Synthesis of an Fmoc-threonine bearing core-2 glycan: A building block for PSGL-1 via Fmoc-assisted solid-phase peptide synthesis

Selectins (L, E, and P) are vascular endothelial molecules that play an important role in the recruitment of leukocytes to inflamed tissue. In this regard, P-Selectin glycoprotein-1 (PSGL-1) has been identified as a ligand for P-Selectin. PSGL-1 binds to P-Selectin through the interaction of core-2 O-glycan expressing sialyl Lewis^x oligosaccharide and the three tyrosine sulfate residues. Herein, we report the synthesis of threonine-linked core-2 O-glycan as an amino acid building block for the synthesis of PSGL-1. This building block was further incorporated in the Fmoc-assisted solid-phase peptide synthesis to provide a portion of the PSGL-1 glycopeptide.     

Protection of membrane cholesterol by sphingomyelin against free radical-mediated oxidation

Although the free radical-mediated oxidation of free cholesterol (FC) is critical in the generation of regulatory sterols and in atherogenesis, the physiological regulation of this process is poorly understood. We tested the hypothesis that sphingomyelin (SM), a major phospholipid of cell membranes, which is closely associated with FC, protects FC against oxidation, because of its unique structure, and affinity to the sterol. We employed phosphatidylcholine (PC) liposomes containing varying amounts of SM, and either radioactive FC or a fluorescent analog, dehydroergosterol (DHE), and determined the oxidative decay of the sterol in presence of 2,2′-azo-bis(2-amidinopropane hydrochloride) (AAPH). Incorporation of 25 mol% of SM in the liposomes inhibited the oxidation of FC or DHE by up to 50%. This inhibition was specific for SM among phospholipids, and was abolished by sphingomyelinase treatment. SM was not degraded during the oxidation reaction, and its effect was not dependent on the nature of the oxidizing agent, because it also inhibited sterol oxidation by FeSO₄/ascorbate, and by cholesterol oxidase. These studies show that SM plays a physiological role in the regulation of cholesterol oxidation by free radicals.     

Bipolar localization of the group II intron Ll.LtrB is maintained in Escherichia coli deficient in nucleoid condensation, chromosome partitioning and DNA replication

Group II introns are mobile genetic elements that invade their cognate intron-minus alleles via an RNA intermediate, in a process known as retrohoming. They can also retrotranspose to ectopic sites at low frequency. In Escherichia coli, retrotransposition of the lactococcal group II intron, Ll.LtrB, occurs preferentially within the Ori and Ter macrodomains of the E. coli chromosome. These macrodomains migrate towards the poles of the cell, where the intron-encoded protein, LtrA, localizes. Here we investigate whether alteration of nucleoid condensation, chromosome partitioning and replication affect retrotransposition frequencies, as well as bipolar localization of the Ll.LtrB intron integration and LtrA distribution in E. coli. We thus examined these properties in the absence of the nucleoid-associated proteins H-NS, StpA and MukB, in variants of partitioning functions including the centromere-like sequence migS and the actin homologue MreB, as well as in the replication mutants DeltaoriC, seqA, tus and topoIV (ts). Although there were some dramatic fluctuations in retrotransposition levels in these hosts, bipolar localization of integration events was maintained. LtrA was consistently found in nucleoid-free regions, with its localization to the cellular poles being largely preserved in these hosts. Together, these results suggest that bipolar localization of group II intron retrotransposition results from the residence of the intron-encoded protein at the poles of the cell.     

Dual-function protein in plant defence: seed lectin from Dolichos biflorus (horse gram) exhibits lipoxygenase activity

Plant-pathogen interactions play a vital role in developing resistance to pests. Dolichos biflorus (horse gram), a leguminous pulse crop of the subtropics, exhibits amazing defence against attack by pests/pathogens. Investigations to locate the possible source of the indomitable pest resistance of D. biflorus, which is the richest source of LOX (lipoxygenase) activity, have led to a molecule that exhibits LOX-like functions. The LOX-like activity associated with the molecule, identified by its structure and stability to be a tetrameric lectin, was found to be unusual. The evidence for the lectin protein with LOX activity has come from (i) MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS, (ii) N-terminal sequencing, (iii) partial sequencing of the tryptic fragments of the protein, (iv) amino acid composition, and (v) the presence of an Mn2+ ion. A hydrophobic binding site of the tetrameric lectin, along with the presence of an Mn2+ ion, accounts for the observed LOX like activity. This is the first ever report of a protein exhibiting both haemagglutination and LOX-like activity. The two activities are associated with separate loci on the same protein. LOX activity associated with this molecule adds a new dimension to our understanding of lectin functions. This observation has wide implications for the understanding of plant defence mechanisms against pests and the cellular complexity in plant-pathogen interactions that may lead to the design of transgenics with potential to impart pest resistance to other crops.     

