Arabidopsis ANTR1 is a thylakoid Na+-dependent phosphate transporter: functional characterization in Escherichia coli

In this study, the putative anion transporter 1 (ANTR1) from Arabidopsis thaliana was shown to be localized to the chloroplast thylakoid membrane by Western blotting with two different peptide-specific antibodies. ANTR1 is homologous to the type I of mammalian Na+-dependent inorganic phosphate (Pi) transporters. The function of ANTR1 as a Na+-dependent Pi transporter was demonstrated by heterologous expression and uptake of radioactive Pi into Escherichia coli cells. The expression of ANTR1 conferred increased growth rates to the transformed cells and stimulated Pi uptake in a pH- and Na+-dependent manner as compared with the control cells. Among various tested effectors, Pi was the preferred substrate. Although it competed with the uptake of Pi, glutamate was not transported by ANTR1 into E. coli. In relation to its function as a Pi transporter, several physiological roles for ANTR1 in the thylakoid membrane are proposed, such as export of Pi produced during nucleotide metabolism in the thylakoid lumen back to the chloroplast stroma and balance of the trans-thylakoid H+ electrochemical gradient storage.     

Production and characterization of laccase from Cyathus bulleri and its use in decolourization of recalcitrant textile dyes

Many fungi (particularly the white rot) are well suited for treatment of a broad range of textile dye effluents due to the versatility of the lignin-degrading enzymes produced by them. We have investigated decolourization of a number of recalcitrant reactive azo and acid dyes using the culture filtrate and purified laccase from the fungus Cyathus bulleri. For this, the enzyme was purified from the culture filtrate to a high specific activity of 4,022 IU mg⁻¹ protein, produced under optimized carbon, nitrogen and C/N ratio with induction by 2,6-dimethylaniline. The protein was characterized as a monomer of 58±5.0 kDa with carbohydrate content of 16% and was found to contain all three Cu(II) centres. The three internal peptide sequences showed sequence identity (80–92%) with laccases of a number of white rot fungi. Substrate specificity indicated highest catalytic efficiency (k _cat/K _M) on guaiacol followed by 2,2′-azino-bis(3-ethylthiazoline-6-sulfonic acid) (ABTS). Decolourization of a number of reactive azo and acid dyes was seen with the culture filtrate of the fungus containing predominantly laccase. In spite of no observable effect of purified laccase on other dyes, the ability to decolourize these was achieved in the presence of the redox mediator ABTS, with 50% decolourization in 0.5–5.4 days.     

Site specific chemical delivery of NSAIDs to inflamed joints: synthesis, biological activity and gamma-imaging studies of quaternary ammonium salts of tropinol esters of some NSAIDs or their active metabolites

Quaternized tropinol ester derivatives of some commonly used non-steroidal anti-inflammatory drugs (NSAIDs) or their active metabolites, were prepared and studied for their anti-inflammatory activity in a chronic inflammation model and for inflamed tissue tropism. The quaternized esters were radiolabeled with 99mTechnetium (99mTc) and their selective localization in the inflamed tissue was traced using scintigraphy. In the chronic arthritis rodent model, most of the quaternized esters exhibited anti-inflammatory effect comparable to their respective parent drugs. In the gamma-imaging studies only the quaternary derivatives exhibited selective accumulation into the inflamed tissue unlike the parent NSAIDs or the unquaternized tropinol esters. This work is a step ahead in the direction of use of quaternary ammonium ester derivatives for site specific chemical delivery of commonly used NSAIDs to the inflamed tissues to minimize their GIT side effect or other systemic toxicities.     

Ranking of binding and nonbinding peptides to MHC class I molecules using inverse folding approach: Implications for vaccine design

T cell recognition of the peptide–MHC complex initiates a cascade of immunological events necessary for immune responses. Accurate T-cell epitope prediction is an important part of the vaccine designing. Development of predictive algorithms based on sequence profile requires a very large number of experimental binding peptide data to major histocompatibility complex (MHC) molecules. Here we used inverse folding approach to study the peptide specificity of MHC Class-I molecule with the aim of obtaining a better differentiation between binding and nonbinding sequence. Overlapping peptides, spanning the entire protein sequence, are threaded through the backbone coordinates of a known peptide fold in the MHC groove, and their interaction energies are evaluated using statistical pairwise contact potentials. We used the Miyazawa & Jernigan and Betancourt & Thirumalai tables for pairwise contact potentials, and two distance criteria (Nearest atom ≫ 4.0 Å & C-beta ≫ 7.0 Å) for ranking the peptides in an ascending order according to their energy values, and in most cases, known antigenic peptides are highly ranked. The predictions from threading improved when used multiple templates and average scoring scheme. In general, when structural information about a protein-peptide complex is available, the current application of the threading approach can be used to screen a large library of peptides for selection of the best binders to the target protein. The proposed scheme may significantly reduce the number of peptides to be tested in wet laboratory for epitope based vaccine design.     

