Highly regioselective methylation of inosine nucleotide: an efficient synthesis of 7-methylinosine nucleotide

Abstract

A facile, straightforward, reliable, and an efficient chemical synthesis of inosine nucleotides such as 7-methylinosine 5′-O-monophosphate, 7-methylinosine 5′-O-diphosphate, and 7-methylinosine 5′-O-triphosphate, starting from the corresponding inosine nucleotide is delineated. The present methylation reaction of inosine nucleotide utilizes dimethyl sulfate as a methylating agent and water as a solvent at room temperature. It is noteworthy that the present methylation reaction proceeds smoothly under aqueous conditions that is highly regioselective to afford exclusive 7-methylinosine nucleotide in good yields with high purity (>99.5%).     

Impact of precursors and plant growth regulators on <Emphasis Type="Italic">in vitro</Emphasis> growth,bioactive lignans,and antioxidant content of <Emphasis Type="Italic">Phyllanthus</Emphasis> species

The production of specific secondary metabolites in vitro can be improved through medium supplementation with secondary metabolite precursors, plant growth regulators (PGRs), and abiotic and biotic elicitors. In the present study, node and internode explants of Phyllanthus amarus and P. urinaria collected from Karkala region, Udupi District, Karnataka, India, were inoculated aseptically onto Murashige and Skoog (MS) medium for callus induction. Uniform calluses were inoculated onto MS medium fortified with one of two precursor’s cinnamic acid (CA) or phenylalanine (PA), or with naphthalene acetic acid (NAA). After 30 d of treatment, calluses from treatment and control groups were harvested and quantitatively analyzed for three lignans (phyllanthin, hypophyllanthin and niranthin) and an antioxidant (ellagic acid). Increased amounts of the lignans and ellagic acid were obtained through supplementation with CA, PA, and NAA, and higher ellagic acid was present at higher amounts than the three lignans. These results demonstrated that the Phyllanthus species collected from Karkala region (designated “Accessions3”) show substantial response to CA, PA, and NAA treatment and represent a potential source of donor plants with higher amounts of lignans and antioxidants. These plants can be cultivated on a large scale both in vitro and in vivo for production of important bioactive compounds. Production of these compounds can be further enhanced through induction of somaclonal variant plants with higher amounts of bioactive molecule production and through production of transgenic plants overexpressing genes related to lignan- and phenolic-compound biosynthesis.     

Rapid definition of five novel HLA-A*3002-restricted human immunodeficiency virus-specific cytotoxic T-lymphocyte epitopes by elispot and intracellular cytokine staining assays

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) play a major role in control of viral replication. To understand the contribution of this antiviral response, an initial step is to fully define the specific epitopes targeted by CTL. These studies focused on CTL responses restricted by HLA-A*3002, one of the HLA-A molecules most prominent in African populations. To avoid the time-consuming effort and expense involved in culturing CTL prior to defining epitopes and restricting alleles, we developed a method combining Elispot assays with intracellular gamma interferon staining of peripheral blood mononuclear cells to first map the optimal epitopes targeted and then define the HLA restriction of novel epitopes. In two A*3002-positive subjects whose CTL responses were characterized in detail, the strongest response in both cases was to an epitope in p17 Gag, RSLYNTVATLY (residues 76 to 86). Using this method, CTL epitopes for which there were no motif predictions were optimized and the HLA restriction was established within 48 to 72 h of receipt of blood. This simple and convenient approach should prove useful especially in the characterization of CTL responses specific to HIV and other viruses, particularly in localities where performing cytotoxicity assays would be problematic.     

Highly Regioselective C-5 Iodination of Pyrimidine Nucleotides and Subsequent Chemoselective Sonogashira Coupling with Propargylamine

An efficient C-5 iodination of pyrimidine-5′-triphosphates and subsequent palladium-catalyzed Sonogashira coupling reaction with propargylamine is described. The iodination reaction is highly regioselective and the coupling reaction is highly chemoselective that furnishes exclusive 5-(3-aminopropargyl)-pyrimidine-5′-triphosphate in good yield with high purity (>99%).     

Extracellular matrix-dependent regulation of angiogenin expression in human placenta

Knowledge of the rapidly developing hierarchy of controls affecting vascular development in placenta is required to understand how the growth factors and their receptor-mediated signals actually produce vessels. At the cell biological level, these events clearly require stable interactions between the cells, and cells with the surrounding ECM. The objective of the study was to understand the role of integrins and ECM on the expression and secretion of angiogenin in placentas and from trophoblasts in culture. Functionally active term placental explant culture and trophoblast cultures were used to demonstrate the differential secretion profile of angiogenin and real-time quantitative RT-PCR to demonstrate the mRNA expression in the presence or absence of ECM proteins. In this study, a significant increase in expression and secretion of angiogenin occurred in the presence of vitronectin (VN) and fibronectin (FN). Using antibody-blocking experiments it was also demonstrated that the angiogenin secretion is mediated by placental integrins, alpha(V)beta3 and alpha5beta1. In addition, exposure to hypoxic conditions resulted in diminished angiogenin secretion in the presence of both ECMs suggesting that angiogenin expression in the presence of ECM is modulated by local O2 concentration. In conclusion, this study provides evidence for the regulatory role of ECM and integrins on the mRNA expression and secretion of angiogenin in human placenta. ECMs may have a pivotal role in enhancing secretion of this peptide necessary for placental angiogenesis and provides the impetus as additional targets for the control of angiogenesis in pathological pregnancy.     

