[Immobilization of organophosphorus hydrolase on the surface of bacillus subtilis spores by the adsorption method]

Objective: Organophosphorus [OPs] compounds are widely used in many pesticides, insecticides and chemical nerve agents. These compounds are hazardous for humans and the environment. Organophosphate hydrolase [OPH] is a homodimeric protein initially isolated from Pseudomonas diminuta MG and Flavobacterium species. This enzyme is able to degrade a broad spectrum of toxic OPs compounds. Using immobilized OPH commonly presents a variety of advantages versus the free form of the enzyme. Advantages include an increase in stability, cost reduction by simple recovery and reutilization of the enzyme, quick and easy separation of the reactant and product in the reaction medium

Methods: Plasmid pET-26b [+] was used to generate the OPH protein under the control of the T7lac promoter. E. coli BL21 [DE3] pLysS was used as the host for expression of the OPH enzyme. Recombinant OPH was secreted into the extracellular medium and the purified enzyme was immobilized on the surface of Bacillus subtilis spores by the adsorption method, for the first time

Results: Approximately 42% to 45% enzymatic activity was determined to be associated with spores. Optimal pH and temperature of the enzyme were not altered by the presence of the spores. Thermo and pH stabilities of the immobilized enzyme was higher than the free form of the enzyme. Conclusion: Bacillus subtilis spores are safe for humans and the environment. Therefore this system can be considered an environmentally friendly biocatalyst for degradation of OPs

Conclusion: Bacillus subtilis spores are safe for humans and the environment. Therefore this system can be considered an environmentally friendly biocatalyst for degradation of OPs.

Human bone marrow mesenchymal stem cell behaviors on PCL/Gelatin nanofibrous scaffolds modified with a collagen IV-derived RGD-containing peptide

We introduce an RGD [Arg-Gly-Asp]-containing peptide of collagen 4 origin that possesses potent cell adhesion and proliferation properties. In this experimental study, the peptide was immobilized on an electrospun nanofibrous polycaprolactone/gelatin [PCL/Gel] hybrid scaffold by a chemical bonding approach to improve cell adhesion properties of the scaffold. An iodine- modified phenylalanine was introduced in the peptide to track the immobilization process. Native and modified scaffolds were characterized with scanning electron microscopy [SEM] and fourier transform infrared spectroscopy [FTIR]. We studied the osteogenic and adipogenic differentiation potential of human bone marrow-derived mesenchymal stem cells [hBMSCs]. In addition, cell adhesion and proliferation behaviors of hBMSCs on native and peptide modified scaffolds were evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide [MTT] assay and 4',6-diamidino-2-phenylindole [DAPI] staining, and the results compared with tissue culture plate, as the control. FTIR results showed that the peptide successfully immobilized on the scaffold. MTT assay and DAPI staining results indicated that peptide immobilization had a dramatic effect on cell adhesion and proliferation. This peptide modified nanofibrous scaffold can be a promising biomaterial for tissue engineering and regenerative medicine with the use of hBMSCs

[ let-7f microRNA functions as a negative regulator of HNF4a expression in human adipose tissue-derived stem cells]

microRNAs [miRNAs] are noncoding RNAs that function as key regulators of diverse biological activities such as cellular metabolism, cell proliferation and cell cycle regulation. Recent studies have indicated the high potential of these small molecules to control stem cell differentiation into desired cells. The aim of present study is to investigate the possible effect of let-7f on expression of hepatic nuclear factor 4 alpha [HNF4a] and some hepatic specific factors such as albumin [ALB], alpha fetoprotein [AFP], cytokeratin18 [CK18] and cytokeratin19 [CK19] in human adipose tissue derived stem cells [hADSCs]. ADSCs were isolated from human adipose tissue using collagenase type I and were transduced by recombinant lentiviruses that contained human inhibitor let-7f and Scramble [negative control]. Afterward, the expressions of HNF4a, ALB, AFP, CK18 and CK19 were evaluated by Real-time PCR at different time points. Transduction efficiency of lentiviral vectors into ADSCs was more than 80% as judged by the expression of the GFP reporter gene. Real-time PCR analysis revealed that inhibition of let-7f in hADSCs resulted in significant up regulation of hepatic specific genes compared with the negative control. The expression level of HNF4a also increased in experimental cells at day 14, which supported the suppression of HNF4a expression by let-7f. The results of this study identified let-7f as a negative regulator of HNF4a expression in hADSCs and increased the expression of hepatocyte specific factors through silencing of let-7f. Therefore, suppression of let-7f could be a considerable tool for hepatic differentiation of hADSCs