Effects of the Booroola (FecB) genotypes on growth performance, ewe's productivity efficiency and litter size in Garole x Malpura sheep

The present study was conducted to evaluate the effects of FecB genotypes on body weight, average daily gain (ADG), ewe's productivity efficiency (EPE) and litter size in FecB introgressed GarolexMalpura (GM) crossbred sheep. A total of 235 GM lambs were selected randomly and screened for FecB mutation using forced RFLP-PCR. The majority (69.8%) of GM individuals were carriers (BB and B+) for the FecB mutation and frequency of the FecB allele was about 0.40. The FecB genotypes were significantly (P<0.01) associated with the lamb's body weights from birth to 12 months of age. The generation wise (F(1), F(2) and F(3)), lamb's body weight did not differ significantly at birth, 6 and 12 months of the age, while it differed significantly (P<0.05) at 3 and 9 months of age. The ADG1 (0-3 months) was significantly associated (P<0.05), but not the ADG2 (3-6 months) and ADG3 (6-12 months) between genotypes. Type of birth and sex significantly (P<0.01) affected the body weight from birth to 12 months of age; and body weight of single born lambs was significantly higher (P<0.01) than that of twins and triplets from birth to 12 months of age. Type of birth significantly (P<0.01) affected the ADG1, but had no significant effect on ADG2 and ADG3. Year of birth did not affect the birth and weaning weights, but it significantly affected (P<0.01) the body weight and ADG's after weaning ages. The EPE was affected significantly (P<0.01) by the FecB genotypes at birth, 3 and 12 months of age. The EPE of B+ and BB ewes were 7.86 kg (36.9%) and 2.32 kg (10.9%) higher as compared to ++ ewes at 12 months of age, respectively. The mean litter size of BB ewes (2.17+/-0.24) was significantly higher (P<0.01) than that of B+ ewes (1.73+/-0.04) and ++ ewes (1.03+/-0.23). The present study indicated that the body weight and ADG of carrier lambs (BB and B+) was comparatively lower than that of non-carriers (++), while EPE of B+ ewes was comparatively higher than that of BB and ++ ewes. Further, it is interesting to note that heterozygous and homozygous state of individuals increased 0.70 and 1.14 extra lambs as compared to non-carriers (++), respectively.     

Treatment of catechol bearing wastewater in an upflow anaerobic sludge blanket (UASB) reactor: sludge characteristics

This paper deals with the characteristics of anaerobic microbial granules grown in an UASB reactor treating catechol bearing synthetic wastewater (SWW). The specific methanogenic activity of the sludge showed an increase in trend with an increase in the organic loading rate and the catechol concentration in the SWW. The settling velocity of individual granules in the size range of 0.5-2.5mm was found to be in the range of 30-75mh(-1). The ash content in the sludge was 11.7% with a sludge volume index of 18-20mlg(-1). The inorganic elemental distribution within the granules showed a decrease except that for phosphorous and cobalt, which increased by approximately 12% and 18%, respectively, after the treatment of SWW. Scanning electron microscopy (SEM) coupled with electron disperse X-ray analysis showed an increase in the sulphur content by approximately 300% after the treatment of SWW. Surface mineral composition of the granules determined by XRD analysis indicated the existence of vuagnatite (CaAlSiO(4)(OH)). SEM observation of the granules showed the predominance of Methanosaeta and Methanobacterium type of species on the surface along with a variety of other species.     

Expression of a synthetic cry1EC gene for resistance against Spodoptera litura in transgenic peanut (Arachis hypogaea L.)

The tobacco cutworm (Spodoptera litura) is a polyphagous foliage insect and a major pest on peanut (Arachis hypogaea L.). S. litura is susceptible to the chimeric delta-endotoxin Cry1EC reported earlier. De-embryonated cotyledon explants of peanut were transformed using Agrobacterium tumefaciens strain EHA101 harboring a synthetic cry1EC gene driven by the CaMV 35S promoter. Transgenic plants of peanut with a single copy insertion of cry1EC were selected in the T(0) generation by Southern blot hybridization. Real-time PCR, Western blot and ELISA analysis indicated that expression of the cry1EC gene was higher in single copy T(1) plants. Immunoassay showed expression of Cry1EC up to 0.13% of total soluble protein in T(1) plants. Leaf feeding bioassay on highly expressing transgenic lines showed 100% killing of larvae at the 2(nd) instar stage of S. litura. This is the first report of transgenic peanut plants with resistance to S. litura.     

Alterations in lipid peroxidation and antioxidant status in pregnancy with preeclampsia

Soy consumption is associated with a lower risk of atherosclerotic disease in the oriental population. Genistein is a soy isoflavone bearing estrogenic properties. Previous experiments in our laboratory demonstrated the potentiation of endothelium-independent relaxation of coronary artery by both estrogen and genistein. The potentiating effects of both estrogen and genistein were mediated through the cAMP-signaling pathway. We hypothesize that genistein could enhance protein kinase A (PKA) activity in porcine coronary artery smooth muscle, thereby offering a mechanism for the potentiation of vascular relaxation by genistein. In our study, a high concentration of genistein (10^−4.5 M) significantly increased PKA activity in porcine coronary artery rings. While genistein at 10^−5.5 M and forskolin at 10⁻⁷ M had no effect on PKA activity, the combination of the two compounds at the prescribed concentrations caused a significant increase in PKA activity. The increase in PKA activity by genistein was abolished by SQ 22536 (adenylate cyclase blocker), but not by NF 449 (Gs protein blocker) or ICI 182780 (estrogen receptor antagonist). Our results suggest that the action of genistein is mediated via adenylate cyclase, but does not appear to involve Gs protein or ICI 182780-sensitive estrogen receptor.