Hesham Saeed

CYP1A1, CYP2E1, and GSTM1 Gene Polymorphisms and Susceptibility to Colorectal and Gastric Cancer Among Lebanese

Mutations in the genes encoding enzymes involved in the metabolism of chemical carcinogens can significantly affect the risk of cell transformation and cancer development. The resident Lebanese population has experienced a sharp increase in cancer incidence within the last few years. The relationship between gene polymorphisms of metabolic enzymes and gastrointestinal (GI) cancer incidence was not previously investigated. The aim of this study was to investigate the relationship between CYP1A1, CYP2E1, and GSTM1 gene polymorphisms and GI cancer incidence among Lebanese. Blood and/or paraffin-embedded biopsy samples were collected from patients and healthy controls. The genotypes were determined by polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism. The results of the present case-control study show that the studied Lebanese population generally resembles Caucasian populations with respect to the considered polymorphisms. Further, the GSTM1*0/*0 genotype is a significant risk factor for gastric (odds ratio = 4.1; 95% confidence interval: 1.2-14.5) and colorectal cancers (odds ratio = 3.8; 95% confidence interval: 1.7-8.5); on the other hand, CYP1A1*2A and CYP2E1*6 alone are not significantly associated with GI cancer development, although CYP1A1*2A was more frequent among patients. A remarkable and statistically significant 36.5-fold increase in the risk of gastric cancer was observed among patients with CYP1A1*2A/*2A combined with GSTM1*0/*0. The investigation of genetic risk factors and susceptibility gene polymorphisms in Lebanese is helpful for better understanding of GI cancer etiology.

Biodegradation of native feather keratin by Bacillus subtilis recombinant strains.

A mixed culture containing two recombinant Bacillus subtilis strains; was used to hydrolyze 1% chicken feather; both were previously transformed with late-expressed and early expressed alkaline protease (aprE) carrying plasmids pS1 and p5.2, respectively. Proteolytic and keratinolytic activities of the mixed culture increased in parallel with those of the culture of B. subtilis DB100 (p5.2), and both were higher than that of B. subtilis (pS1) cultures. On the other hand, data indicated that degradation of feather by the recombinant strains B. subtilis DB100 (p5.2), was greatly enhanced when using a previously optimized medium. High levels of free amino groups as well as soluble proteins were also obtained. The concentration of amino acids was considerably increased during the fermentation process. It was found that, the amino acids Phe, Gly and Tyr were the major amino acids liberated in the cultures initiated by both strains. Results render these recombinant strains suitable for application in feather biodegradation large scale processes.

Enhanced production of lipase by the thermophilic Geobacillus stearothermophilus strain-5 using statistical experimental designs

Statistically based experimental designs were applied to optimize the cultural conditions for the production of a glycerol-inducible lipase from the thermophilic Geobacillus stearothermophilus strain-5. The effect of nineteen culture conditions on enzyme production was evaluated using Plackett-Burman factorial design. Tween 80, K(2)HPO(4), glycerol and glucose were the most significant factors in improving enzyme production. The selected parameters were then further investigated using central composite design to define the optimal process conditions. Maximal enzyme activity (578 U/ml) was reached under the following conditions: glycerol, 2.24% (v/v); Tween 80, 0.76% (v/v); glucose, 0.76% (w/v) and K(2)HPO(4), 0.38% (w/v) which is about five folds the activity in basal medium. A verification experiment was carried out to examine model validation and revealed more than 98% validity.

Evidence for the presence of active cytochrome P450 systems in Schistosoma mansoni and Schistosoma haematobium adult worms

Extracts of the adult worms of both Schistosoma mansoni and Schistosoma haematobium can metabolise some typical P450 substrates but to differing degrees. S. mansoni worm extracts displayed a ∼12‐fold higher specific activity for an aminopyrine substrate than rat liver microsomes. At 4 mM substrate concentration the demethylation reaction with N‐nitrosodimethylamine (NDMA) (5 nmol HCHO/mg protein/min) was only half that of rat liver microsomes, whereas in extracts of S. haematobium, no detectable activity was found towards NDMA. Using ethylmorphine as substrate the demethylation activity of S. mansoni extracts (1.82 nmol HCHO/mg protein/min) was 5.5‐fold lower than that of rat liver microsomes. Benzphetamine demethylase activity was also readily detectable in S. mansoni worm extracts at 6.79 nmol HCHO/mg protein/min compared with 10.20 nmol HCHO/mg protein/min in the case of rat liver microsomes. When aniline was used as substrate, surprisingly, no activity was found in worm extracts of either S. mansoni or S. haematobium, whereas rat liver microsomes showed high activity towards this amine. The anti‐P450 2E1 and 2B1/2 cross‐reacted with both worm homogenates and gave a specific band corresponding to a protein of molecular weight of ∼50.0 kDa. A study with anti‐P450 IVA antibody revealed that while this protein was strongly expressed in S. haematobium worm extracts, no immunoreactivity was observed with extracts of S. mansoni. Immunoblotting analyses with anti‐P450 IIIA and P450 1A1 did not detect immunoreactive protein in either S. mansoni or S. haematobium.

