International Journal of Biological Macromolecules, May 1, 2016
A serine metallokeratinase Keratinase enzyme (30kDa) produced by a newly isolated Bacillus strain... more A serine metallokeratinase Keratinase enzyme (30kDa) produced by a newly isolated Bacillus strain (Bacillus pumilus NRC21) cultivated under optimized conditions in medium containing chicken feather meal was purified and characterized in a set of biochemical assays. The purification was carried out using two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-100 columns. The purified enzyme showed a specific activity of 2000 units/mg protein against 170 units/mg protein for crude extract with 12 fold purification. The enzymatic activity of the keratinase stimulated by (Na+, K+, Mg2+), Hg+2 had no effect, and inhibited by entire tested cations, serine and metalloproteinase inhibitors, therefore it can be considered as a serine metalloenzymes. The optimum pH and temperature for the purified enzyme were (7.5, 8.5) and (50, 45°C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5-10) and (20-60°C), respectively and its thermoactivity and thermostability were enhanced in the presence of 5mMMg(+2).These results suggest that the purified keratinase may be used in several industrial applications.
En esta tesis se presenta: la identificacion y clonacion del ADNc que codifica para el homologo p... more En esta tesis se presenta: la identificacion y clonacion del ADNc que codifica para el homologo porcino de la proteina asociada a integrina (IAP o CD47), la localizacion cromosomica del gen correspondiente y la caracterizacion funcional de dicha molecula. Se genero una sonda molecular especifica mediante amplificacion por PCR de ADNc de lelucocitos porcinos, utilizando cebadores basados en regiones consensus entre las secuencias conocidas de CD47 procedentes de diferentes especies. El rastreo (screening) de una genoteca de ADNc de musculo liso de aorta de cerdo identifico seite clones conteniendo todos ellos secuencias identicas. Los clones comprendian una fase abierta de lectura (ORF) que codificaban un peptido senal putativo de 18 aminoacidos, una secuencia de 122 aminoacidos correspondiente a un dominio extracelular de tipo inmunoglobulina variable (IgV) seguida por una region de 147 aminoacidos conteniendo cinco dominios que atraviesan la membrana y una cola citoplasmica de 16 aminoacidos. Dicha secuencia de aminoacidos presentaba un 73% de homologia con la secuencia humana de IAP, por lo que se la denomino IAP porcina o CD47 porcino. La RT-PCR mostro que el CD47 porcino se expresa en una gran variedad de tejidos. Mediante citometria de flujo, se ha mostrado que el anticuerpo monoclonal BRIC 126, anti-CD47 humano, reacciona tanto con plaquetas como con celulas ovaricas de hamster chino (celulas CHO) transfectadas con el ADNc que codifica para el CD47 porcino. El analisis por Western-blot de CD47 de eritrocitos y plaquetas de cerdo revelo un peso molecular de 43-50 kDa y de 55-65 kDa, respectivamente, en condiciones no reductoras. El CD47 porcino se expresaba de modo estable en las celulas CHO transfectadas y se unia a Trombospondina humana (TSP). El anticuerpo BRIC-126, anti-CD47 humano, inhibia la union de plaquetas y de celulas transfectadas con CD47 a TSP humana y a fibrinogeno porcino, en ta
Rhipicephalus (Boophilus) annulatus is a bloodsucking ectoparasite that causes severe production ... more Rhipicephalus (Boophilus) annulatus is a bloodsucking ectoparasite that causes severe production losses in the cattle industry. This study aims to evaluate the in vitro effects of tannic acid, hematin (GST inhibitors) and different plant extracts (rich in tannic acid) on the activity of the recombinant glutathione S-transferase enzyme of the Egyptian cattle tick R. annulatus (rRaGST), in order to confirm their ability to inhibit the parasitic essential detoxification enzyme glutathione S-transferase. Extraction with 70% ethanol of Hibiscus