A Prof. Gad | Taif University, Kingdom of Saudi Arabia (original) (raw)

Papers by A Prof. Gad

Oncogene, 2006

p21-activated kinase 1 (PAK1) is a mediator of downstream signaling from the small GTPases Rac an... more p21-activated kinase 1 (PAK1) is a mediator of downstream signaling from the small GTPases Rac and Cdc42. In its inactive state, PAK1 forms a homodimer where two kinases inhibit each other in trans. The kinase inhibitory domain (KID) of one molecule of PAK1 binds to the kinase domain of its counterpart and keeps it inactive. Therefore, the isolated KID of PAK1 has been widely used to specifically inhibit and study PAK function. Here, we show that the isolated KID induced a cell cycle arrest with accumulation of cells in the G1 phase of the cell cycle with an inhibition of cyclin D1 and D2 expression. This cell cycle arrest required the intact KID and was also induced by a mutated KID unable to block PAK1 kinase activity. Furthermore, the KID-induced cell cycle arrest could not be rescued by the expression of a constitutively active PAK1-T423E mutant, concluding that this arrest occurs independently of PAK1 kinase activity. Our results suggest that PAK1 through its KID inhibits cyclin D expression and thereby enforces a cell cycle arrest. Our results also call for serious precaution in the use of KID to study PAK function.

IIUM Engineering Journal, 1970

Phytase gene obtained from Bacillus subtilis ASUIA243 was cloned into a medium vector and transfo... more Phytase gene obtained from Bacillus subtilis ASUIA243 was cloned into a medium vector and transformed into E. coli. Restriction enzyme digestion was conducted to get blunt-ended phytase gene and ligated into the Pichia expression vector, pPICZαA. The recombinant vector, pPICZαA-243HPp was then linearized with PmeI and transformed into P. pastoris strain X33. Screening for multi copy gene number of transformants was done by re-plating the selected colonies on increasing concentration of zeocin. One positive clone, X243HPp#2 was then grown in BMGY media as the starting culture, followed by induction in BMMY media for protein expression study. The supernatant was then analysed by SDS-PAGE and Western blot method to check the protein expression.ABSTRAK: Gen fitase yang didapati daripada Bacillus subtilis ASUIA243 diklonkan sebagai vektor perantara dan berubah menjadi E. coli. Sekatan pencernaan enzim dijalankan untuk mendapatkan gen fitase berhujung tumpul dan diligatkan dengan vektor e...

British Journal of Cancer, 1969

The Egyptian Journal of Remote Sensing and Space Science, 2010

Acta cardiologica, Jan 1, 2010

A collaborative trial study has been conducted for validation of an extraction method and a subse... more A collaborative trial study has been conducted for validation of an extraction method and a subsequent PCR for the detection of transgenic rice sold in Saudi Arabia. The tests were carried out in Saudi Arabia using Real-Time PCR and the positive samples were validated in another lab in Malaysia using PCR and agarose gel visualization. The samples were tested for the existence of the NOS Terminator. A total of 150 samples were tested out of which three samples tested positive as GM-rice which were retested in Malaysia. The presence of GMO rice in Saudi Arabia supports the necessity of developing precise quantitative and qualitative ways for routine analyses and detection of GMO products in the Saudi Arabian market. With the discovery of GM products in the Saudi Arabian market it would be of no surprise that other Middle Eastern nations also knowingly or unknowingly import GM crops.

Phytases catalyze the hydrolysis of phosphomonoester bonds in phytate, thereby releasing lower fo... more Phytases catalyze the hydrolysis of phosphomonoester bonds in phytate, thereby releasing lower forms of myo-inositol phosphates and inorganic phosphate. Phytase enzyme preparations have a wide range of applications in animal and human nutrition. The addition of phytate-degrading enzyme can improve the nutritional value of plant-based foods by enhancing protein digestibility and mineral availability through phytate hydrolysis during digestion in stomach or during food processing. 30 strains of potential phytate-degrading enzymes bacteria isolated from Malaysian maize plantation were cultivated in Luria Bertani (LB) and Luria Bertani + Rice Bran (LBRB) media for 5 days and were analyzed for phytase activity. The 6 strains with highest activity were chosen for species identification. Two set of broad-range 16S rRNA PCR primers were used for genotypic identification. ASUIA279 was the strain that had has the highest phytase activity in LBRB followed by ASUIA271, ASUIA138, ASUIA260, ASUIA243 and ASUIA30. The genotypic technique revealed Pantoea stewartii ASUIA271, Enterobacter sakazakii ASUIA279, Bacillus cereus ASUIA260, Bacillus subtilis ASUIA243, P. stewartii ASUIA138 and B. cereus ASUIA30.