Associations between Single Nucleotide Polymorphisms of COX-2 and MMP-2 Genes and Colorectal Cancer Susceptibility in the Saudi Population

BACKGROUND It has been reported that COX-2 expression is associated with MMP-2 expression in thyroid and breast cancers, suggesting that MMPs are linked to COX-2-mediated carcinogenesis. Several polymorphisms within the MMP2 promoter region have been reported in cases with oncogenesis and tumor progression, especially in colorectal carcinogenesis. MATERIALS AND METHODS This research evaluated risk of association of the SNPs, including genes for COX-2 (A/G transition at +202) and MMP-2 (C/T transition at-1306), with colorectal cancer in 125 patients and 125 healthy controls. RESULTS AND CONCLUSIONS Our data confirmed that MMP2 C-1306 T mutations were significantly more common in colon cancer patients than in our control Saudi population; p=0.0121. On the other hand in our study, there was no significant association between genotype distribution of the COX2 polymorphism and colorectal cancer; p=0.847. An elevated frequency of the mutated genotype in the control group as compared to the patients subjects indeed suggested that this polymorphism could decrease risk in the Saudi population. Our study confirmed that the polymorphisms that could affect the expressions of MMP-2 and COX-2 the colon cancer patients were significantly higher than that in the COX-2 negative group. The frequency of individuals with MMP2 polymorphisms in colon cancer patients was higher than individuals with combination of COX2 and MMP2 polymorphisms. Our study confirmed that individuals who carried the polymorphisms that could affect the expressions of COX2 are more susceptible to colon cancer. MMP2 regulatory polymorphisms could be considered as protective; further studies need to confirm the results with more samples and healthy subjects.

Matrix Metalloproteinase-2 (-1306 C>T) Promoter Polymorphism and Risk of Colorectal Cancer in the Saudi Population

BACKGROUND Matrix metalloproteinase-2 (MMP-2) is an enzyme with proteolytic activity against matrix proteins, particularly basement membrane constituents. A single nucleotide polymorphism (SNP) at -1306, which disrupts a Sp1-type promoter site (CCACC box), results in strikingly lower promoter activity with the T allele. In the present study, we investigated whether this MMP-2 genetic polymorphism might be associated with susceptibility to colorectal cancer (CRC) in the Saudi population. We also analyzed MMP-2 gene expression level sin CRC patients and 4 different cancer cell lines. MATERIALS AND METHODS TaqMan allele discrimination assays and DNA sequencing techniques were used to investigate the C-1306T SNP in the MMP-2 gene of Saudi colorectal cancer patients and controls. The MMP-2 gene expression level was also determined in 12 colon cancer tissue samples collected from unrelated patients and histologically normal tissues distant from tumor margins. RESULTS AND CONCLUSIONS The MMP-2 C-1306T SNP in the promoter region was associated with CRC in our Saudi population and the MMP-2 gene expression level was found to be 10 times higher in CRC patients. The MMP-2 C-1306T SNP is significantly associated with CRC in the Saudi population and this finding suggested that MMP-2 variants might help predict CRC progression risk among Saudis. We propose that analysis of this gene polymorphism could assist in identification of patient subgroups at risk of a poor disease outcome.