cannabinus (kenaf flowers), Punica granatum (red and white pomegranate peel), Musa acuminata (banana peel) (Musaceae), Medicago sativa (alfalfa seeds), Tamarindus indicus (seed) and Cuminum cyminum (cumin seed) were used to assess: (i) inhibitory capacities of rRaGST and (ii) their phenolic and flavonoid contents. Ethanol extraction of red pomegranate peel contained the highest content of phenolic compounds (29.95mg gallic acid/g dry tissue) compared to the other studied plant extracts. The highest inhibition activities of rRaGST were obtained with kenaf and red pomegranate peel (P. granatum) extracts with IC50 values of 0.123 and 0.136mg dry tissue/ml, respectively. Tannic acid was the more effective inhibitor of rRaGST with an IC50 value equal to 4.57μM compared to delphinidine-HCl (IC50=14.9±3.1μM). Gossypol had a weak inhibitory effect (IC50=43.7μM), and caffeic acid had almost no effect on tick GST activity. The IC50 values qualify ethacrynic acid as a potent inhibitor of rRaGST activity (IC50=0.034μM). Cibacron blue and hematin showed a considerable inhibition effect on rRaGST activity, and their IC50 values were 0.13μM and 7.5μM, respectively. The activity of rRaGST was highest for CDNB (30.2μmol/min/mg protein). The enzyme had also a peroxidatic activity (the specific activity equals 26.5μmol/min/mg protein). Both tannic acid and hematin inhibited rRaGST activity non-competitively with respect to GSH and competitively with respect to CDNB. While red pomegranate extracts inhibited rRaGST activity competitively with respect to GSH, uncompetitive inhibition was observed with respect to CDNB.
International Journal of Biological Macromolecules, Apr 1, 2013
Novel keratinolytic enzyme (32kDa) secreted by a newly isolated Bacillus strain (Bacillus subtili... more Novel keratinolytic enzyme (32kDa) secreted by a newly isolated Bacillus strain (Bacillus subtilis NRC3) cultivated in medium containing chicken feather meal was purified and partially characterized in a set of biochemical assays. The purification was carried out by applying a protocol of two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-75 columns. The purified enzyme showed a specific activity of 5233units/mg protein against 169units/mg protein for crude extract with 31 fold purification. The enzymatic activity of the purified keratinolytic enzyme stimulated by Na(+), K(+), Mg(2+), Ba(2+), Ca(2+), and inhibited by entire tested cations and metalloproteinase inhibitors, indicating that it belongs to metallo-keratinase enzymes. The optimum pH and temperature for the purified enzyme were (7.5, 8.0) and (50, 40°C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5-10) and (20-60°C), respectively. These results suggest that this keratinase may be a useful alternative and ecofriendly route for handling the abundant amount of waste feathers.
Echinacea (E.) purpurea herb is commonly known as the purple coneflower, red sunflower and rudbec... more Echinacea (E.) purpurea herb is commonly known as the purple coneflower, red sunflower and rudbeckia. In this paper, we report the curative efficacy of an Echinacea ex-tract in γ-irradiated mice. E. purpurea was given to male mice that were divided into five groups (control, treated, irradiated, treated before irradiation & treated after irra-diation) at a dose of 30 mg/kg body weight for 2 weeks be-fore and after irradiation with 3 Gy of γ-rays. The results reflected the detrimental reduction effects of γ-rays on pe-ripheral blood hemoglobin and the levels of red blood cells, differential white blood cells, and bone marrow cells. The thiobarbituric acid-reactive substances (TBARs) lev-el, Superoxide dismutase (SOD) and glutathione perox-idase (GSPx) activities and DNA fragmentation were also investigated. FT-Raman spectroscopy was used to explore the structural changes in liver tissues. Significant changes were observed in the microenvironment of the major con-stituents, including ...
