Probing the structure of Leishmania donovani chagasi DHFR-TS: comparative protein modeling and protein–ligand interaction studies

Dihydrofolate reductase (DHFR) has been used successfully as a drug target in the area of anti-bacterial, anti-cancer and anti-malarial therapy. It also acts as a drug target for Leishmaniasis. Inhibition of DHFR leads to cell death through lack of thymine (nucleotide metabolism). Although the crystal structures of Leishmania major and Trypanosoma cruzi DHFR-thymidylate synthase (TS) have been resolved, to date there is no three-dimensional (3D)-structural information on DHFR-TS of Leishmania donovani chagasi, which causes visceral leishmaniasis. Our aim in this study was to model the 3D structure of L. donovani chagasi DHFR-TS, and to investigate the structural requirements for its inhibition. In this paper we describe a highly refined homology model of L. donovani chagasi DHFR-TS based on available crystallographic structures by using the Homology module of Insight II. Structural refinement and minimization of the generated L. donovani chagasi DHFR-TS model employed the Discover 3 module of Insight II and molecular dynamic simulations. The model was further validated through use of the PROCHECK, Verify_3D, PROSA, PSQS and ERRAT programs, which confirm that the model is reliable. Superimposition of the model structure with the templates L. major A chain, L. major B chain And T. cruzi A chain showed root mean square deviations of 0.69 Å, 0.71 Å and 1.11 Å, respectively. Docking analysis of the L. donovani chagasi DHFR-TS model with methotrexate enabled us to identify specific residues, viz. Val156, Val30, Lys95, Lys75 and Arg97, within the L. donovani chagasi DHFR-TS binding pocket, that play an important role in ligand or substrate binding. Docking studies clearly indicated that these five residues are important determinants for binding as they have strong hydrogen bonding interactions with the ligand.     

Screening of chemopreventive effect of naringenin-loaded nanoparticles in DMBA-induced hamster buccal pouch carcinogenesis by FT-IR spectroscopy

The aim of the present study is to investigate the chemopreventive effects of the prepared naringenin-loaded nanoparticles (NARNPs) relative to efficacy of free naringenin (NAR) in modifying the functional, structural, and compositional changes at the molecular level during 7, 12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis by Fourier transform infrared (FT-IR) spectroscopy. The results revealed that a significant increase in the amount of proteins and nucleic acid contents and a decrease in the amount of lipids and glycogen contents are observed in DMBA-induced tumor tissues. In addition, in tumor tissues a decrease in lipid order and a significant increase in membrane dynamics were noticed. Further, the composition and secondary structure of proteins were found to be altered, which indicates some important structural alterations in the existing proteins and/or the expression of new types of proteins occurring under the tumor transformation. Furthermore, oral administration of free NAR and NARNPs significantly increased lipids and their order as well as increased the glycogen contents and decreased the levels of proteins and nucleic acid contents. On a comparative basis, NARNPs were found to have a more potent antitumor effect than free NAR in completely preventing the formation of squamous cell carcinoma and in improving the biochemical constituents to a normal range in DMBA-induced HBP carcinogenesis. The present study further shows a great potential of FT-IR spectroscopy as a complimentary tool for the screening of various anticancer drugs and follow-up, which may allow faster response to critical problems arising during treatment.     

丛枝菌根真菌促进南美蟛蜞菊生长及对难溶磷的吸收

为了解2种丛枝菌根真菌(AMF)摩西管柄囊霉(Funneliformis mosseae, FM)和地表球囊霉(Glomus versiforme, GV)对入侵植物南美蟛蜞菊(Wedelia trilobata)的生长和对难溶性磷酸盐利用的影响,采用沙培盆栽方式,研究了南美蟛蜞菊在接种AMF与添加难溶性磷酸盐的生长和磷含量的变化。结果表明,在磷限制环境下FM对南美蟛蜞菊的侵染率达55%～69%,GV的侵染率达到63%～80%。添加难溶性磷酸盐后,2种AMF均促进了南美蟛蜞菊茎的伸长(FM:+46%; GV:+65%)、总生物量的增加(FM:+27.2%; GV:+40%)和磷含量的增加(FM:+36.6%; GV:+40.7%)。对比FM,GV对植物利用难溶性磷有更显著的促进作用。因此,南美蟛蜞菊与2种AMF形成的共生体系可以促进植物生长和对营养资源的利用,提高对难溶性磷的吸收效率可能使得南美蟛蜞菊在营养贫乏的环境中更好地建立种群。     

Oligonucleotide Conjugates: Alteration of the Pharmacokinetic Properties of Antisense Agents

Abstract

Cholic acid, cholesterol, several polyamines and polyethylene glycols were conjugated to antisense oligonucleotides targeted to human or murine intercellular adhesion molecule-1 (ICAM-1) mRNA to study their effects on cellular absorption.     

Solid Phase Conjugation Chemistry: Use of Alloc as a Protecting Group for 2′-Aminolinker Containing Oligonucleotides

Abstract

The drug properties of antisense and antigene oligonucleotides can he enhanced by strategic positioning of ligands capable of ameliorating these properties.^1,2 Certain ligands may improve the cellular delivery of oligomers and increase their affinity for the target gene and resistance to nucleases. The 2′-O-position is an attractive modification site.³ Oligonucleotides possessing the 2′-O-akyl modifications exhibit higher chemical stability under depurination conditions, higher stability to enzymatic cleavage by both endo- and exonucleases, and increased affinity for target mRNA. In addition, they form highly stable triple helices. Thus they promise to be versatile compounds in controlling gene expression by antisense and antigene technologies.     

Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars

For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacterium-mediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l^?1 citric acid, 100 µM acetosyringone, and 100 mg l^?1 l-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l^?1 hygromycin and the embryos were germinated in basal medium containing 20 mg l^?1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into soybean has been developed.     

The role of charged residues in the transmembrane helices of monocarboxylate transporter 1 and its ancillary protein basigin in determining plasma membrane expression and catalytic activity

Monocarboxylate transporters MCT1-MCT4 require basigin (CD147) or embigin (gp70), ancillary proteins with a glutamate residue in their single transmembrane (TM) domain, for plasma membrane (PM) expression and activity. Here we use site-directed mutagenesis and expression in COS cells or Xenopus oocytes to investigate whether this glutamate (Glu₂₁₈ in basigin) may charge-pair with a positively charged TM-residue of MCT1. Such residues were predicted using a new molecular model of MCT1 based upon the published structure of the E. coli glycerol-3-phosphate transporter. No evidence was obtained for Arg₃₀₆ (TM 8) of MCT1 and Glu₂₁₈ of basigin forming a charge-pair; indeed E218Q-basigin could replace WT-basigin, although E218R-basigin was inactive. No PM expression of R306E-MCT1 or D302R-MCT1 was observed but D302R/R306D-MCT1 reached the PM, as did R306K-MCT1. However, both were catalytically inactive suggesting that Arg₃₀₆ and Asp₃₀₂ form a charge-pair in either orientation, but their precise geometry is essential for catalytic activity. Mutation of Arg₈₆ to Glu or Gln within TM3 of MCT1 had no effect on plasma membrane expression or activity of MCT1. However, unlike WT-MCT1, these mutants enabled expression of E218R-basigin at the plasma membrane of COS cells. We propose that TM3 of MCT1 lies alongside the TM of basigin with Arg₈₆ adjacent to Glu₂₁₈ of basigin. Only when both these residues are positively charged (E218R-basigin with WT-MCT1) is this interaction prevented; all other residue pairings at these positions may be accommodated by charge-pairing or stabilization of unionized residues through hydrogen bonding or local distortion of the helical structure.     

Recent advances in barley transformation

Barley, an important member of the cereals, has been successfully transformed through various methods such as particle bombardment, Agrobacterium tumefaciens, DNA uptake, and electroporation. Initially, the transformation in barley concentrated on developing protocols using marker genes such as gus, bar, and hpt. Immature embryos and callus derived from immature embryos were targeted for transformation. Subsequently, genes of agronomic and malting importance have been deployed in barley. Particle bombardment appears to be the preferred choice for barley transformation in the majority of the reports, although Agrobacterium-mediated transformation is being used more often. The current review focuses on the challenges encountered in barley transformation such as somaclonal variation, development of transformation systems for commercial cultivars, gene expression, stability and inheritance, and gene flow. Newer markers such as the green fluorescent protein (gfp), firefly luciferase, and phosphomannose isomerase were found to be useful in the selection of transgenic plants. Tissue-specific promoters such as those for B1-hordein and D-hordein genes, and spike-specific promoters, are increasingly used to drive gene expression. The review also describes recent research on gene-tagging through transformation, insertion of disease resistance, and abiotic stress resistance genes, transformation with genes for improved malting quality, nutrient content, feed quality, and the production of feed enzymes and pharmaceutical compounds.     

Studies on nitrosyl hemes in Ni(II)–Fe(II) hybrid hemoglobins

Subunit heterogeneity within a particular subunit in hemoglobin A have been explored with electron paramagnetic resonance spectroscopy using the nitrosyl hemes in Ni-Fe hybrid Hb under various solution conditions. Our previous studies on the crystal structure of NiHb demonstrated the presence of subunit heterogeneity within alpha-subunit. To further cross check this hypothesis, we made a hybrid Hb in which either the alpha- or beta-subunit contains iron, which alone can bind to NO. By this way dynamic exchange between penta- and hexa-coordinated forms within a subunit was confirmed. Upon the addition of inositol hexa phosphate (IHP) to these hybrids, R to T state transition is observed for [alpha(2)(Fe-NO)beta(2)(Ni)] but such a direct transformation is less marked in [alpha(2)(Ni)beta(2)(Fe-NO)]. Hence the bond between N(epsilon) and Fe is fundamental to the structure-function relation in Hb, as the motion of this nitrogen triggers the vast transformation, which occurs in the whole molecule on attachment of NO.     

Membrane degradation, accumulation of Phosphatidic acid, stimulation of catalase activity and nuclear DNA fragmentation during 2,4-d-induced leaf senescence in mustard

We investigated 2,4-D-induced leaf senescence in young mustard seedlings. A set of morphometric, biochemical and molecular parameters were analyzed to characterize senescence markers. In accordance with earlier reports, chloroplast-membrane degradation marked the early phase of leaf senescence based on the analysis of the galactolipid fraction. Degradation of grana occurred earlier to that of the envelope, as revealed by the relative level of their specific galactolipids, namely, monogalactosyl diglyceride and digalactosyl diglyceride. Phospholipids showed extensive degradation resulting in the accumulation of lyso-derivatives of major phospholipids and phosphatidic acid (PA) in senescing leaves. Catalase activity was stimulated by 2,4-D and reflected scavenging of reactive oxygen species. Nuclear DNA degradation, a previously known death signal that represented a point of no return from progression of senescence, occurred late on the 4th day subsequent to 2,4-D supplementation. AgNO₃, an inhibitor of ethylene biosynthesis, inhibited leaf senescence by ca. 54% based on PA content Involvement of 2,4-D, ethylene and abscisic acid in leaf senescence is discussed in relation to hormonal interplay.