diffusion [DAD] method. ESBL production was screened by phenotypic test that including disk diffusion agar and combined disk as recommended by the Clinical and Laboratory Standards Institute [CLSI] Screened isolates were investigated by PCR assay for detection of CTX-M-I group genes. The results show that out of 188 E.coli isolates identified, 56 [29.8%] were producing ESBls by phenotypic test. All isolates were sensitive to imipenem. Overall, 49 [87.5%] isolates were confirmed as CTX-M-I producer by PCR. The results of this study showed that about 30% of the identified E.coli were producing ESBL Therefore, we recommend to use molecular methods in such researches

Cisplatin resistant patterns in ovarian cell line using FTIR and principle component analysis

Cisplatin is a common chemotherapeutic agent that used for treatment of many solid cancers. Rapid identification of chemotherapy resistance is very important and may lead to effective treatment plan. Spectroscopy techniques, such as infrared spectroscopy, which are sensitive to biochemical composition of samples, have shown potentials to discriminate tissues. Developing in Fourier transform infrared [FTIR] as a diagnostic tool support conventional technique in investigating cell phenotype. By this goal three different cell lines, two cisplatin resistant OV2008-DDP [C13] and A2780-CP ovarian cell lines and one cisplatin sensitive A2780 cell line were investigated by FTIR spectroscopy. Data were subjected to principle component analysis [PCA] to obtain FTIR pattern for cisplatin resistance. Using FTIR spectroscopy on these cells in the range of 400-4000 cm[-1] was shown dramatic change in cells. Results shows that Cisplatin resistance pattern is characterized in spectrum with the alteration of conformation in secondary structure of proteins and a shift toward the high wave numbers of CH2 stretching vibration. The FTIR data set between 1000 and 3000 cm[-1] could be consumed as biochemical typicality spectra among resistant and sensitive cell lines while correctly classified by PCA model. Our work supports the promise of PCA analysis of FTIR data as a powerful combined approach for the development of automated methods to recognize resistant to cisplatin in experimental cell lines. One of the advantages of this tool is to investigate the resistant percent of cancer cells .Such technique may bring new tool in cancer diagnosis and stage definition in cancerous tissues

Cytotoxic effects of two Iranian scorpions odontobuthusdoriae and bothutus saulcyi on five human cultured cell lines and fractions of toxic venom

Scorpion venom toxicity is of major concern due to its influence on human activities and public health. We investigated the in-vitro process of cell death caused by two Iranian scorpions Odontobuthus doriae and Bothutus salceyi venom on human cell lines. The aim of this study was to provide further information about triggering cell death and suggestion of methods for the elimination of unwanted cells such as tumor cells. The cytotoxicity and apoptosis induced by effect of scorpion venoms on five established eukaryotic cell lines are analyzed on different human cell lines. All cultured cell lines were incubated with varying doses of scorpion venom for 24 h at 37°C. Control culture was treated with an equal amount of SFM. The percentage of cell survival was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium [MTT] colorimetric assay. Our data demonstrated that Bothutus saulcyi, does not show cytotoxic effect on any of the used cell lines. Odontobuthus doriae, however, has resulted a dose dependent cytotoxic effect with maximum at 1 ug/mL on 1321N1 glioma like cell line. Then the cytotoxic venom of O. doriae was fractionated using Sephadex G50 gel chromatography. The toxic fractions on mouse used to Cytotoxicity assay on 1321 N1 cell line and data demonstrated that, the fraction F3 showed a dose dependent Cytotoxicity assay. Further studies to explode the mode of action of these venoms are recommended and purification of the toxic fraction should be done

Patterns prediction of chemotherapy sensitivity in cancer cell lines using FTIR spectrum, neural network and principal components analysis