The incidence of hepatocellular carcinoma (HCC) and HCC-related deaths has increased over the las... more The incidence of hepatocellular carcinoma (HCC) and HCC-related deaths has increased over the last few decades. There are several risk factors of HCC such as viral hepatitis (B, C), cirrhosis, tobacco and alcohol use, aflatoxin-contaminated food, pesticides, diabetes, obesity, nonalcoholic fatty liver disease (NAFLD), and metabolic and genetic diseases. Diagnosis of HCC is based on different methods such as imaging ultrasonography (US), multiphasic enhanced computed tomography (CT), magnetic resonance imaging (MRI), and several diagnostic biomarkers. In this review, we examine the epidemiology of HCC worldwide and in Egypt as well as risk factors associated with the development of HCC and, finally, provide the updated diagnostic biomarkers for the diagnosis of HCC, particularly in the early stages of HCC. Several biomarkers are considered to diagnose HCC, including downregulated or upregulated protein markers secreted during HCC development, circulating nucleic acids or cells, metab...
BACKGROUND: The recently emerged SARS-CoV-2 caused a global pandemic since the last two years. Th... more BACKGROUND: The recently emerged SARS-CoV-2 caused a global pandemic since the last two years. The urgent need to control the spread of the virus and rapid application of the suitable health measures raised the importance of available, rapid, and accurate diagnostic approaches. OBJECTIVE: The purpose of this study is to describe a rapid in-house optimized ELISA based on the expression of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in a prokaryotic system. METHODS: We show the expression of the 30 kDa recombinant SARS-CoV-2 RBD-6×His in four different E. coli strains (at 28∘C using 0.25mM IPTG) including the expression strain E. coli BL21 (DE3) Rosetta Gami. SARS-CoV-2 rRBD-6×His protein was purified, refolded, and used as an antigen coat to assess antibody response in human sera against SARS-CoV-2 infection. RESULTS: The assessment was carried out using a total of 155 human sero-positive and negative SARS-CoV-2 antibodies. The ELISA showed 69.5% sensitivity, 88...
International Journal of Biological Macromolecules, May 1, 2016
A serine metallokeratinase Keratinase enzyme (30kDa) produced by a newly isolated Bacillus strain... more A serine metallokeratinase Keratinase enzyme (30kDa) produced by a newly isolated Bacillus strain (Bacillus pumilus NRC21) cultivated under optimized conditions in medium containing chicken feather meal was purified and characterized in a set of biochemical assays. The purification was carried out using two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-100 columns. The purified enzyme showed a specific activity of 2000 units/mg protein against 170 units/mg protein for crude extract with 12 fold purification. The enzymatic activity of the keratinase stimulated by (Na+, K+, Mg2+), Hg+2 had no effect, and inhibited by entire tested cations, serine and metalloproteinase inhibitors, therefore it can be considered as a serine metalloenzymes. The optimum pH and temperature for the purified enzyme were (7.5, 8.5) and (50, 45°C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5-10) and (20-60°C), respectively and its thermoactivity and thermostability were enhanced in the presence of 5mMMg(+2).These results suggest that the purified keratinase may be used in several industrial applications.
