Genome mapping,molecular markers and marker-assisted selection in crop plants

Molecular Breeding -     

Time-resolved fluoroimmunoassay of potato virus M with monoclonal antibodies

Mouse monoclonal antibodies (MAbs) specific for potato virus M (PVM) were prepared and the properties of three of them were studied. MAb M4C1 is IgG2b, it binds with high affinity to PVM coat protein, to purified virus preparations and recognises PVM in infected potato leaves and tubers. MAb M6D5 is IgG2a and also reacts with PVM coat protein, purified PVM and with PVM in potato leaf and tuber extracts. In double-antibody sandwich ELISA (DAS ELISA) MAbs M4C1 and M6D5 reacted with all 17 PVM isolates tested. MAb M7 is IgG2b and recognises PVM only in indirect dot ELISA on nitrocellulose filters and viral coat protein on Western blots. MAbs against PVM were used as capture antibodies and europium-labelled MAbs as conjugates in time-resolved fluoroimmunoassay (EuTRFIA). The standard EuTRFIA curve of PVM detection is approximately linear over a range of PVM concentrations from 0.5 ng/ml to 1000 ng/ml. The lowest PVM concentration detectable in EuTRFIA was 0.5 ng/ml and correspondingly 6 ng/ml in DAS ELISA. The use of the europium chelate label allows PVM detection in potato leaf and tuber sap at dilutions greater than 10^--⁴ with very low background fluorescence. EuTRFIA with MAbs, with either one or two incubations is about 10–20 times more sensitive for PVM detection than is DAS ELISA. PVM and PVX, mixed with healthy potato tuber sap, were simultaneously tested in a single sample at concentrations lower than 10 ng/ml by double-label TRFIA using europium-labelled MAbs to PVM and samarium-labelled MAbs to PVX.     

Purification and characterization of the MUC1 mucin-type glycoprotein, epitectin, from human urine: structures of the major oligosaccharide alditols

The MUC1 glycoprotein, epitectin, a component of the human bladder epithelium, was purified from human urine. Sedimentation equilibrium analysis and gel filtration using polysaccharide or protein standards revealed a polydisperse preparation with molecular weights ranging from about 0.9 to 1.3×106. This suggests that in the native state epitectin exists as aggregates of three or four monomer units of 350–400 kDa. Epitectin was found to have significant affinity to hexyl-, octyl- or phenyl agarose indicating that hydrophobic interactions and possibly carbohydrate-carbohydrate interactions may be responsible for the self-association. Chemical and enzymic deglycosylation of [125I]-labeled urine epitectin and metabolically labeled H.Ep.2 epitectin resulted in extremely polydisperse products. The buoyant densities of epitectin purified from urine and H.Ep.2 cells were found to be 1.39–1.40 g ml–1, suggesting that the total carbohydrate content of these preparations is not significantly different. The O-linked saccharides of epitectin were fractionated by HPLC and analyzed by permethylation and FAB-MS. The neutral saccharides from both sources 001contain three common structures, namely Gal13GalNAc, GlcNAc16 (Gal13) GalNAc and Gal14 GlcNAc6 (Gal13)GalNAc. The sialic acid of urine epitectin consisted entirely of N-acetylneuraminic acid. The two sources of epitectin, in vitro labeled on sialic acid, were found to have the same sialyl oligosaccharides but in different proportions. Metabolic labeling and N-glycanase susceptibility experiments firmly established the presence of N-linked saccharides in epitectin as minor components. The remarkable similarities in the total carbohydrate content, the carbohydrate composition and structures of saccharides between epitectin from urine, a non-malignant source, and H.Ep.2 cells is surprising in view of the prevailing view that MUC1 glycoproteins of cancer cells are underglycosylated compared to those produced by non-malignant cells.     

