Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465 / Expressão e caracterização de Escherichia coli de alfa-amilase de Geobacillus thermodenitrificans DSM-465

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% ß-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Escherichia coli expression and characterization of alfa-amylase from Geobacillus thermodenitrificans DSM-465 / Expressão e caracterização de Escherichia coli de alfa-amilase de Geobacillus thermodenitrificans DSM-465

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% β-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (∆G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Escherichia coli expression and characterization of -amylase from Geobacillus thermodenitrificans DSM-465

Abstract Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% -helices, 15% -sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

Resumo A alfa-amilase, que catalisa a hidrólise do amido, é uma enzima ubíqua com imensas aplicações industriais. Um gene de 1698 pb que codifica a amilase de 565 aminoácidos foi amplificado por PCR, a partir de Geobacillus thermodenitrificans DSM-465, clonado no plasmídeo pET21a (+), expresso na cepa BL21 (DE3) de E. coli e caracterizado. A enzima recombinante exibiu peso molecular de 63 kDa, pH ótimo igual a 8, temperatura ótima de 70° C e valor KM de 157,7 µM. Em escala piloto, a enzima purificada removeu com eficiência até 95% de amido do tecido de algodão, indicando sua capacidade de desengomagem em alta temperatura. O modelo 3D da enzima construída por Raptor-X e validada por Ramachandran plot apareceu como um monômero com 31% de hélices alfa, 15% de folhas beta e 52% de loops. Os estudos de docking mostraram melhor afinidade de ligação da enzima com amilopectina (G: - 10,59). De acordo com nossos resultados, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276 e Arg175 constituem o sítio ativo potencial da enzima.

Exogenous melatonin-mediated regulation of K+ /Na+ transport, H+ -ATPase activity and enzymatic antioxidative defence operate through endogenous hydrogen sulphide signalling in NaCl-stressed tomato seedling roots.

Melatonin (Mel) and hydrogen sulphide (H2 S) have emerged as potential regulators of plant metabolism during abiotic stress. Presence of excess NaCl in the soil is one of the main causes of reduced crop productivity worldwide. The present investigation examines the role of exogenous Mel and endogenous H2 S in tomato seedlings grown under NaCl stress. Effect of 30 µm Mel on endogenous synthesis of H2 S was examined in roots of NaCl-stressed (200 mm) tomato seedlings. Also, the impact of treatments on the oxidative stress markers, transport of K+ and Na+ , and activity of H+ -ATPase and antioxidant enzymes was assessed. Results show that NaCl-stressed seedlings supplemented with 30 µm Mel had increased levels of endogenous H2 S through enhanced L-cysteine desulfhydrase activity. Mel in association with H2 S overcame the deleterious effect of NaCl and induced retention of K+ that maintained a higher K+ /Na+ ratio. Use of plasma membrane inhibitors and an H2 S scavenger revealed that Mel-induced regulation of K+ /Na+ homeostasis in NaCl-stressed seedling roots operates through endogenous H2 S signalling. Synergistic effects of Mel and H2 S also reduced the generation of ROS and oxidative destruction through the enhanced activity of antioxidant enzymes. Thus, it is suggested that the protective function of Mel against NaCl stress operates through an endogenous H2 S-dependent pathway, wherein H+ -ATPase-energized secondary active transport regulates K+ /Na+ homeostasis.

Escherichia coli expression and characterization of α-amylase from Geobacillus thermodenitrificans DSM-465.

