A study of the oxidation-induced conformational and functional changes in neuroserpin

Background: Neuroserpin, a member of the Serine Proteinase Inhibitor (Serpin) superfamily, is known to be a neuroprotective factor in the focal ischemic stroke followed by reducing the microglial activation. Neuroserpin is a protein rich of methionine residues that can scavenge the free radical species which may increase its neuroprotective effect. On the other hand, the oxidative modifications of the amino acid residues in neuroserpin may lead to changes in its conformation and function. In this study, it was investigated the changes in the conformation and the function of the oxidized neuroserpin. Methods: Neuroserpin expressed in E. coli, BL21 or M15 harboring plasmid pQE81L containing neuroserpin cDNA. Expressed neuroserpin was purified by resin sulfopropyl A50 precharged with 0.1 M NiSO4 under denaturing condition. Neuroserpin was oxidized under oxidative stress condition in the presence of different concentration of hydrogen peroxide. The oxidation of neuroserpin was conveniently detected by a carbonyl content assay using 2, 4 dinitrophenylhydrazine. Changes in tertiary structure of neuroserpin were monitored by spectrofluorimeter to study the alteration of intrinsic fluorescence and also fluorescence of 8-anilinonaphthalin-1 sulfonic acid (ANS) in native and oxidized form of neuroserpin. Results: Total expressed neuroserpin was estimated 4-5 mg/lit in 2XYT culture media. SDS-PAGE analysis of purified neuroserpin showed a single band which reflects the efficiency of the resin SP A50 for purification of the proteins containing 6xHis tag. Carbonyl content of oxidized and native neuroserpin was estimated 12.3 ± 0.3 and 0.45 ± 0.05, respectively. The inhibitory activity of oxidized neuroserpin decreased up to 40-60 as compared with native form of neuroserpin. Intrinsic fluorescence and also the emission of ANS bind to the hydrophobic region of the protein altered from 380 to 85 and in the case of ANS from 105 to 150 in oxidized and native form of neuroserpin, respectively. Conclusion: The decreased intrinsic fluorescence intensity, an enhancement in the fluorescence of ANS, and loss of the inhibitory activity up to 40-60 in neuroserpin, all suggested a conformational modification in the protein under the oxidative stress condition. Remaining the inhibitory activity of neuroserpin reflects that the protein tolerates the oxidative stress condition effectively

Stability Study of Iriba Brucellosis Full-dose and Reduced-dose Vaccine Produced by Razi Institute in Iran

Stability study of biological products plays an important role for determination of product changes in maintenance period and ensuring of safety and efficacy of vaccines. In this research, accelerated and long-term stability study performed for six batches of full and reduced-dose cattle Iriba strain brucellosis vaccine that manufactured by Razi vaccine and serum research institute as a new vaccine. After sampling, the vaccines were tested for accelerated stability, after four days storage at 22 0C and tested intervals in three months until 24 months for long-term stability after storage at 2-8 0C. The result indicated all batches of vaccines in accelerated stability met the specification recommended by OIE 2012 and the mean loss of activity for full-dose was 16.68, 18.87 and 17.79 % and for reduced-dose was 38.85, 36.06 and 34.98 %. In long term stability, the quality control tests including colony forming unit, purity, dissociation and physicochemical tests in all samples until 24 months, met the specification recommended by OIE 2012. The full-dose vaccines showed a mean loss of activity of 30.73, 25.53 and 32.45 % and the reduced-dose vaccines showed 63.51, 58.60 and 60.83 %. The mean increasing of moisture content was, 187.85, 214.13 and 160.77 % for full-dose and 142.35, 110.23 and 164.47 % for reduced-dose. So, the results of this research indicated in spite of moisture content increasing in second year, the brucellosis vaccines with this strain are stable at least 24months if the cold chain considered properly but the best expiry date for the vaccine is one year

Assessment of <i>MC1R</i> and <i>&#945;</i><i>-MSH</i> gene sequences in Iranian vitiligo patients

Background: Vitiligo is an acquired pigmentary disorder of the skin that is caused by unknown factors and is characterized by white and depigmented patches that enlarge and become more numerous with time. Genetic factors, oxidative stress, autoimmunity, and neurochemical agents, such as catecholamines might also contribute to vitiligo. Cutaneous pigmentation is determined by the amounts of eumelanin and pheomelanin synthesized by the epidermal melanocytes and interference of melanocortin-1 receptor (MC1R), a G-protein coupled receptor, its normal agonist, alpha-melanocyte stimulating hormone (&#945;-MSH), and key enzymes, such as tyrosinase, to protect against sun-induced DNA damage. The MC1R, a 7 pass trans-membrane G-protein coupled receptor, is a key control point in melanogenesis. Loss-of-function mutations at the MC1R are associated with a switch from eumelanin to pheomelanin production, resulting in a red or yellow coat color. Aim: In this research, we aim to examine the genetic variety of MC1R and &#945;-MSH gene in 20 Iranian vitiligo patients and 20 healthy controls. Materials and Methods: Analysis of the MC1R coding gene was performed with direct sequencing. Results: We found the following 9 MC1R coding region variants: Arg163Gl (G488A), Arg227Leu (G680A), Val 97Phe (G289T), Asp184Asn (G550A), Arg227Lys (G680A), Arg142His (G425A), Val60Leu (G178T), Val247Met (C739A), and Val174Ile (G520A). We also found 2 frameshift changes: one of them was the Insertion of C (frameshift in Pro136, stop at Trp148) and the other, Insertion of G (frameshift in Pro256, stop at Trp 333). Of all the changes, the most common was Val60Leu at 5&#x0025; in patients vs 20&#x0025; in controls, Val247Met at 15&#x0025; in patients vs 0&#x0025; in controls and Val174Ile at 15&#x0025; in controls and 0&#x0025; in patients. The other variants showed a frequency &lt;5&#x0025; in both patients and controls. Also in this study, we have examined the frequency of single nucleotide polymorphisms within the &#945;-MSH genes with direct sequencing in 20 patients and 20 healthy subjects but found no changes along this gene. Conclusion: We could not find any relationship between MC1R and &#945;-MSH genes and their effect on the disease in Iranian vitiligo patients

Impact of end of lease contracts’ option on joint pricing and inventory decisions of remanufacturable leased products

Leasing currently plays an important role for the global economy. The equipment leasing earning acquired through leasing rather than cash or credit, has reached a dominant level. With this regards, this paper represents a basic mixed-integer non-linear programming model. The study deliberates a firm that leases new products and remanufactured leased merchandises. The proposed study considers the end of lease contract, which contains several options: Return the leased product, return the used product and purchase other remanufactured product and buying the leased product. The primary objective is to maximize the discrepancy between the revenue and the costs of a firm, which leases new products as well as selling remanufactured ones. The product deteriorates with time and the difference between a new and used good is obvious. The product must undergo a remanufacturing procedure before being sold as a remanufactured product