Spectroscopic and biological activity studies of the chromium-binding peptide EEEEGDD

While trivalent chromium has been shown at high doses to have pharmacological effects improving insulin resistance in rodent models of insulin resistance, the mechanism of action of chromium at a molecular level is not known. The chromium-binding and transport agent low-molecular-weight chromium-binding substance (LMWCr) has been proposed to be the biologically active form of chromium. LMWCr has recently been shown to be comprised of a heptapeptide of the sequence EEEEDGG. The binding of Cr³⁺ to this heptapeptide has been examined. Mass spectrometric and a variety of spectroscopic studies have shown that multiple chromic ions bind to the peptide in an octahedral fashion through carboxylate groups and potentially small anionic ligands such as oxide and hydroxide. A complex of Cr and the peptide when administered intravenously to mice is able to decrease area under the curve in intravenous glucose tolerance tests. It can also restore insulin-stimulated glucose uptake in myotubes rendered insulin resistant by treating them with a high-glucose media.     

Characterization and biological activities of cyclic (1 → 3, 1 → 6)-β-glucans from <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis>

Objective

To isolate cyclic (1 → 3, 1 → 6)-β-glucan from Bradyrhizobium japonicum MTCC120, to characterize its structure and to study its biological activities.

Results

The degree of polymerization of cyclic (1 → 3, 1 → 6)-β-glucan varied between 10 and 13 and with substituents acetyl, succinyl and phosphocholine. The cyclic glucans showed bimodal particle size distribution, with hydrodynamic diameters of 1.92 and 231 nm corresponding to monomeric and aggregated cyclic glucans, respectively. SEM and TEM images showed that the glucans formed aggregates of nanorods. The glucans were biocompatible, exhibited good antioxidant activity and had the abilities to bind to Aniline Blue dye to form a fluorescence complex which was concentration dependent.

Conclusion

The glucans isolated are cyclic and have good antioxidant activities, hence have potential application in food and pharmaceutical industries. Their dye binding ability could be exploited in medical imaging to reduce the cytotoxicity of the dyes.

     

Cardiomyocyte-specific knockout of endothelin receptor a attenuates obesity cardiomyopathy

Endothelin (ET)-1 is implicated in the pathophysiology of cardiovascular diseases although its role in obesity anomalies has not been fully elucidated. This study was designed to examine the impact of ET-1 receptor A (ET_A) ablation on obesity-induced changes in cardiac geometry and contractile function, as well as the mechanisms involved with a focus on autophagy. Cardiomyocyte-specific ET_A receptor knockout (ETAKO) and WT mice were fed either low-fat (10% calorie from fat) or high-fat (45% calorie from fat) diet for 24?weeks. Glucose tolerance test was examined to confirm insulin resistance. High-fat diet intake compromised myocardial geometry (enlarged left ventricular diameters in systole and diastole), morphology (cardiac hypertrophy, increased wall thickness and interstitial fibrosis), contractile function (reduced fractional shortening, ejection fraction and cardiomyocyte shortening) and intracellular Ca²⁺ handling, the effect of which was significantly attenuated by ETAKO. TUNEL staining revealed overt apoptosis in high-fat-fed group, the effect was reverted by ETAKO. Western blot analysis noted that high-fat intake downregulated leptin receptor and PPARγ, insulin signaling (elevated basal/dampened insulin-stimulated phosphorylation of Akt and IRS1), phosphorylation of AMPK, ACC, upregulated GATA-4, ANP, NFATc3, PPARα, m-TOR/p70s6k signaling, which were attenuated by ETAKO with the exception of AMPK/ACC. Furthermore, high-fat intake suppressed cardiac autophagy, which was abrogated by ETAKO. In cultured murine cardiomyocytes, palmitic acid challenged mimicked high-fat diet-induced hypertrophic and autophagic responses, the effect of which were abolished by the ET_A receptor antagonist BQ123 or mTOR inhibitor rapamycin. These results suggest that inhibition of ET_A rescues high-fat intake-induced cardiac anomalies possibly through autophagy regulation.