Alpha amylase, catalyzing the hydrolysis of starch is a ubiquitous enzyme with tremendous industrial applications. A 1698 bp gene coding for 565 amino acid amylase was PCR amplified from Geobacillus thermodenitrificans DSM-465, cloned in pET21a (+) plasmid, expressed in BL21 (DE3) strain of E. coli and characterized. The recombinant enzyme exhibited molecular weight of 63 kDa, optimum pH 8, optimum temperature 70°C, and KM value of 157.7µM. On pilot scale, the purified enzyme efficiently removed up to 95% starch from the cotton fabric indicating its desizing ability at high temperature. 3D model of enzyme built by Raptor-X and validated by Ramachandran plot appeared as a monomer having 31% α-helices, 15% ß-sheets, and 52% loops. Docking studies have shown the best binding affinity of enzyme with amylopectin (∆G -10.59). According to our results, Asp 232, Glu274, Arg448, Glu385, Asp34, Asn276, and Arg175 constitute the potential active site of enzyme.

Disclosure of cancer information among Saudi cancer patients.

BACKGROUND: Cancer has been perceived as an incurable disease, and therefore, disclosure of cancer could harm patients. Therefore, the aim of this study was to explore Saudi cancer patients' views regarding disclosure of cancer information and the impact of gender, regions, and level of education on their desire to be disclosed of the diagnosis and prognosis. MATERIALS AND METHODS: A face-to-face interview was conducted during patient's evaluation at our hospital prior to their knowledge of being diagnosed with cancer. RESULTS: Of 420 patients, 97.62% wanted to know all information about cancer and more than 96% wished to know the benefits and side effects of chemotherapy. None of the patients wanted to be treated without knowing their diagnosis, and almost all of them (94.76%) wanted to know about the prognosis. Patients from eastern region and male patients wanted to know their diagnosis more frequently than those from other regions and female patients (98.2% vs. 74.47%, P = 0.001; 97.90% vs. 92.17%, P = 0.008, respectively). Patients from Eastern Province, male patients, and educated patients wished to know their prognosis more frequently than those from other regions, female patients, and illiterate patients (94.98% vs. 68.79%, P = 0.001; 94.74% vs. 86.09%, P = 0.003; and 94.23% vs. 98.58%, P = 0.016, respectively). CONCLUSION: This study indicates that almost all the Saudi cancer patients wanted to know about their cancer diagnosis, prognosis, benefits and side effects of chemotherapy. Regions, gender, and level of education may affect patient's desire for cancer information.

Management Of Metastatic Breast Cancer (MBC).

UNLABELLED: Chemotherapy of metastatic breast carcinoma so far, is not curative using the currently available chemotherapeutic, hormonal or biologic agents. The treatment of metastatic breast cancer is aimed mainly at alleviation of symptoms rather than cure. The first choice of therapy is dependent on patient age, performance status, hormone receptor status, human epidermal growth factor receptor-2 (HER-2), involvement of the viscera, or enrollment of patients in investigational trials. Combination of chemotherapeutic drugs showed an advantage for survival, tumor response and time to progression with adverse effects of these agents. It is very important, therefore, to balance between the benefits of treatment and the adverse effects and complication of therapy. KEYWORDS: chemotherapy, metastatic, HER-2, breast, combination.

Gastrointestinal stromal tumours (GISTs): case report and review of the literature.

Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract, the majority of which is KIT (CD117) positive. In this case report, we describe a case of recurrent and metastatic GIST who presented with hepatic and brain metastases. Despite the patient's GISTs was negative for c-Kit (CD 117), he responded to imatinib mesylate (Glivec) treatment with complete resolution of his liver and brain lesions. The patient has been and still in complete remission for 18 months of follow-up.

Interleukin 17A and F and asthma in Saudi Arabia: gene polymorphisms and protein levels.