The aims of the study were to observe the effects of different concentration of rice bran in diff... more The aims of the study were to observe the effects of different concentration of rice bran in different media on phytase synthesis and to optimize the temperature and pH of the media for phytase production by those bacterial strains. Three bacterial strain isolates obtained from the soil of Malaysian maize plantation were used to produce phytase. In this study, the effects of different rice bran concentration, incubation temperature and initial pH-values of the media on phytase production were evaluated. Incorporation of 7.5% rice bran has the inducible effect on all the bacterial tested. In respect to phytase production, the best cultivation media and cultivation time for Bacillus cereus ASUIA260 was PFE with 7.5% rice bran after 3 days, whilst for Pantoea stewartii ASUIA271 and Enterobacter sakazakii ASUIA279, it was LB with 7.5% rice bran after 3 days and 5 days, respectively. The arrangement of those isolates according to their ability to produce phytases were E. sakazakii ASUIA279 > P. stewartii ASUIA271 > B. cereus ASUIA260. Production of phytase by those bacteria was triggered by the high content of organic phytate in the rice bran. Optimum temperature for phytase production of B. cereus ASUIA260 was 41 ºC compared to P. stewartii ASUIA271 and E. sakazakii ASUIA279 with 33 ºC and 37 ºC, respectively. Optimum initial pH for phytase production of B. cereus ASUIA 260 was pH 7.2, while P. stewartii ASUIA271 and E. sakazakii ASUIA 279 were both at pH 6.0.

Phytate or myo-inositol hexakisphosphates (IP 6 ) is widely distributed in plants like rice bran.... more Phytate or myo-inositol hexakisphosphates (IP 6 ) is widely distributed in plants like rice bran. The production of myo-inositol phosphate intermediates has received much attention due to the remarkable potential health benefits offered by the compounds. In this study, the cytotoxicity of the partially purified myo-inositol phosphate fractions and commercial IP 1 and IP 6 were investigated against MCF-7 breast cancer cell lines. The study showed that the commercial standard IP 1 and IP 6 showed good inhibition towards the MCF-7 cell line. The MCF-7 cells growth was inhibited in minimum concentration of myo-inositol phosphates (<1000 µg/ml). However, no inhibition observed on the MCF-7 cell line by the myo-inositol phosphates fractions partially purified from rice bran at concentration <1000 µg/ml. The inhibition of MCF-7 was only observed at concentration more than 30 mg/ml with more than 40% cells were inhibited. This indicates that the partially purified rice bran myo-inositol phosphates degraded by ASUIA279 phytase on MCF-7 breast cancer cells exhibit positive results towards the inhibition of cancer cells growth at relatively high concentration.

Phytate is largely unavailable to monogastric animal such as swine, poultry and fish, as they lac... more Phytate is largely unavailable to monogastric animal such as swine, poultry and fish, as they lack of sufficient endogenous enzymatic activity to hydrolyze phytate. The result is the elimination of precious nutrients that would be beneficial to their growth; furthermore, they will excrete most of the indigestive phytate which can contribute to phosphorus being over applied to the land. Phosphorus has a beneficial impact on vegetative growth on land as well as marine vegetation, causing an increased growth of weeds. This enhanced vegetation consumes large amounts of oxygen, resulting in the loss of aquatic life and ultimately contributes to water pollution and eutrophication of ground water and aquatic environment. Phytase, a type of histidine acid phosphatase hydrolyzes phytin phosphorus and when present in an animal's digestive tract, benefits the animal while reducing total phosphorus levels in manure. Computer modeling has been used to identify and examine the active site of phytase. The factors influencing the ligand binding strength in the active site were analyzed and computational site directed mutagenesis experiments were carried out to evaluate the effects of mutations on the binding strength before and after mutation. From the directive results of computational studies, point mutation was introduced by site directed mutagenesis using polymerase chain reaction (PCR). The activity was measured by kinetic characterization with phytate as a substrate. Decrease in K M notable in all functional mutants indicates that all mutant shows increment in substrate binding. Two functional mutants showed improvement in phytase activity and thermostability.