Drug resistance enables cancer cells to break away from cytotoxic effect of anticancer drugs. Identification of resistant phenotype is very important because it can lead to effective treatment plan. There is an interest in developing classifying models of resistance phenotype based on the multivariate data. We have investigated a vibrational spectroscopic approach in order to characterize a sensitive human ovarian cell line, A2780, and its cisplatin-resistant derivative, A2780-cp. In this study FTIR method have been evaluated via the use of principal components analysis [PCA], ANN [artificial neuronal network] and LDA [linear discriminate analysis]. FTIR spectroscopy on these cells in the range of 400-4000 cm[-1] showed alteration in the secondary structure of proteins and a CH stretching vibration. We have found that the ANN models correctly classified more than 95% of the cell lines, while the LDA models with the same data sets could classify 85% of cases. In the process of different ranges of spectra, the best classification of data set in the range of 1000-2000 cm[-1] was done using ANN model, while the data set between 2500-3000 cm[-1] was more correctly classified with the LDA model. PCA of the spectral data also provide a good separation for representing the variety of cell line spectra. Our work supports the promise of ANN analysis of FTIR spectrum as a supervised powerful approach and PCA as unsupervised modeling for the development of automated methods to determine the resistant phenotype of cancer classification

Cytotoxicity of diimine palladium [II] complexes of alkyldithiocarbamate derivatives on human lung, ovary and liver cells

Three new Complexes of formula [pd[bpy][R-NH-CSS]] Cl [where bpy is 2/2'- bipyridine, and R-NH-CSS is butylamine, hexylamine- and octyamine-dithiocabamate anion] have been synthesized by University of Sistan and Blachostan. These complexes have been characterized by spectroscopic methods such as ultraviolet-visible, infrared and [1]H-NMR as well as conductivity measurements and chemical analysis. In these complexes, each of the dithiocarbamate ligands coordinates to Pd [II] center as bidentate with two sulfur atoms. We have found a 1:1 electrolyte in water conductivity test for the above mentioned compounds. To measure the biologic activity and potential anticancer efficacy of these compounds, they have been compared with cisplatin and its palladium analogue of [Pd [NH[3]][2] Cl[2]] on three different cell lines of human hepatocarcinoma HepG2, human ovarian carcinoma OV2008, and human lung adenocarcinoma A549. Clonogenic assay has shown LD[50]s in the range of 0.131 +/- 0.025 to 0.934 +/- 0.194 for these compounds on above cell lines. In comparison, cisplatin has shown LD[50]s of 0.838 +/- 0.074, 2.196 +/- 0.220, and 2.799 +/- 0.733 on OV2008, HepG2 and A549 cell lines, respectively. As a conclusion, above three new complexes have shown higher cytotoxicities compared to cisplatin on three different human cell lines. Based on biological tests, these compounds may potentially be considered as good anticancer candidates for further pharmacological studies

[Study of antibacterial properties of adiantum capillus-veneris extract on eight species of gram positive and negative bacteria]

Adiantum capillus-veneris L. is a traditional medicinal plant which was used in the treatment of bronchitis and coughs, and also to prevent hair loss. Methanolic extract of this plant has demonstrated significant antimicrobial activities. In this study the antibacterial properties of Adiantum capillus-veneris L. extract on eight species of Gram positive and negative bacteria were evaluated. The herbal sample of Adiantum capillus-veneris was collected during the summer [June-July] from the north region of Iran called Condoluse and identified by herbarium laboratory in Faculty of Pharmacy, Tehran University of Medical Sciences, where a voucher specimen is deposited. The sample was pretreated and extracted with methanol 96% by percolation method and then concentrated and stored in a safe bottle until the experiments started. By using dilution method different dilutions of the extract have been prepared [10%, 5%, 2.5% and 1.25%]. Antimicrobial activity of the methnolic extract of Adiantum capillus-veneris were evaluated against 8 strain of Gram positive and Gram negative bacteria using agar disk diffusion and agar well plate methods. Our results demonstrated that prepared dilutions of the Adiantum capillus-veneris extract had significant effects on Staphylococcus aureus, Escherichia coli and Helicobacter pylori strains. Also the results indicated no significant inhibitory effects on Salmonella typhi, Shigella sonnei, Pseudomonas aeroginosa, Proteus vulgaris and Streptococcus pyogenes strains. Consistent with the other studies, our investigation demonstrated some antimicrobial effects of Adiantum capillus-veneris extract. With respect to various and multiple chemical properties of this plant, it is suggested that Adiantum capillus-veneris can be used for more medical and therapeutic purposes