[Study of hBMSC adhesion and proliferation on RGD-modified polycaprolactone/gelatin nanofibrous scaffold]

In this study we introduced an RGD-containing peptide of collagen IV origin that possesses potent cell adhesion and proliferation properties. This peptide was immobilized on a nanofibrous polycaprolactone/gelatin scaffold after which we analyzed human bone marrow-derived mesenchymal stem cells [hBMSCs] adhesion and proliferation on this peptide-modified scaffold. Nanofibrous scaffold was prepared by electrospinning. The peptide was synthesized by solid-phase peptide synthesis and immobilized on electrospun nanofibrous a polycaprolactone/gelatin scaffold by chemical bonding. Native and modified scaffolds were characterized with Scanning Electron Microscope [SEM] and Fourier-Transform Infra-red Spectroscopy [FTIR]. Adhesion and proliferation of hBMSCs on native and modified scaffolds were analyzed by the Methylthiazol Tetrazolium [MTT] assay. SEM images showed that electrospun scaffolds had homogenous morphology and were 312 +/- 89 nm in diameter. There was no significant difference in scaffold morphology before and after peptide immobilization. FTIR results showed that the peptide was successfully immobilized on the scaffold. Based on MTT assay, cell adhesion studies indicated that peptide immobilization improved cell adhesion on RGD-modified scaffolds at all corresponding time points [p<0.05]. RGD immobilization led to increased cell proliferation potential of the scaffold compared with tissue culture plate and native scaffold [P<0.05]. This novel peptide and modified nanofibrous scaffold, having improved cell adhesion and proliferation properties, can be used for tissue engineering and regenerative medicine by using hBMSCs.

Expression and purification of functionally active recombinant human alpha 1-antitrypsin in methylotrophic yeast pichia pastoris

Human alpha 1-antitrypsin [AAT] cDNA was obtained from HepG2 cell lines. After PCR and construction of expression vector pPICZ?-AAT, human AAT was expressed in the yeast Pichia pastoris [P.pastoris] in a secretary manner and under the control of inducible alcohol oxidase 1 [AOX1] promoter. The amount of AAT protein in medium was measured as 60 mg/l 72 hr after induction with methanol. Results indicated the presence of protease inhibitory function of the protein against elastase. Purification was done using His-tag affinity chromatography. Due to the different patterns of glycosylation in yeast and human, the recombinant AAT showed different SDS-PAGE patterns compared to that of serum-derived AAT while pI shifted from 4.9 in native AAT compared to 5.2 in recombinant AAT constructed in this study

[Serratia marcescens B4A chitinase product optimization using Taguchi approach]

Chitinase production by newly isolated Serratia marcescens B4A was optimized following Taguchi's array methods. Twenty-three bacterial isolates were screened from shrimp culture ponds in the South of Iran. A chitinase-producing bacterium was isolated based on it's ability to utilize chitin as the sole carbon source. The isolate designated as B4A, was identified as Serratia marcescens based on its 16S rRNA sequence and key morphological, physiological and biochemical characteristics. The cultivation of Serratia marcescens B4A in the appropriate liquid medium resulted in production of high levels of chitinase. The malt extract and colloidal chitin represented the best nitrogen and carbon sources, respectively. Chitinase production by Serratia marcescens B4A was optimized following the Taguchi orthogonal array [OA] for the design of experiments [DOE]. Statistical experimental design via the Taguchi method was applied to determine the optimal levels of physical parameters and key media components in the medium, such as temperature, pH, NaCl and chitin concentrations. The results of this study showed that temperature of 30§C, pH 7.9, NaCl 0.1% [w/v] and chitin 1% [w/v] are optimal conditions for this protocol