BACKGROUND: Asthma is a multifactorial disorder, and both genetic and environmental factors contribute to its development. We investigated the possible association between asthma and 5 single-nucleotide polymorphisms (SNPs) in the interleukin 17 (IL17) gene--rs17880588 (G/A) and rs17878530 (C/T) in IL17A and rs763780 (T/C), rs11465553 (T/C), and rs2397084 (G/A) in IL17F--and compared levels of the proteins IL17A and IL17F in asthma patients with those of controls. PATIENTS AND METHODS: The study group included 100 asthma patients and 102 ethnically matched controls. Genotyping was performed on purified DNA using reverse transcriptase-polymerase chain reaction with specific primers and probes. Levels of IL17A and IL17F were measured in plasma using enzyme-linked immunosorbent assay. RESULTS: Genotyping showed that AG heterozygotes of rs17880588 in IL17A were significantly more common in the control group than among the asthma patients (P < .05); no significant associations were observed for any of the other SNPs examined. Levels of IL17A and IL17F were both higher in asthma patients (IL17A, 2.242 [0.099] vs 2.752 [0.287] pg/mL; IL17F, 236.01 [38.28] vs 700 [201.078] pg/mL). The difference was statistically significant for IL17F (P = .025, t test). Levels of IL17A and IL17F were positively and significantly correlated in the asthma patients CONCLUSION: Of all the SNPs analyzed, only rs17880588 showed a significant association with asthma in the Saudi population we studied. Levels of IL17A and IL17F were significantly upregulated in the asthma patients. The morphology of IL17F appeared to affect expression levels.

Castleman's disease: a study of a rare lymphoproliferative disorder in a university hospital.

Castleman's disease (CD) is a group of rare lymphoproliferative disorders sharing characteristic clinical and histological features, and usually accompanied by a marked systemic inflammatory response. Two histological patterns of lymph nodes were described: the hyaline-vascular and plasma-cell types. The former is more common (80-90%) and tends to be localized. The plasma cell type is more aggressive and usually multicentric. It is interesting that the inflammatory manifestations seem to be related to a lymph node lesion, because the systemic symptoms and inflammatory activity can return to normal after surgical excision or successful medical treatment of the disease. We report here our 15-year experience with this rare disease in King Fahd Hospital of the University, Al-Khobar, Saudi Arabia, focusing on the clinical features, therapy, and patients'outcome.

Cancer patients' desire for information: a study in a teaching hospital in Saudi Arabia.

Relatives of cancer patients in Saudi Arabia tend to believe that telling the truth to the patient could lead to harm and suffering. This study assessed the attitudes of Saudi Arabian cancer patients towards disclosure of cancer diagnosis and prognosis, and benefits and adverse effects of therapy. A questionnaire survey was administered to patients attending the oncology service between January 2002 and December 2005 before they knew their diagnosis. Of 114 patients interviewed, all except 1 (99%) wished to know all the information about their disease and 100% rejected withholding information. Most of the patients (77%) wanted their family to know the diagnosis but few (17%) wanted their friends to be informed. Almost all patients wanted to know the benefits and adverse effects of therapy (98% and 99% respectively). All patients wanted to know about the prognosis of their disease.

Cancer patients' desire for information: a study in a teaching hospital in Saudi Arabia

Relatives of cancer patients in Saudi Arabia tend to believe that telling the truth to the patient could lead to harm and suffering. This study assessed the attitudes of Saudi Arabian cancer patients towards disclosure of cancer diagnosis and prognosis, and benefits and adverse effects of therapy. A questionnaire survey was administered to patients attending the oncology service between January 2002 and December 2005 before they knew their diagnosis. Of 114 patients interviewed, all except 1 [99%] wished to know all the information about their disease and 100% rejected withholding information. Most of the patients [77%] wanted their family to know the diagnosis but few [17%] wanted their friends to be informed. Almost all patients wanted to know the benefits and adverse effects of therapy [98% and 99% respectively]. All patients wanted to know about the prognosis of their disease

Inpatient preseptal cellulitis: experience from a tertiary eye care centre.

