Isolation of a Penicillin Acylase Producing E.coli and Kinetic Characterization of the Whole Cell Enzyme Activity

ABSTRACT Penicillin acylase (EC 3.5.1.11) has been a target of study for a long time because of its pivotal role in the deacylation of the penicillin into the 6-aminopenicillanic acid (6-APA) and the side-chain organic acids. This property of penicillin acylase has been exploited commercially for large scale production of 6-APA, which is the key intermediate in the manufacture of semi-synthetic penicillins. Due to the worldwide demand for semi-synthetic penicillins, production of 6-APA has been increased up to 7000 tons in recent years. In this study, Sixty-five strains of E. coli were investigated for penicillin acylase activity using fluorescamine method. The 6-aminopenicillanic acid formed in the reaction mixture was developed on thin layer chromatography. One-minute beta-lactamase test was carried out to follow any trace of penicillinase activity. Only one sample designated as E.coli PPA78 was found to be penicillin acylase producer. The optimal pH and temperature of penicillin acylase activity of the whole cells were determined to be 8.0 and 57°C, respectively. Km value and activation energy of the enzymatic hydrolysis reaction of penicillin G by intracellular enzyme were estimated as 0.004 mmol and 6.2 Kcal/mol, respectively. Iran. Biomed. J. 6 (2 & 3): 93-96, 200

Effect of Dexamethasone on Striatal Neurotransmissions in the Rats Subjected to Parkinson’s Disease Animal Model

Objective(s)The aim of this study was to evaluate the effects of dexamethasone on striatal dopaminergic, glutamatergic and gamma amino butyric acid (GABA) ergic neurotransmission in normal and parkinsonian rats.Materials and MethodsDexamethasone (0.15, 0.30, 0.60 and 0.8 mg/kg) was administered to normal or parkinsonian rats (i.p.) followed by the analysis of the striatal neurotransmitters concentrations. Additionally, the effect of dexamethasone on the damaged Substantia nigra pars compata (SNc) neurons has been investigated. ResultsDexamethasone resulted in decreased level of striatum glutamatergic-GABAergic and enhanced dopaminergic neurotransmission in normal and parkinsonian rats. In addition, acute treatment with dexamethasone did not improve the lesion at all. ConclusionThese findings suggest the new therapeutic mechanism of action for dexamethasone in Parkinson’s disease animal model

Pegylated niosomal nanoparticles loaded with vincristine: Characterization and in vitro evaluation

Purpose: To investigate the effect of pegylated niosomal vincristine (VCR) on enhanced performance, drug resistance and prolonged blood circulation time.Methods: Pegylated niosomal VCR was synthesized by reverse phase evaporation. The mean diameter, size distribution, and zeta potential of pegylated niosomal VCR were evaluated using a Zetasizer. The half-maximal concentration (IC50) values of pegylated niosomal VCR and standard VCR were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The impact of pegylated niosomal VCR on apoptosis and cell cycle of BCL1 lymphoma cancer cells were investigated.Results: The mean diameter, size distribution and zeta potential of pegylated niosomal VCR were 220 nm, 0.4, and –18.8 mV, respectively. Cell proliferation was evaluated using the MTT assay. The IC50 values of pegylated niosomal VCR and standard VCR were 1.6 and 3.5 μg/mL, respectively, after a 24-h incubation. The cytotoxicity of pegylated niosomal VCR was twice that of standard VCR. Furthermore, flow cytometric analysis of the cell cycle showed that pegylated niosomal VCR induced greater mitotic arrest than did standard VCR.Conclusions: The findings demonstrate the effective antitumor activity of pegylated niosomal VCR compared with standard VCR.Keywords: Niosome, Anti-tumour, Polyethylene glycol, Vincristine,   Encapsulation, Lymphom

Meningococcal vaccines: past, present, and future perspectives

Meningococcal disease remains a significant global public health issue and is unique among causes of bacterial meningitis and sepsis where not only does it cause sporadic disease but also outbreaks. The prevention of meningococcal disease also presents a serious challenge . Although polysaccharide vaccines have been available for serogroup A, C, Y, and W135 for many years, serogroup C polysaccharide- protein conjugate vaccine has only recently been licensed in many countries. More recently, a conjugate vaccine for a combination of serogroup A, C, Y, and W135 has been made available. The major hurdle in achieving the goal of eradication is the development of a safe and immunogenic vaccine against serogroup B infections. Outer membrane vesicle vaccines are already used in some countries, and will likely be used more widely in the next few years, but efficacy forendemic disease in children has so far been disappointing. Through the recent availability of the meningococcal genome sequence, many new vaccine candidates are being identified and there is increasing optimism that a solution to the problem can be found. However, none of these has yet been presented as the "universal" protective antigen and work in this field continues to be held back by our limited knowledge concerning the mechanisms of natural protection against serogroup B

An Investigation into Improvement of Stability and Efficacy of Intravesical BCG Formulations Using Freeze-Drying Technique: Stability and efficacy of intravesical BCG formulations