Estimation of atlatoxin-albumin adduct in serum sample by optimized HPLC-fluorescence

With consideration of lethal effects of aflatoxins specially Bl on human health. Estimation of aflatoxin-albumin adduct, as an important marker of aflatoxin exposure, seems essential. The aim of this study is optimization of HPLC-fluorescence method for measurement of this important marker in blood serum. In this study, blood serum of three groups of rats as A] positive controls [treated with AFB1], B] negative controls [without treatment] and standard rats [treated with radiolabeled AFBl] were used. After albumin isolation using ammunium sulphate and acetic acid, purity of albumin was tested by SDS-PAGE electrophoresis and albumin concentration was quantified by bradford method. Then albumin was hydrolysed by pronase and aflatoxin bound to albumin was released as aflatoxin-lysine. Pronase was precipitated and albumin was digested by aceton in cold, the volume of supernatant was reduced by freeze-drier and injected into HPLC system. Aflatoxin was quantified in comparison to standard rats samples. The purity of this isolated albumin was confirmed by SDS-PAGE electrophoresis. Albumin concentration in positive, negative and standard samples were 10, 13 and 12.5 mg/ml, respectively. Detection limit [20 pg/mg Alb] for measurement of aflatoxin was determined by HPLC method, specificity and sensitivity of method were 92% and 100% respectively. The mean concentration of AF-Alb adducts in serum of positive control rats was 10 ng/mg Alb and the reproducibility of the method after several repeat was very good. In this study, for AF-Alb adduct quantification by HPLC method, mobile phase, I percentage of solvents and run time were changed and the affinity chromatography before HPLC, was deleted. Therefor HPLC- fluorescence which is a precise and specific method, and since it is fast, highly reproducible and cost effective, also with improvement made, could easily be used for the quantification of this important marker in serum

Enhanced cytochrome C oxidase activity in WBC of COPD patients

Chronic obstructive pulmonary disease [COPD] is characterized by decreased expiratory flow rates, increased pulmonary resistance and hyperinflation. Cytochrome C Oxidase [COX] as a key oxidative enzyme modulates oxygen uptake and catalyzes the oxidation of reduced cytochrome C by molecular oxygen. In vitro studies indicate that the activity of COX can be directly regulated by the presence of molecular oxygen. Thus, a better understanding of the role of COX in patients with COPD can provide an important link between the availability of oxygen to tissues and the regulation of oxygen uptake and energy production in these patients. We studied 42 COPD patients [36 males, 6 females] with clinically stable conditions and 50 [42 males, 8 females] healthy sedentary volunteers of similar age. Whole blood was collected by venipuncture in sodium citrate tubes and WBCs were separated by Ficoll according to standard protocol and lysed with microtube pestle homogenizer. The homogenates were centrifuged and the supernatants were used as a cell extract for COX activity determination. Aliquots of this were assayed for total protein content and COX activity. Analysis of COX activity was performed using COX assay kit. Absolute specific COX activity was normalized for total protein. Relative activities were determined by dividing absolute specific COX activity on absolute specific citrate synthase activity. Mitochondrial COX activity and specific activity [absolute and relative] significantly increased in WBCs of patients with COPD in comparison with control samples [p< 0.05] These results indicated that the activity of COX was increased in WBCs of patients with COPD but whether this is a primary or secondary change relevant to hypoxic condition in these patients is not clear and needs further investigation.