Phytase gene obtained from Bacillus subtilis ASUIA243 was cloned into a medium vector and transfo... more Phytase gene obtained from Bacillus subtilis ASUIA243 was cloned into a medium vector and transformed into E. coli. Restriction enzyme digestion was conducted to get blunt-ended phytase gene and ligated into the Pichia expression vector, pPICZαA. The recombinant vector, pPICZαA-243HPp was then linearized with PmeI and transformed into P. pastoris strain X33. Screening for multi copy gene number of transformants was done by re-plating the selected colonies on increasing concentration of zeocin. One positive clone, X243HPp#2 was then grown in BMGY media as the starting culture, followed by induction in BMMY media for protein expression study. The supernatant was then analysed by SDS-PAGE and Western blot method to check the protein expression.

An extracellular phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279 was purifie... more An extracellular phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279 was purified to homogeneity using FPLC anion exchange chromatography and gel filtration. The enzyme was purified about 66fold with a recovery of 27%. Its molecular mass was estimated to be 43 kDa by SDS-PAGE. The Michaelis constant (K M ) and turnover number (k cat ) for sodium phytate at pH 5.0 and 50°C were calculated from the Lineweaver-Burk plot to be 760 µM and 4.14s -1 , respectively. The enzyme showed narrow substrate specificity and not phytate, but GTP was dephosphorylated with the highest relative rate of hydrolysis. However, according to the k cat /K M values, phytate was concluded to be the in vivo substrate of the enzyme. Optimal activity was determined at pH 4.5 and 45-55°C. The enzyme was strongly inhibited by Fe 3+ , Cu 2+ , Zn 2+ , molybdate, vanadate, fluoride and phosphate (1 mM).

The growth and phosphorus utilization of plants in sterile media when supplied with inositol hexa... more The growth and phosphorus utilization of plants in sterile media when supplied with inositol hexaphosphate, glucose 1-phosphate or inorganic phosphate. Plant and Soil 220: 165-174.

Oncogene, 2006

p21-activated kinase 1 (PAK1) is a mediator of downstream signaling from the small GTPases Rac an... more p21-activated kinase 1 (PAK1) is a mediator of downstream signaling from the small GTPases Rac and Cdc42. In its inactive state, PAK1 forms a homodimer where two kinases inhibit each other in trans. The kinase inhibitory domain (KID) of one molecule of PAK1 binds to the kinase domain of its counterpart and keeps it inactive. Therefore, the isolated KID of PAK1 has been widely used to specifically inhibit and study PAK function. Here, we show that the isolated KID induced a cell cycle arrest with accumulation of cells in the G1 phase of the cell cycle with an inhibition of cyclin D1 and D2 expression. This cell cycle arrest required the intact KID and was also induced by a mutated KID unable to block PAK1 kinase activity. Furthermore, the KID-induced cell cycle arrest could not be rescued by the expression of a constitutively active PAK1-T423E mutant, concluding that this arrest occurs independently of PAK1 kinase activity. Our results suggest that PAK1 through its KID inhibits cyclin D expression and thereby enforces a cell cycle arrest. Our results also call for serious precaution in the use of KID to study PAK function.

IIUM Engineering Journal, 1970

Phytase gene obtained from Bacillus subtilis ASUIA243 was cloned into a medium vector and transfo... more Phytase gene obtained from Bacillus subtilis ASUIA243 was cloned into a medium vector and transformed into E. coli. Restriction enzyme digestion was conducted to get blunt-ended phytase gene and ligated into the Pichia expression vector, pPICZαA. The recombinant vector, pPICZαA-243HPp was then linearized with PmeI and transformed into P. pastoris strain X33. Screening for multi copy gene number of transformants was done by re-plating the selected colonies on increasing concentration of zeocin. One positive clone, X243HPp#2 was then grown in BMGY media as the starting culture, followed by induction in BMMY media for protein expression study. The supernatant was then analysed by SDS-PAGE and Western blot method to check the protein expression.ABSTRAK: Gen fitase yang didapati daripada Bacillus subtilis ASUIA243 diklonkan sebagai vektor perantara dan berubah menjadi E. coli. Sekatan pencernaan enzim dijalankan untuk mendapatkan gen fitase berhujung tumpul dan diligatkan dengan vektor e...