Bacillus Calmette–Guérin (BCG) has been used as an intravesical product for the treatment of intermediate and high risk, non-muscle-invasive bladder cancer (NMIBC). Freeze drying technique is highly recommended for product development, however, the microorganism sensitivity to freezing and drying processes is a major chalenge which may lead to poor survival. To overcome this problem, the use of cryoprotectants in intravesical BCG formulation is required. This study was, therefre, planned to design a new formulation, using an attenuated strain of Mycobacterium bovis, which could be produced by freeze drying technique with the aim of prolonging its storage stability and increasing its efficacy as well as the ease of administration. For this purpose, sodium L-glutamate monohydrate (a commonly used stabilizer in domestic BCG suspension formulations) was replaced by lactose monohydrate. New intravesical BCG formulations, both in lyophilized and liquid forms, were eventually evaluated by moisture content assay, viable count assay, bacterial and fungal contamination, safety test and determination of bacterial concentration and O2 consumption. The results were compared with the data obtained for the conventional lyophilized and liquid products. Maximum survival rate was achieved in the presence of 10 % w/v lactose monohydrate for both liquid and lyophilized formulations when stored at less than -10 and 2-8C, respectively. In summary, the freeze-dried formulations developed with lactose monohydrate met the requirements of intravesical BCG in high viability and stability during storage. HIGHLIGHTS Lyophilization technique and lactose monohydrate as a protecting sugar were used to improve the stability, storage conditions, shelf-life and effcicay of intravesical BCG product. Maximum survival rate was attained for both liquid and lyophilized lactose-based formulations prepared with 10 %w/v lactose monohydrate and stored at less than -10 and 2-8°C, respectively. Freeze-dried products possessed higher stability and efficacy in comparison with corresponding liquid preparations. &nbsp

Investigation of purification process stresses on erythropoietin peptide mapping profile

Background: Full compliance of recombinant protein peptide mapping chromatogram with the standard reference material, is one of the most basic quality control tests of biopharmaceuticals. Changing a single amino acid substitution or side chain diversity for a given peptide changes protein hydrophobicity and causes peak shape or retention time alteration in a peptide mapping assay. In this work, the effect of different stresses during the recombinant erythropoietin (EPO) purification process, including pH 4, pH 5, and room temperature were checked on product peptide mapping results. Materials and Methods: Cell culture harvest was purified under stress by different chromatographic techniques consisting of gel filtration, anionic ion exchange, concentration by ultrafiltration, and high resolution size exclusion chromatography. To induce more pH stresses, the purified EPO was exposed to pH stress 4 and 5 by exchanging buffer by a 10 KDa dialysis sac overnight. The effects of temperature and partial deglycosylation (acid hydrolysis) on purified EPO were also studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and peptide mapping analysis. Removal of sialic acid by mild hydrolysis was performed by exposure to two molar acetic acid at 80°C for 3 h. Results: No significant effect was observed between intact and stressed erythropoietin peptide mapping profiles and SDS-PAGE results. To validate the sensibility of the technique, erythropoietin was partially acid hydrolyzed and significant changes in the chromatographic peptide map of the intact form and a reduction on its molecular weight were detected, which indicates some partial deglycosylation. Conclusions: Purification process does not alter the peptide mapping profile and purification process stresses are not the cause of peptide mapping noncompliance

Enhanced antitumor activity of bovine lactoferrin through immobilization onto functionalized nano graphene oxide: an in vitro/in vivo study

This study aims to improve the anticancer activity of bovine lactoferrin through enhancing its stability by immobilization onto graphene oxide. Bovine lactoferrin was conjugated onto graphene oxide and the conjugation process was confirmed by FT-IR, SDS-PAGE, and UV spectrophotometry. Physical characterization was performed by DLS analysis and atomic force microscopy. The cytotoxicity and cellular uptake of the final construct (CGO-PEG-bLF) was inspected on lung cancer TC-1 cells by MTT assay and flow cytometry/confocal microscopy. The anticancer mechanism of the CGO-PEG-bLF was studied by cell cycle analysis, apoptosis assay, and western blot technique. Finally, the anticancer activity of CGO-PEG-bLF was assessed in an animal model of lung cancer. Size and zeta potential of CGO-PEG-bLF was obtained in the optimum range. Compared with free bLF, more cytotoxic activity, cellular uptake and more survival time was obtained for CGO-PEG-bLF. CGO-PEG-bLF significantly inhibited tumor growth in the animal model. Cell cycle arrest and apoptosis were more induced by CGO-PEG-bLF. Moreover, exposure to CGO-PEG-bLF decreased the phospho-AKT and pro-Caspase 3 levels and increased the amount of cleaved caspase 3 in the treated cells. This study revealed the potential of CGO-PEG as a promising nanocarrier for enhancing the therapeutic efficacy of anticancer agents

Oxidative stress and leaf senescence

Abstract Background Senescence is an important developmental process that leads to the cell death through highly regulated genetically controlled processes in plants. Biotic and abiotic Oxidative stresses can also artificially induce senescence and increase the production of reactive oxygen species (ROS) specifically in chloroplast. One of the important oxidative stresses is paraquat that induces deviation of electron from photosynthesis electron chain and lead to the production of more ROS in chloroplast. Plants have evolved special adoptive mechanism to reallocate nutrient to reproductive and juvenile organs in senescence and different oxidative stresses. Rubisco seems to be the most abundant protein in plants and is involved in many changes during senescence. Results In the present study, the effects of ROS on Rubisco during senescence and oxidative stresses were evaluated by measuring photosynthesis factors such as net photosynthesis rate (Pn), stomatal conductance (G), evaporation rate (E), intra cellular CO2 concentration (Ci), fluorescence and total protein during three stages of development. Our results showed that in paraquat treated plants, CO2 assimilation is the most effective factor that refers to Rubisco damages. The highest correlation and regression coefficient belonged to Ci, while correlation coefficient between photosynthesis rate and total protein was much smaller. Conclusion It appears in the early stage of oxidative stresses such as exposing to paraquat, ROS has the most effect on Rubisco activity that induces more susceptibility to Rubisco specific protease. Moreover, Rubisco deactivation acts as an initiative signal for Rubisco degradation.</p