Simultaneous detection of caga and cage of helicobacter pylori strains recovered from Iranian patients with different gastroduodenal diseases

To asses the status of two representative genes of cag PAl i.e cagA and cagE of Helicobacter pylori strains infecting Iranian patients suffered from various clinical outcomes using one-step PCR. A total of 120 H. pylori infected patients including non-ulcer dyspepsia, NUD [n=81], peptic ulcer disease, PUD [n=17], and gastric carcinoma, GC [n=22] referred for endoscopy or gastric resection to Amir Alam Hospital or Cancer Institute from 2005 to 2008 were assessed. The status of cagA and cagE genes was determined by gene specific PCR. 84.2% and 90.8% of the tested strains were positive for cagA and cage, respectively. 81.7% strains were positive for both cagA and cagE genes, whereas 8 [6.7%] were found double negative. The prevalence of cagA in GC patients [100%] was slightly higher than PUD patients [94.1%]. All of GC cases were infected with cagA-positive strains. The same distribution pattern was indicated for cagE gene in GC and PUD patients. The cagA-positive strains were significantly associated with GC as compared with NUD [P< 0.05] but this association did not gain statistical significance when cagE gene was assessed. The concurrent detection of cagA/cagE genes allowed rapid and specific clarification of cag PAI status. The strains with cagA/cagE genotype are predominant in Iran regardless of clinical outcome and create a distinct cluster pattern from those in the West and similar to those of East Asian countries. The current study also demonstrated that cagE gene can be explored as a better indication of cag-PAI in Iranian H. pylori strains

Frequency of Helicobacter pylori vacA genotypes in Iranian patients with gastric and duodenal ulcer

Helicobacter pylori infection is a risk factor for developing chronic peptic ulcers and gastric cancer. The purpose of this study was to investigate the frequency of Helicobacter pylori vacA genotypes in patients with gastric and duodenal ulcer. A total of 100 biopsy specimens of patients with gastric [n = 50] and duodenal [n = 50] ulcer were collected. The specimens were cultured on selective media and incubated in a microaerophilic atmosphere at 37°C for 5-10 days. The isolates were characterized to species level by conventional biochemical tests. The extracted DNA from isolates was used to perform a polymerase chain reaction based, simultaneous analysis of the cagA status, allelic variation of the signal regions [s1, s2] and the middle regions [m1, m2] of the vacA gene. H. pylori isolated from 50 specimens of patients and the vacA gene was detected in all isolates. Among vacA genotypes the s1/m1 was the most common in H. pylori isolates from patients with gastric ulcer [56%] and duodenal ulcer [68%]. This study demonstrated that vacA slml is common genotype of H. pylori in patients with peptic ulcer and the vacA allele s1 of this bacterium is associated with ulcer

Prevalence of helicobacter pylori Cag E genotypes, in patients with non-ulcer dyspepsia, peptic ulcer and gastric carcinoma