OBJECTIVE: To describe causes of preseptal cellulitis (PSC) and outcome of treatment in patients admitted to a tertiary eye-care centre. METHODS: A 15-year (January 1991 to December 2005) review of inpatients with clinical signs and symptoms or radiological evidence suggestive of PSC was conducted. Patients with infection anterior to the orbital septum which is characterised by acute onset of eyelid oedema, tenderness, erythema, warmth and chemosis were included in the study. RESULTS: Among the 104 patients (male:female 64:40) fulfilling the diagnostic criteria for PSC, acute dacryocystitis (ADC) was the most common predisposing cause in 32.6% patients, followed by sinusitis/upper-respiratory infection (URI) in 28.8% and trauma/recent surgery in 27.8% patients. Fifty-per cent required surgical intervention including dacryocystorhinostomy/probing/stenting in 74% and abscess/chalazian drainage in 28.8%. In 38.5% of the patients who had surgical intervention, microbiological investigations were carried out, cultures were positive in 90%. Most common micro-organisms recovered included Staphylococcus and Streptococcus species followed by Haemophilus influenzae and Klebsiella pneumonia. Blood cultures were positive in two of the 34 patients in whom blood was drawn. Most patients responded to systemic antibiotics with resolution of PSC. Seven patients developed late complications which included subacute lid abscesses, eyelid necrosis and cicatricial ectropion. CONCLUSIONS: Sinusitis/URI, ADC and recent history of trauma/surgery were the most common cause of PSC in admitted patients. Although most patients responded to systemic antibiotics, surgical intervention was necessary in some patients to prevent associated complications.

Heroin profiling: mannitol hexaacetate as an unusual ingredient of some illicit drug seizures.

Mannitol hexaacetate (MHA) has been detected by GC-MS in some brown illicit drug seizures in which diacetylmorphine (DAM) was completely lacking. The presence of MHA as a genuine ingredient of the drug seizures rather than a storage- or an analytical artifact, formed by transacetylation of mannitol with the DAM in the heroin, has been verified. It is argued that MHA was formed as a result of the addition of mannitol, as diluent, before the acetylating step in the process of the heroin preparation. This early dilution in the production and distribution chain of the illicit drug is deemed as peculiar, nonetheless it may be highly indicative of a specific production process of heroin in a trafficking organization.

The GC-MS detection and characterization of reticuline as a marker of opium use.

Reticuline (a precursor of opium alkaloids) was detected and characterised as its trimethylsilyl ethers, acetyl esters and methyl ethers by GC-EIMS and GC-CIMS in opium and the urine of opium users after hydrolysis by acid or beta-glucuronidase as coextractive of morphine. Because this compound cannot be detected in heroin and poppy seeds, it is suggested as a differentiating marker between opium and heroin use, opium and poppy seeds use, or opium and "pharmaceutical" codeine use in cases when opiate use has been confirmed by detection of morphine and codeine in the urine. As well as being a constituent of opium, reticuline in the urine of opium users may also result from the metabolic demethylation of the three other benzyltetrahydroisoquinoline opium alkaloids: codamine, laudanosine and laudanine.

The GC-MS detection and characterization of reticuline as a marker of opium use.

Reticuline (a precursor of opium alkaloids) was detected and characterised as its trimethylsilyl ethers, acetyl esters and methyl ethers by GC-EIMS and GC-CIMS in opium and the urine of opium users after hydrolysis by acid or beta-glucuronidase as coextractive of morphine. Because this compound cannot be detected in heroin and poppy seeds, it is suggested as a differentiating marker between opium and heroin use, opium and poppy seeds use, or opium and "pharmaceutical" codeine use in cases when opiate use has been confirmed by detection of morphine and codeine in the urine. As well as being a constituent of opium, reticuline in the urine of opium users may also result from the metabolic demethylation of the three other benzyltetrahydroisoquinoline opium alkaloids: codamine, laudanosine and laudanine.

The use of cyclohexanone as a "derivatizing" reagent for the GC-MS detection of amphetamines and ephedrines in seizures and the urine.