Determination and occurrence of polybrominated diphenyl ethers in maternal adipose tissue from inhabitants of Singapore

Polybrominated diphenyl ethers (PBDEs), as a specific group of brominated flame retardants (BFR), are used in a variety of consumer products including electronics and household furnishings. In recent years, a marked increase in the levels of PBDEs in human biological tissues and fluids, especially breast milk, has been reported in several countries. However, few data are available from countries in the Asia-pacific region, including Singapore. This study presents a validated method procedure and the first available data of the concentrations of PBDE congeners: PBDE-47 (2,2,4,4-Tetrabromodiphenyl ether), PBDE-99 (2,2',4,4',5-Pentabromodiphenyl ether), PBDE-100 (2,2',4,4',6-Pentabromodiphenyl ether), PBDE-153 (2,2',4,4',5,5'-Hexabromodiphenyl ether), PBDE-154 (2,2',4,4',5,6'-Hexabromodiphenyl ether) in maternal adipose tissue collected from inhabitants of Singapore. Microwave-assisted extraction (MAE) of PBDEs spiked adipose tissues coupled with GC-MS analysis achieved comparable recoveries to a conventional Soxhlet Extraction (SE) procedure of between 70 and 130%. MAE also yielded comparable precision data (variance less than 13%) relative to the SE procedure. Spiked Carbon-13 PBDE congeners were also used as surrogates for MAE quality assurance and confirmed the efficiency of the procedure. PBDE congeners were detected in all of 16 maternal adipose tissues collected in Singapore, where levels were comparable to available data from Belgium.     

Precise blood lead analysis using a combined internal standard and standard addition approach with disposable screen-printed electrodes

We report here a highly accurate and efficient method for blood lead analysis (BLA) through the use of a disposable electrode. A new type of mercury-plated preanodized screen-printed carbon electrode, together with a thallium(III) internal standard, simplifies calibration and gives easily quantifiable signals for accurate BLA. A preanodization procedure improves the preconcentration ability of the working electrode through the effect of lead (Pb) complexing with an electrogenerated surface functional group, mainly >C=O. Under optimized conditions, the ratio of the anodic stripping peak currents of Pb and Tl (i.e., i(Pb)/i(Tl)) is linear against [Pb] in the window of 1-300 ppb with a correlation coefficient and detection limit (signal-to-noise ratio=3) of 0.999 and 0.23 ppb, respectively. This approach was used to analyze blood samples (n=55) from workers at a local battery factory. The results are consistent with those obtained from graphite furnace atomic absorption spectroscopy and confirm the applicability of the proposed method.     

Foraging patterns and kleptoparasitism among three sympatric cormorants (Phalacrocorax spp.) from the Delhi region, North India

Cormorants, described as ‘foot-propelled pursuit divers’, constitute an important component of aquatic food webs and exhibit unique foraging behaviour patterns, which can be properly understood through a comparative study. Since, after a foraging dive they surface to ingest the prey, the intensity of kleptoparasitic attacks on the surface can have a major impact upon the net energetic gain for each individual. Inspite of the fact that cormorants and their habitats are severely threatened in India, their foraging behaviour has not been adequately studied. Such considerations prompted us to undertake field studies on three sympatric cormorants (Phalacrocorax spp.) at 60 different sites in the Delhi region of North India, during 2004–2007. By means of video photography, some key foraging parameters including group size, prey size and patterns of kleptoparasitic attacks were quantified. Along a loose body size gradient, we observed significant differences among the three species with respect to not only their preference for wetland size but also prey size. The frequency of a kleptoparasitic attacks depended upon the group size and foraging behaviour of each species. It was observed that several foraging bouts were abruptly terminated due to human disturbances, mostly at sites lying outside the protected areas. This observation points towards the need to conserve small waterbodies in the countryside, currently threatened by pollution and urbanization, for the benefit of a variety of waterbirds including cormorants.     

Convenient strategies for the synthesis of 1,4-phenylene spaced sugars

Two synthetic strategies have been developed for the synthesis of spaced sugars with lipophilic 1,4-phenylene core. A building block combining the usefulness of Weinreb amide functionality and modified Julia olefination reaction has been explored towards this objective. This building block offers complete flexibility in attaching any desired sugar derivative across phenylene spacer via C-C bond formation. The other strategy uses carbohydrate based building blocks for the synthesis of symmetrical as well as unsymmetrical 1,4-phenylene spaced sugars. This is the first report for the synthesis of 1,4-phenylene spaced sugars via C-C bond formation.