Helicobacter pylori are a bacterial pathogen evolved to chronically colonize the gastric epithelium and causes gastritis, peptic ulcers, and even gastric malignancies in few infected humans. More recently, a pathogenicity island has been identified within the H. pylori genome that contains a cluster of genes, including cagE. The aim of the current study was to investigate the prevalence of cagE genotypes of H. pylori isolates from patients with NUD[Non Ulcer Dyspepsia], peptic ulcer and cancer. 150 Gastric biopsy specimens obtained from patients were inoculated onto a Brucella Columbia Agar containing 5% sheep for 3 to 5 days at 37°C under micro aerobic conditions [5% O2, 5% CO2, and 90% N2]. After DNA extraction, genotyping of the cagE gene was performed by PCR amplification using the primers. PCR products were separated by 1% agarose gel electrophoresis and examined under UV illumination. Of 92 positive cultures, 34, 28, 20, and 10 isolations were obtained from patients with NUD, duodenal ulcers [DU], gastric ulcers [GU] and gastric cancer [GC], respectively. The frequency of cagE gene was 88/24%, 100%, 85%, 100% and within isolates of patients with NUD, DU, GU and GC, respectively. The presence of cagE in patients with Helicobacter pylori infection is not a marker for predicting or diagnosing the resultant diseases

[Cloning and Sequencing of sacol a novel gene from Staphylococcus aureus]

Natural staphylococcal infections and vaccines based whole bacteria lead to poor antibody responses, but recent research reveals that specific antibodies based on recombinant staphylococcal antigens are much more protective. Sacol is a novel antigen that its structural and immunological traits poorly characterized. This research aimed to clone of sacol, a novel gene from Staphylococcus aureus. The specific primers with suitable restriction sites were designed and sacol amplified by PCR. The sacol and plasmid were produced as sticky ends by restriction enzymes NdeI and XhoI. To amplify the recombinant plasmid the pET21 sacol transferred into competent cell E.coliTOP10. The recombinant plasmid harvested from the host and analyzed by restriction enzymes and sequencing. Finally, sacol gene analyzed by bioinformatics tools. The sacol gene has 723bp which amplified, cloned and sequenced successfully. Sacol is highly conserved in Staphylococcus aureus strains. Moreover, software analysis shows that sacol encodes a protein with 32KDa molecular weight [267 amino acids] which has similarity with C51 peptidase in N-terminal with one alpha helix and 14 beta sheets. The sacol gene is conserved in majority of Staphylococcus aureus strains and may exist and express in most of staphylococcal infections. The role and regulation of the gene is thus of great interest

[Assessment the relationship of cagA gene with different gastroduodenal diseases in Helicobacter pylori infected patients]

The gastric pathogen Helicobacter pylori is introduced as an etiologic agent of gastritis and peptic ulcer and is associated with development of gastric adenocarcinoma. One of the most studied virulence marker of H. pylori is cytotoxin-associated gene A [cagA] with significant geographical heterogeneity around the world. This study was undertaken to assess the status of cagA gene of H .pylori strains infecting Iranian patients suffering from various gastrointestinal diseases and to evaluate the detection of this gene as a screening marker of high-risk patients. In this study, 180 patients [Mean age: 44 years] with upper gastrointestinal manifestations referred for endoscopy to Amir-Alam Hospital or Cancer Institute in Tehran were included. Among one hundred twenty H. pylori infected patients 81, 17 and 22 had non-ulcer dyspepsia [NUD], peptic ulcer disease [PUD], and gastric carcinoma [GC] respectively. Tissue samples were homogenized and incubation was performed up to 5 days. Identification was based on morphology under Gram staining and biochemical tests. The status of conserved region of cagA gene was determined by gene specific PCR. For statistical analysis, chi square test was used. Among the 180 of studied patients, 120 H. pylori strains were isolated. One hundred and one [84.2%] of the tested strains were positive for cagA and the remaining strains [15.8%] were negative. All of gastric cancer cases were infected with cagA -positive strains. The cagA -positive strains were significantly associated with GC as compared with NUD [p < 0.05] but this association did not gain statistical significance for other clinical outcomes. Although the possession of cagA is associated with GC when compared to NUD, due to the uniform distribution of cagA in all other disease categories detection of cagA alone can not be considered as a discriminative marker for a specific clinical outcome. Hence, the study of other virulence determinants and functional characteristics of cagA gene might be necessary for screening high risk patients