Comparison of bacterial and plant genes participating in proline biosynthesis with Osmotin gene, with respect to enhancing salinity tolerance of transgenic tobacco plants

In an attempt to increase tolerance to salinity stress in tobacco plants, the genes encoding the mutant form of glutamyl kinase (proB), pyrroline-5-carboxylate synthetase, and osmotin were cloned into three different shuttle vectors and were separately introduced into the tobacco plants. The transgenic lines were compared for their ability to produce shoots and grow in MS medium containing 320 mM NaCl; it was shown that the transgenic lines containing genetically handled osmotin gene are more resistant to salinity. The amount of chlorophyll a was used to show continuing growth of plant lines. The results showed that only the tobacco lines transformed with the modified osmotin gene exhibited greater tolerance to salinity. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 1, pp. 122–127. The text was submitted by the authors in English.     

A Novel Approach for Bioleaching of Sulfur,Iron, and Silica Impurities from Coal by Growing and Resting Cells of Rhodococcus spp

Coal is one of the most important sources of fossil energy on earth. However, direct combustion of coal with a high sulfur content can cause various environmental problems. Other constituents of coal that can cause environmental problems include iron oxide (hematite), iron hydroxide, and silica. In this study, growing and resting cells of Rhodococcus erythropolis strains PD1, R1, and FMF, and R. qingshengii were used in heterotrophic removal of sulfur and bioleaching of iron and silica from coal. All of the mentioned strains have an ability of dibenzothiophene (DBT) desulfurization via 4-S pathway. 2-hydroxybiphenyl, sulfate, and ferric ions (Fe³⁺) were assayed by Gibb’s test, barium chloride (BaCl₂), and thiocyanate ions (SCN^?), respectively. FTIR and XRF analyzer were used for detection of the coal bioleaching process by the selected strain of R. erythropolis (PD1). Results indicated that all strains have the ability to grow on coal as the sulfur source. Among them, strain PD1 produced the highest optical density and continued to grow even after 150-h incubation. In both growing- and resting-cells experiments, strain PD1 desulfurized coal most readily compared to other strains. Results of XRF showed that growing cells of strain PD1 had high desulfurizing ability of coal (46%) compared to resting cells in the absence of any carbon sources (24%). Growing cells of strain PD1 also leached 46% of the iron and 14% of the silicate after 7?days of incubation. Resting cells of PD1 leached 32% of the iron as determined by XRF analysis. Also, growing cells of PD1 removed most SiO₂ from coal as detected and confirmed by FTIR and XRF. To the best of our knowledge, this is the first report on bioleaching of iron and silica from coal by R. erythropolis strain PD1, making it a suitable candidate for coal bioremediation.     

Cold-induced physiological and biochemical responses of three grapevine cultivars differing in cold tolerance

In this study the cold tolerance potential of three Vitis vinifera cultivars including ‘Red Sultana’, ‘White Sultana,’ and ‘Flame Seedless’ was evaluated under greenhouse condition. After 15 leaves stage in average, the grapevine plants were subjected to cold stress regimes (4, 0 and ? 4 °C) and compared with control plants (24 °C). A clear increase in leaf electrolyte leakage (EL), thiobarbituric acid reactive substances (TBARS), and H₂O₂ concentrations was observed with decreasing temperature from 4 to ? 4 °C in all grapevine cultivars. Chilled plants showed marked increases in their abscisic acid (ABA), soluble sugars, and proline contents in compared to control vines. Upon exposure to cold stress, the EL, TBARS, H₂O₂, and relative water content of ‘Red Sultana’ were found to be lower compared to ‘White Sultana’ and ‘Flame Seedless’. Under 0 °C condition, ‘Red Sultana’ had the highest superoxide dismutase, guaiacol peroxidase and catalase activities, which was approximately twofold higher than those of all other cultivars. Soluble sugars such as glucose, fructose, and sucrose increased from 4 to ? 4 °C. These increments were higher in ‘Red Sultana’ compared to other cultivars which was concomitant with higher accumulation of endogenous ABA concentration in this cultivar. Higher accumulation of ABA and soluble sugars in ‘Red Sultana’ confirmed the key roles of these compounds in cold tolerance which could be applied as a cold tolerance marker for early selection of grapevine cultivars with the aim to establish vineyards in cold winter regions.     