British Journal of Cancer, 1969

The Egyptian Journal of Remote Sensing and Space Science, 2010

Acta cardiologica, Jan 1, 2010

A collaborative trial study has been conducted for validation of an extraction method and a subse... more A collaborative trial study has been conducted for validation of an extraction method and a subsequent PCR for the detection of transgenic rice sold in Saudi Arabia. The tests were carried out in Saudi Arabia using Real-Time PCR and the positive samples were validated in another lab in Malaysia using PCR and agarose gel visualization. The samples were tested for the existence of the NOS Terminator. A total of 150 samples were tested out of which three samples tested positive as GM-rice which were retested in Malaysia. The presence of GMO rice in Saudi Arabia supports the necessity of developing precise quantitative and qualitative ways for routine analyses and detection of GMO products in the Saudi Arabian market. With the discovery of GM products in the Saudi Arabian market it would be of no surprise that other Middle Eastern nations also knowingly or unknowingly import GM crops.

Phytases catalyze the hydrolysis of phosphomonoester bonds in phytate, thereby releasing lower fo... more Phytases catalyze the hydrolysis of phosphomonoester bonds in phytate, thereby releasing lower forms of myo-inositol phosphates and inorganic phosphate. Phytase enzyme preparations have a wide range of applications in animal and human nutrition. The addition of phytate-degrading enzyme can improve the nutritional value of plant-based foods by enhancing protein digestibility and mineral availability through phytate hydrolysis during digestion in stomach or during food processing. 30 strains of potential phytate-degrading enzymes bacteria isolated from Malaysian maize plantation were cultivated in Luria Bertani (LB) and Luria Bertani + Rice Bran (LBRB) media for 5 days and were analyzed for phytase activity. The 6 strains with highest activity were chosen for species identification. Two set of broad-range 16S rRNA PCR primers were used for genotypic identification. ASUIA279 was the strain that had has the highest phytase activity in LBRB followed by ASUIA271, ASUIA138, ASUIA260, ASUIA243 and ASUIA30. The genotypic technique revealed Pantoea stewartii ASUIA271, Enterobacter sakazakii ASUIA279, Bacillus cereus ASUIA260, Bacillus subtilis ASUIA243, P. stewartii ASUIA138 and B. cereus ASUIA30.

The aims of the study were to observe the effects of different concentration of rice bran in diff... more The aims of the study were to observe the effects of different concentration of rice bran in different media on phytase synthesis and to optimize the temperature and pH of the media for phytase production by those bacterial strains. Three bacterial strain isolates obtained from the soil of Malaysian maize plantation were used to produce phytase. In this study, the effects of different rice bran concentration, incubation temperature and initial pH-values of the media on phytase production were evaluated. Incorporation of 7.5% rice bran has the inducible effect on all the bacterial tested. In respect to phytase production, the best cultivation media and cultivation time for Bacillus cereus ASUIA260 was PFE with 7.5% rice bran after 3 days, whilst for Pantoea stewartii ASUIA271 and Enterobacter sakazakii ASUIA279, it was LB with 7.5% rice bran after 3 days and 5 days, respectively. The arrangement of those isolates according to their ability to produce phytases were E. sakazakii ASUIA279 > P. stewartii ASUIA271 > B. cereus ASUIA260. Production of phytase by those bacteria was triggered by the high content of organic phytate in the rice bran. Optimum temperature for phytase production of B. cereus ASUIA260 was 41 ºC compared to P. stewartii ASUIA271 and E. sakazakii ASUIA279 with 33 ºC and 37 ºC, respectively. Optimum initial pH for phytase production of B. cereus ASUIA 260 was pH 7.2, while P. stewartii ASUIA271 and E. sakazakii ASUIA 279 were both at pH 6.0.