En esta tesis se presenta: la identificacion y clonacion del ADNc que codifica para el homologo p... more En esta tesis se presenta: la identificacion y clonacion del ADNc que codifica para el homologo porcino de la proteina asociada a integrina (IAP o CD47), la localizacion cromosomica del gen correspondiente y la caracterizacion funcional de dicha molecula. Se genero una sonda molecular especifica mediante amplificacion por PCR de ADNc de lelucocitos porcinos, utilizando cebadores basados en regiones consensus entre las secuencias conocidas de CD47 procedentes de diferentes especies. El rastreo (screening) de una genoteca de ADNc de musculo liso de aorta de cerdo identifico seite clones conteniendo todos ellos secuencias identicas. Los clones comprendian una fase abierta de lectura (ORF) que codificaban un peptido senal putativo de 18 aminoacidos, una secuencia de 122 aminoacidos correspondiente a un dominio extracelular de tipo inmunoglobulina variable (IgV) seguida por una region de 147 aminoacidos conteniendo cinco dominios que atraviesan la membrana y una cola citoplasmica de 16 aminoacidos. Dicha secuencia de aminoacidos presentaba un 73% de homologia con la secuencia humana de IAP, por lo que se la denomino IAP porcina o CD47 porcino. La RT-PCR mostro que el CD47 porcino se expresa en una gran variedad de tejidos. Mediante citometria de flujo, se ha mostrado que el anticuerpo monoclonal BRIC 126, anti-CD47 humano, reacciona tanto con plaquetas como con celulas ovaricas de hamster chino (celulas CHO) transfectadas con el ADNc que codifica para el CD47 porcino. El analisis por Western-blot de CD47 de eritrocitos y plaquetas de cerdo revelo un peso molecular de 43-50 kDa y de 55-65 kDa, respectivamente, en condiciones no reductoras. El CD47 porcino se expresaba de modo estable en las celulas CHO transfectadas y se unia a Trombospondina humana (TSP). El anticuerpo BRIC-126, anti-CD47 humano, inhibia la union de plaquetas y de celulas transfectadas con CD47 a TSP humana y a fibrinogeno porcino, en ta
Rhipicephalus (Boophilus) annulatus is a bloodsucking ectoparasite that causes severe production ... more Rhipicephalus (Boophilus) annulatus is a bloodsucking ectoparasite that causes severe production losses in the cattle industry. This study aims to evaluate the in vitro effects of tannic acid, hematin (GST inhibitors) and different plant extracts (rich in tannic acid) on the activity of the recombinant glutathione S-transferase enzyme of the Egyptian cattle tick R. annulatus (rRaGST), in order to confirm their ability to inhibit the parasitic essential detoxification enzyme glutathione S-transferase. Extraction with 70% ethanol of Hibiscus cannabinus (kenaf flowers), Punica granatum (red and white pomegranate peel), Musa acuminata (banana peel) (Musaceae), Medicago sativa (alfalfa seeds), Tamarindus indicus (seed) and Cuminum cyminum (cumin seed) were used to assess: (i) inhibitory capacities of rRaGST and (ii) their phenolic and flavonoid contents. Ethanol extraction of red pomegranate peel contained the highest content of phenolic compounds (29.95mg gallic acid/g dry tissue) compared to the other studied plant extracts. The highest inhibition activities of rRaGST were obtained with kenaf and red pomegranate peel (P. granatum) extracts with IC50 values of 0.123 and 0.136mg dry tissue/ml, respectively. Tannic acid was the more effective inhibitor of rRaGST with an IC50 value equal to 4.57μM compared to delphinidine-HCl (IC50=14.9±3.1μM). Gossypol had a weak inhibitory effect (IC50=43.7μM), and caffeic acid had almost no effect on tick GST activity. The IC50 values qualify ethacrynic acid as a potent inhibitor of rRaGST activity (IC50=0.034μM). Cibacron blue and hematin showed a considerable inhibition effect on rRaGST activity, and their IC50 values were 0.13μM and 7.5μM, respectively. The activity of rRaGST was highest for CDNB (30.2μmol/min/mg protein). The enzyme had also a peroxidatic activity (the specific activity equals 26.5μmol/min/mg protein). Both tannic acid and hematin inhibited rRaGST activity non-competitively with respect to GSH and competitively with respect to CDNB. While red pomegranate extracts inhibited rRaGST activity competitively with respect to GSH, uncompetitive inhibition was observed with respect to CDNB.