Expression and characterization of <Emphasis Type="Italic">Escherichia coli</Emphasis> derived hepatitis C virus ARFP/F protein

Genome of the hepatitis C virus (HCV) contains a long open reading frame encoding a polyprotein that is cleaved into 10 proteins. Recently, a novel, so called “ARFP/F”, or “core+1,” protein, which is expressed through a ribosomal frame shift within the capsid-coding sequence, has been described. Herein, to produce and characterize a recombinant form of this protein, the DNA sequence corresponding to the ARFP/F protein (amino acid 11–161) was amplified using a frame-shifted forward primer exploiting the capsid sequence of the lb-subtype as a template. The amplicon was cloned into the pET-24a vector and expressed in different Escherichia coli strains. The expressed protein (mostly as insoluble inclusion bodies) was purified under denaturing conditions on a nickel-nitrilotriacetic acid (Ni-NTA) affinity column in a single step with a yield of 5 mg/L of culture media. After refolding steps, characterization of expressed ARFP/F was performed by SDS-PAGE and Western blot assay using specific antibodies. Antigenic properties of the protein were verified by ELISA using HCV-infected human sera and by its ability for a strong and specific interaction with sera of mice immunized with the peptide encoding a dominant ARFP/F B-cell epitope. The antigenicity plot revealed 3 major antigenic domains in the first half of the ARFP/F sequence. Immunization of BALB/c mice with the ARFP/F protein elicited high titers of IgG indicating the relevance of produced protein for induction of a humoral response. In conclusion, possibility of ARFP/F expression with a high yield and immunogenic potency of this protein in a mouse model have been demonstrated.     

Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells

Humans use two sodium-ascorbate cotransporters (hSVCT1 and hSVCT2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this research used a human hepatic cell line (HepG2), confirming certain results with primary human hepatocytes and determined the initial rate of ascorbic acid uptake to be Na(+) gradient, pH dependent, and saturable as a function of concentration over low and high micromolar ranges. Additionally, hSVCT2 protein and mRNA are expressed at higher levels in HepG2 cells and native human liver, and the cloned hSVCT2 promoter has more activity in HepG2 cells. Results using short interfering RNA suggest that in HepG2 cells, decreasing hSVCT2 message levels reduces the overall ascorbic acid uptake process more than decreasing hSVCT1 message levels. Activation of PKC intracellular regulatory pathways caused a downregulation in ascorbic acid uptake not mediated by a single predicted PKC-specific amino acid phosphorylation site in hSVCT1 or hSVCT2. However, PKC activation causes internalization of hSVCT1 but not hSVCT2. Examination of other intracellular regulatory pathways on ascorbic acid uptake determined that regulation also potentially occurs by PKA, PTK, and Ca(2+)/calmodulin, but not by nitric oxide-dependent pathways. These studies are the first to determine the overall ascorbic acid uptake process and relative expression, regulation, and contribution of the hSVCT systems in human liver epithelial cells.     

Investigating the Structural Stability of RADA16-I Peptide Conjugated to Gold Nanoparticles

International Journal of Peptide Research and Therapeutics - RADA 16-I is an amphiphilic peptide which can form macroscopic scaffolds through self-assembly and has found many applications in tissue...     

Development of Acid-Resistant Alginate/Trimethyl Chitosan Nanoparticles Containing Cationic β-Cyclodextrin Polymers for Insulin Oral Delivery

In this study, the use of trimethylchitosan (TMC), by higher solubility in comparison with chitosan, in alginate/chitosan nanoparticles containing cationic β-cyclodextrin polymers (CPβCDs) has been studied, with the aim of increasing insulin uptake by nanoparticles. Firstly, TMCs were synthesized by iodomethane, and CPβCDs were synthesized within a one-step polycondensation reaction using choline chloride (CC) and epichlorohydrine (EP). Insulin–CβCDPs complex was prepared by mixing 1:1 portion of insulin and CPβCDs solutions. Then, nanoparticles prepared in a three-step procedure based on the iono-tropic pregelation method. Nanoparticles screened using experimental design and Placket Burman methodology to obtain minimum size and polydispercity index (pdI) and the highest entrapment efficiency (EE). CPβCDs and TMC solution concentration and pH and alginate and calcium chloride solution concentrations are found as the significant parameters on size, PdI, and EE. The nanoparticles with proper physicochemical properties were obtained; the size, PdI, and EE% of optimized nanoparticles were reported as 150.82 ± 21 nm, 0.362 ± 0.036, and 93.2% ± 4.1, respectively. The cumulative insulin release in intestinal condition achieved was 50.2% during 6 h. By SEM imaging, separate, spherical, and nonaggregated nanoparticles were found. In the cytotoxicity studies on Caco-2 cell culture, no significant cytotoxicity was observed in 5 h of incubation, but after 24 h of incubation, viability was decreased to 50% in 0.5 mμ of TMC concentration. Permeability studies across Caco-2 cells had been carried out, and permeability achieved in 240 min was 8.41 ± 0.39%, which shows noticeable increase in comparison with chitosan nanoparticles. Thus, according to the results, the optimized nanoparticles can be used as a new insulin oral delivery system.KEY WORDS: alginate, cationic β-cyclodextrin, insulin nanoparticle, oral delivery, trimethyl chitosan