Phytate or myo-inositol hexakisphosphates (IP 6 ) is widely distributed in plants like rice bran.... more Phytate or myo-inositol hexakisphosphates (IP 6 ) is widely distributed in plants like rice bran. The production of myo-inositol phosphate intermediates has received much attention due to the remarkable potential health benefits offered by the compounds. In this study, the cytotoxicity of the partially purified myo-inositol phosphate fractions and commercial IP 1 and IP 6 were investigated against MCF-7 breast cancer cell lines. The study showed that the commercial standard IP 1 and IP 6 showed good inhibition towards the MCF-7 cell line. The MCF-7 cells growth was inhibited in minimum concentration of myo-inositol phosphates (<1000 µg/ml). However, no inhibition observed on the MCF-7 cell line by the myo-inositol phosphates fractions partially purified from rice bran at concentration <1000 µg/ml. The inhibition of MCF-7 was only observed at concentration more than 30 mg/ml with more than 40% cells were inhibited. This indicates that the partially purified rice bran myo-inositol phosphates degraded by ASUIA279 phytase on MCF-7 breast cancer cells exhibit positive results towards the inhibition of cancer cells growth at relatively high concentration.

Phytate is largely unavailable to monogastric animal such as swine, poultry and fish, as they lac... more Phytate is largely unavailable to monogastric animal such as swine, poultry and fish, as they lack of sufficient endogenous enzymatic activity to hydrolyze phytate. The result is the elimination of precious nutrients that would be beneficial to their growth; furthermore, they will excrete most of the indigestive phytate which can contribute to phosphorus being over applied to the land. Phosphorus has a beneficial impact on vegetative growth on land as well as marine vegetation, causing an increased growth of weeds. This enhanced vegetation consumes large amounts of oxygen, resulting in the loss of aquatic life and ultimately contributes to water pollution and eutrophication of ground water and aquatic environment. Phytase, a type of histidine acid phosphatase hydrolyzes phytin phosphorus and when present in an animal's digestive tract, benefits the animal while reducing total phosphorus levels in manure. Computer modeling has been used to identify and examine the active site of phytase. The factors influencing the ligand binding strength in the active site were analyzed and computational site directed mutagenesis experiments were carried out to evaluate the effects of mutations on the binding strength before and after mutation. From the directive results of computational studies, point mutation was introduced by site directed mutagenesis using polymerase chain reaction (PCR). The activity was measured by kinetic characterization with phytate as a substrate. Decrease in K M notable in all functional mutants indicates that all mutant shows increment in substrate binding. Two functional mutants showed improvement in phytase activity and thermostability.

Phytase gene obtained from Bacillus subtilis ASUIA243 was cloned into a medium vector and transfo... more Phytase gene obtained from Bacillus subtilis ASUIA243 was cloned into a medium vector and transformed into E. coli. Restriction enzyme digestion was conducted to get blunt-ended phytase gene and ligated into the Pichia expression vector, pPICZαA. The recombinant vector, pPICZαA-243HPp was then linearized with PmeI and transformed into P. pastoris strain X33. Screening for multi copy gene number of transformants was done by re-plating the selected colonies on increasing concentration of zeocin. One positive clone, X243HPp#2 was then grown in BMGY media as the starting culture, followed by induction in BMMY media for protein expression study. The supernatant was then analysed by SDS-PAGE and Western blot method to check the protein expression.

An extracellular phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279 was purifie... more An extracellular phytate-degrading enzyme produced by Enterobacter sakazakii ASUIA279 was purified to homogeneity using FPLC anion exchange chromatography and gel filtration. The enzyme was purified about 66fold with a recovery of 27%. Its molecular mass was estimated to be 43 kDa by SDS-PAGE. The Michaelis constant (K M ) and turnover number (k cat ) for sodium phytate at pH 5.0 and 50°C were calculated from the Lineweaver-Burk plot to be 760 µM and 4.14s -1 , respectively. The enzyme showed narrow substrate specificity and not phytate, but GTP was dephosphorylated with the highest relative rate of hydrolysis. However, according to the k cat /K M values, phytate was concluded to be the in vivo substrate of the enzyme. Optimal activity was determined at pH 4.5 and 45-55°C. The enzyme was strongly inhibited by Fe 3+ , Cu 2+ , Zn 2+ , molybdate, vanadate, fluoride and phosphate (1 mM).

The growth and phosphorus utilization of plants in sterile media when supplied with inositol hexa... more The growth and phosphorus utilization of plants in sterile media when supplied with inositol hexaphosphate, glucose 1-phosphate or inorganic phosphate. Plant and Soil 220: 165-174.