Preliminary report of NAD[+]-dependent amino acid dehydrogenase producing bacteria isolated from soil

Amino acid dehydrogenases [L-amino acid: oxidoreductase deaminating; EC 1.4.1.X] are members of the wider superfamily of oxidoreductases that catalyze the reversible oxidative deamination of an amino acid to its keto acid and ammonia with the concomitant reduction of either NAD[+], NADP[+] or FAD. These enzymes have been received much attention as biocatalysts for use in biosensors or diagnostic kits to screen amino acid metabolism disorders such as phenylketonuria [PKU], maple syrup urine disease [MSUD], homocystinuria [HCY] and hyperprolinemia. This study was aimed to isolation and screening of novel amino acid dehydrogenases from soil bacteria. The enzyme producing bacteria were selected among L-methionine and L-phenylalanine utilizers isolated from soil by thin layer chromatography, activity staining and confirmed by enzyme assay. Bacterial strains were identified by phenotypic and biochemical characteristics. The steady-state kinetic studies of enzymes were also performed. In total of 230 tested strains, four of them were recognized as amino acid dehydrogenase producers that belong to species of Pseudomonas, Citrobacter and Proteus. They exhibited the desired NAD[+]-dependent dehydrogenase activities toward L-isoleucine, L-methionine, L-cysteine, L-serine and L-glutamine in oxidative deamination reaction. The specific activity of L-isoleucine dehydrogenase, L-methionine dehydrogenase and L-glutamine dehydrogenase for oxidative deamination of L-isoleucine, L-methionine and L-glutamine were 1.59, 1.2 and 0.73 U/mg, respectively. The Kcat /Km [s-1.mM -1] values in these strains were as follows: L-isoleucine, 113.6, L-methionine, 62.05 and L-glutamine, 95.83. This is the first report of occurrence a specific isoleucine dehydrogenase, glutamine dehydrogenase and methionine dehydrogenase in bacteria

Alteration in antioxidant capacity in patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease [COPD] is a major public health problem that needs greater attention. Variability in the susceptibility to develop COPD is related to both genetic and environmental factors. Oxidative stress and inflammation are the major hallmarks of COPD and antioxidant status can be used as a biomarker to assess the risk of chronic diseases. We used the FRAP [ferric reducing ability of plasma] assay as a simple and powerful test for determination of the total antioxidant capacity of plasma of patients and normal subjects. The patients were selected by cross-sectional method. The mean average age +/- SD of normal subjects and patients was 56 +/- 4 and 60 +/- 2 years respectively. The spectrophotometeric method was used for this assay. The means of the FRAP assays in the patients were higher [about twice] than those of normal subjects. The differences were significant [p < 0.01]. The high levels of antioxidant capacity in the patient group indicated that the antioxidant defense system had been activated due to the oxidative stress and hypoxic condition. A though, FRAP assay can probably be used for demarcation of severity and risk of developing COPD, clinical follow-up and further investigation are required for the assessment of this hypothesis

Study of alpha1-antitrypsin phenotypes frequencies in patients with primary antibody deficiency

Primary antibody deficiencies are the most frequent primary immunodeficiency disorders. Bronchiectasis as a feature of these disorders may be developed due to some factors such alpha-1-antitrypsin deficiency. In order to determine the prevalence of two common alpha-1-antitrypsin deficiency alleles [PI*Z and PI*S] in Iranian patients with antibody deficiency, this study was performed. The prevalence of PI*M, PI*S, and PI*Z allele combinations was determined in 40 patients with primary antibody deficiency [with and without bronchiectasis] and compared with 60 healthy control subjects. Phenotyping was performed by isoelectric focusing. The phenotype frequencies among patients were as follow: M in 92.5%, S in 2.5% and Z in 5%. There was not any significant difference in distribution of alleles or phenotypes between patients and control subjects. Moreover, no significant difference was found between patients with and without bronchiectasis. We did not find evidence to support an association between alpha-1-antitrypsin phenotypes and primary antibody deficiencies in a small, controlled study. Larger studies will be required to clarify the relationship between alpha-1-antitrypsin genotype and susceptibility to bronchiectasis in patients with antibody deficiency