Regulation of the activity and fatty acid specificity of lecithin-cholesterol acyltransferase by sphingomyelin and its metabolites, ceramide and ceramide phosphate

Sphingomyelin (SM), the second most abundant phospholipid in plasma lipoproteins, was previously shown to be a physiological inhibitor of the lecithin-cholesterol acyltransferase (LCAT) reaction. In this study, we investigated the effects of its metabolites, ceramide and ceramide phosphate, on the activity and fatty acid specificity of LCAT in vitro. Treatment of SM-containing substrate with SMase C, which hydrolyzes SM to ceramide, abolished the inhibitory effect of SM, whereas treatment with SMase D, which hydrolyzes it to ceramide phosphate, increased the level of inhibition. Although incorporation of ceramide into the substrate in the absence of SM activated the LCAT reaction only modestly, its co-incorporation with SM neutralized the inhibitory effect of SM. Ceramide phosphate, on the other hand, inhibited the LCAT reaction more strongly than SM. The effects of the sphingolipids on the phospholipase A and cholesterol esterification reactions of the enzyme were similar, indicating that they regulate the binding of phosphatidylcholine (PC) to the active site, rather than the esterification step. Incorporation of ceramide into the substrate stimulated the synthesis of unsaturated cholesteryl esters at the expense of saturated esters. However, these effects on fatty acid specificity disappeared when the PC substrates were incorporated into an inert diether PC matrix, suggesting that ceramide increases the availability of polyunsaturated PCs to the enzyme by altering the macromolecular structure of the substrate particle. Since the plasma ceramide levels are increased during inflammation, these results indicate that the activity and fatty acid specificity of LCAT may be altered during the inflammatory response.     

Mutant and Overexpression Analysis of a C2H2 Single Zinc Finger Gene of Arabidopsis

The goal of this work was to characterize an Arabidopsis C2H2 single zinc finger gene, named AtZFP11, that is similar to SUPERMAN (SUP) and RABBIT EARS (RBE). No altered phenotype was observed in mutants analyzed that were derived through TILLING nor a T-DNA insertion into the exon of AtZFP11. Plants derived by intercrossing AtZFP11 to SUP and RBE alleles did not display any phenotype that could be attributed to AtZFP11. Transgenic plants constitutively overexpressing the AtZFP11 protein displayed severe abnormalities. Phenotypically normal transgenic plants that contained an ecdysone-inducible promoter system fused to the AtZFP11 gene were recovered. These transgenic plants displayed similar abnormalities once induced with methoxyfenozide. Microarray and RT-PCR analysis of gene expression in plants overexpressing AtZFP11 after induction revealed changes in gene expression of a number of genes involved in auxin, jasmonic acid, and stress responses. These results suggest that AtZFP11 plays a role in regulation of gene expression and may be functionally redundant or that conditions needed to observe phenotypic differences in the mutant plants were not provided in the course of these experiments.     

Characterization of temperature inducible promoters from a novel rolling circle replicating plasmid of Enterococcus faecium DJ1

Limited studies have been performed on the characterization of small size plasmids of Enterococcus faecium with the intention of evaluating the strength of their promoters in Escherichia coli. The complete nucleotide sequence (3.825 Kb) and structural organization of E. faecium DJ1 cryptic plasmid pNJAKD is presented. Seven promoter sequences from the pNJAKD plasmid of E. faecium have been identified. The regions coding for the putative promoters were either amplified using PCR based techniques or chemically synthesized as oligonucleotides of different sizes. These were subsequently cloned in the pEGFP vector at the Pvu II site. The efficiency of putative promoter fragments were measured using the intensity of eGFP fluorescence in E. coli JM101, DH5α and BL21(DE3), among which AKD3 exhibited moderate to strongest promoter activity at temperatures of 30, 37, and 42°C.     

Synthesis and evaluation of novel 2-pyridone derivatives as inhibitors of phosphodiesterase3 (PDE3): A target for heart failure and platelet aggregation

Twenty-six 2-pyridone derivatives (8a-8z), which are structurally analogous to amrinone and milrinone two important cardiotonic drugs, are synthesized and characterized. The synthesis of 2-pyridone derivatives involves addition, followed by cyclization between Baylis-Hillman acetates (7a-7k) and enamino esters or nitriles (3a-3e). Thus synthesized pyridones were subjected to PDE3 inhibitory activity, 14 pyridones were found to be hits out of 26 pyridones synthesized and out of 14 hits, there are 5 pyridones found to be lead compounds having excellent PDE3 inhibitory activity. Further we have carried out computational analysis to understand protein/enzyme and 2-pyridone derivative interactions to identify amino acid residues involved in the vicinity of binding and compared with milrinone drug.