Cytochrome P450 1A1, 2E1 and GSTM1 Gene Polymorphisms and Susceptibility to Colorectal Cancer in the Saudi Population

BACKGROUND The Saudi population has experienced a sharp increase in colorectal and gastric cancer incidences within the last few years. The relationship between gene polymorphisms of xenobiotic metabolizing enzymes and colorectal cancer (CRC) incidence has not previously investigated among the Saudi population. The aim of the present study was to investigate contributions of CYP1A1, CYP2E1, and GSTM1 gene polymorphisms. MATERIALS AND METHODS Blood samples were collected from CRC patients and healthy controls and genotypes were determined by polymerase chain reaction restriction fragment length polymorphism and sequencing. RESULTS AND CONCLUSIONS CYP2E1*6 was not significantly associated with CRC development (odd ratio=1.29; confidence interval 0.68-2.45). A remarkable and statistically significant association was observed among patients with CYP1Awt/*2A (odd ratio=3.65; 95% confidence interval 1.39-9.57). The GSTM1*0/*0 genotype was found in 2% of CRC patients under investigation. The levels of CYP1A1, CYP2E1 and GSTM1 mRNA gene expression were found to be 4, 4.2 and 4.8 fold, respectively, by quantitative real time PCR. The results of the present case-control study show that the studied Saudi population resembles Caucasians with respect to the considered polymorphisms. Investigation of genetic risk factors and susceptibility gene polymorphisms in our Saudi population should be helpful for better understanding of CRC etiology.

Expression of alkaline proteinase gene in two recombinant Bacillus cereus feather-degrading strains

Two Bacillus cereus feather-degrading strains (23/1 and 6/2) were transformed using a recombinant plasmid p5.2 carrying the alkaline proteinase gene (aprE). A high level of the aprE gene expression was observed when the recombinant strains were grown on sporulation medium. The expression of the aprE gene proceeded during the early stationary phase and the p5.2 plasmid was segregationally and structurally stable in both strains. The two recombinant strains grown on a mineral medium with 1 % chicken feather as source of energy, carbon and nitrogen exhibited higher proteolytic activity (≈6-fold and 2.4-fold higher for strains 23/1 (p5.2) and 6/2 (p5.2), respectively. Keratinolytic activity increased ≈3.5-fold and 4.15-fold, respectively. The keratinolytic activity further increased when an optimized medium with yeast extract and corn oil was used. Considerable amounts of free amino acids were obtained after the biodegradation of feather which makes the new strains promising for application in feather-waste treatment to, e.g., transformation to animal feedstuff.

Molecular cloning, structural modeling and production of recombinant Aspergillus terreus l. asparaginase in Escherichia coli

l-Asparaginase (EC 3.5.1.1) is an important medical enzyme that catalysis the hydrolysis of l-asparagine to aspartic acid and ammonium. For over four decades l. asparaginase utic agent for the treatment of a variety of lymphoproliferative disorders and lymphoma such as acute lymphoblastic leukemia. In the present study A. terreus full length l. asparaginase gene, 1179bp was optimized for expression in Escherichia coli BL21 (DE3) pLysS. The full length A. terreusl. asparaginase gene encoding a protein of 376 amino acids with estimated molecular weight of 42.0kDa and a theoretical isoelectric point (pI) of 5.0. BLAST and phylogeny analysis revealed that the A. terreusl. asparaginase shared high similarity with other known fungal l. asparaginase (75% homology with A. nomius and 71% with A. nidulans). The recombinant protein was overexpressed in the form of amorphous submicron proteinaceous inclusion bodies upon induction with 1mM IPTG at 37°C for 18h.

The Arabian camel Camelus dromedarius heat shock protein 90α: cDNA cloning, characterization and expression

Heat shock protein 90 (Hsp90) is a highly conserved ubiquitous molecular chaperone contributing to assisting folding, maintenance of structural integrity and proper regulation of a subset of cytosolic proteins. In the present study, a heat shock protein 90α full length coding cDNA was isolated and cloned from the Arabian one-humped camel by reverse transcription polymerase chain reaction (RT-PCR). The full length cDNA sequence was submitted to NCBI GeneBank under the accession number KF612338. The sequence analysis of the Arabian camel Hsp90α cDNA showed 2202bp encoding a protein of 733 amino acids with estimated molecular mass of 84.827kDa and theoretical isoelectric point (pI) of 5.31. Blast search analysis revealed that the C. dromedarius Hsp90α shared high similarity with other known Hsp90α. Comparative analyses of camel Hsp90α protein sequence with other mammalian Hsp90s showed high identity (85-94%). Heterologous expression of camel Hsp90α cDNA in E. coli JM109 (DE3) gave a fusion protein band of 86.0kDa after induction with IPTG for 4h.