International Journal of Biological Macromolecules, Apr 1, 2013
Novel keratinolytic enzyme (32kDa) secreted by a newly isolated Bacillus strain (Bacillus subtili... more Novel keratinolytic enzyme (32kDa) secreted by a newly isolated Bacillus strain (Bacillus subtilis NRC3) cultivated in medium containing chicken feather meal was purified and partially characterized in a set of biochemical assays. The purification was carried out by applying a protocol of two successive chromatographic steps; cation exchange chromatography on CM-cellulose and gel filtration on sephadex G-75 columns. The purified enzyme showed a specific activity of 5233units/mg protein against 169units/mg protein for crude extract with 31 fold purification. The enzymatic activity of the purified keratinolytic enzyme stimulated by Na(+), K(+), Mg(2+), Ba(2+), Ca(2+), and inhibited by entire tested cations and metalloproteinase inhibitors, indicating that it belongs to metallo-keratinase enzymes. The optimum pH and temperature for the purified enzyme were (7.5, 8.0) and (50, 40°C) when using keratin azure and azocasein as substrates, respectively. The purified enzyme was highly stable at broad pH and temperature ranged (5-10) and (20-60°C), respectively. These results suggest that this keratinase may be a useful alternative and ecofriendly route for handling the abundant amount of waste feathers.
Echinacea (E.) purpurea herb is commonly known as the purple coneflower, red sunflower and rudbec... more Echinacea (E.) purpurea herb is commonly known as the purple coneflower, red sunflower and rudbeckia. In this paper, we report the curative efficacy of an Echinacea ex-tract in γ-irradiated mice. E. purpurea was given to male mice that were divided into five groups (control, treated, irradiated, treated before irradiation & treated after irra-diation) at a dose of 30 mg/kg body weight for 2 weeks be-fore and after irradiation with 3 Gy of γ-rays. The results reflected the detrimental reduction effects of γ-rays on pe-ripheral blood hemoglobin and the levels of red blood cells, differential white blood cells, and bone marrow cells. The thiobarbituric acid-reactive substances (TBARs) lev-el, Superoxide dismutase (SOD) and glutathione perox-idase (GSPx) activities and DNA fragmentation were also investigated. FT-Raman spectroscopy was used to explore the structural changes in liver tissues. Significant changes were observed in the microenvironment of the major con-stituents, including ...
The incidence of hepatocellular carcinoma (HCC) and HCC-related deaths has increased over the las... more The incidence of hepatocellular carcinoma (HCC) and HCC-related deaths has increased over the last few decades. There are several risk factors of HCC such as viral hepatitis (B, C), cirrhosis, tobacco and alcohol use, aflatoxin-contaminated food, pesticides, diabetes, obesity, nonalcoholic fatty liver disease (NAFLD), and metabolic and genetic diseases. Diagnosis of HCC is based on different methods such as imaging ultrasonography (US), multiphasic enhanced computed tomography (CT), magnetic resonance imaging (MRI), and several diagnostic biomarkers. In this review, we examine the epidemiology of HCC worldwide and in Egypt as well as risk factors associated with the development of HCC and, finally, provide the updated diagnostic biomarkers for the diagnosis of HCC, particularly in the early stages of HCC. Several biomarkers are considered to diagnose HCC, including downregulated or upregulated protein markers secreted during HCC development, circulating nucleic acids or cells, metab...
BACKGROUND: The recently emerged SARS-CoV-2 caused a global pandemic since the last two years. Th... more BACKGROUND: The recently emerged SARS-CoV-2 caused a global pandemic since the last two years. The urgent need to control the spread of the virus and rapid application of the suitable health measures raised the importance of available, rapid, and accurate diagnostic approaches. OBJECTIVE: The purpose of this study is to describe a rapid in-house optimized ELISA based on the expression of the receptor binding domain (RBD) of the SARS-CoV-2 spike protein in a prokaryotic system. METHODS: We show the expression of the 30 kDa recombinant SARS-CoV-2 RBD-6×His in four different E. coli strains (at 28∘C using 0.25mM IPTG) including the expression strain E. coli BL21 (DE3) Rosetta Gami. SARS-CoV-2 rRBD-6×His protein was purified, refolded, and used as an antigen coat to assess antibody response in human sera against SARS-CoV-2 infection. RESULTS: The assessment was carried out using a total of 155 human sero-positive and negative SARS-CoV-2 antibodies. The ELISA showed 69.5% sensitivity, 88...
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Papers by Yasser E Shahein