A GC-MS method has been developed for the detection of amphetamine, methamphetamine, and the ephedrines, in seizures and the urine, based on on-GC condensation (derivatization) with cyclohexanone. The method is simple: the dried seizure material or the urine extract was mixed with cyclohexanone and injected into the GC-MS. The method was found to be superior to the methods based on acyl and trimethylsilyl (TMS) derivatization. Unlike for the acyl and TMS derivatives, the molecular and fragment ions of the cyclohexanone condensation products (cyclohexanone derivatives) were of substantial abundance, a useful property in unambiguous compound characterization. Furthermore, the high stability of the "derivatizing" reagent, cyclohexanone, compared with acyl and TMS derivatizing reagents, is a useful property in method development. The present method has proved selective and, tentatively, sensitive enough in the following areas (where methods based on acyl and TMS derivatization, as tested in this laboratory, have failed): (a) detection of amphetamine as a metabolite of methamphetamine; (b) detection of norpseudoephedrine as a metabolite of pseudoephedrine; (c) detection of amphetamine as an impurity of methamphetamine; (d) detection of cathine (norephedrine) as a constituent of Khat leaves; and (e) differentiation of Khat use from phenylpropanolamine use.

Investigation into some aspects of EMIT d.a.u., TLC, and GC-MS urinalysis of bromazepam.

Among the different 1,4-benzodiazepine urinary metabolites, those of bromazepam possess distinctive chemical features that may be used in their selective isolation and detection. The detection of bromazepam metabolites in urine was carried out using EMIT d.a.u., thin-layer chromatography (TLC), and gas chromatography-mass spectrometry (GC-MS). The positive EMIT d.a.u. benzodiazepine assay for bromazepam was found to be due to the 3-hydroxybromazepam (3HOB) metabolite. The detection by TLC and GC-MS was carried out after enzyme or acid hydrolysis of the glucuronide conjugates. Both the 2-amino-3-hydroxy5-bromobenzoylpyridine (AHBBP) metabolite and the acid hydrolysis product of 3-HOB, 2-amino-5-bromobenzoylpyridine (ABBP), were selectively detected by TLC. The bromazepam metabolites in urine could be both isolated and detected selectively by GC-MS in the presence of the metabolites of other 1,4-benzodiazepines that were sometimes used in combination with bromazepam. Both 3-HOB and AHBBP were detected by GC-MS only after trimethylsilyl (TMS) derivatization and not as the free compounds or the acetyl derivatives. Only ABBP was detected in three forms: ABBP, the TMS derivative, and the acetyl derivative. Evidence has been obtained from the enzyme hydrolysis and the TLC studies for the formation of the glucuronide conjugate of AHBBP at the 3-OH group rather than at the 2-NH2 group. All the results have been validated using reference 3-HOB and AHBBP.

A GC-MS method for the detection of toluene and ethylbenzene in volatile substance abuse.

The interference of some substances with the gas chromatography-flame ionization detection and gas chromatography-Fourier transform infrared detection of toluene and ethylbenzene in volatile substance abuse poses problems. A gas chromatography-mass spectrometry (GC-MS) method that will overcome such interference has been developed for the detection of toluene and/or ethylbenzene in the headspace of preparations and products containing these substances and in the headspace of blood samples in the cases of volatile substance abuse. The method is based on converting toluene to benzoic acid via the formation of benzotrichloride. The latter compound was obtained upon the reaction of toluene with chlorine gas under direct sunlight conditions. In the presence of water, benzotrichloride was converted to benzoic acid. Ethylbenzene was converted to benzoic acid and two phenylethanols via the formation of side chain chloro-substituted phenylethanes followed by reaction with water. The chloro-substituted phenylethanes were obtained by the reaction of ethylbenzene with chlorine under direct sunlight conditions. The benzoic acid resulting from toluene and/or ethylbenzene and the two phenylethanols resulting from ethylbenzene were detected by GC-MS as their trimethylsilyl (TMS) derivatives. For the method to be viable for the detection of volatile substance abuse, the chlorination reactions were effected in the gaseous state.