Two-dimensional electrophoresis of surface glycoproteins of normal BHK cells and ricin resistant mutants

The surface glycoproteins of baby hamster kidney (BHK) cells were iodinated by lactoperoxidase and submitted to a two-dimensional electrophoresis procedure involving isoelectric focusing in the first dimension and SDS gel electrophoresis in the second dimension. After autoradiography a complex but reproducible pattern was obtained. The technique was then applied to the study of three ricin-resistant mutant clones with reduced rates of cell-cell and/or cell-substratum adhesion. Abnormal patterns were observed in all three mutant clones indicating different mechanisms of ricin resistance and identifying glycoproteins which may be involved in cellular interactions.     

Immunocytochemical localization of NAD-dependent glyceraldehyde-3-phosphate dehydrogenase in soybean nodules

Polyclonal antibodies raised against NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase [phosphorylating], EC 1.2.1.12) from the plant cytosolic fraction of soybean [ Glycine max (L.) Merr. cv. Williams] nodules were used to study the subcellular location of the enzyme and its relative distribution between infected, interstitial and cortical cells of soybean (cv. Lincoln) nodules. Post-embedding immunogold labelling was carried out on nodules harvested 5, 12, 19 and 25 days after the first sign of nodulation. Labelling for glyceraldehyde-3-phosphate dehydrogenase was observed over the cytoplasm and nuclei of infected and uninfected cells, as well as over the nucleoid regions of bacteroids. In 5-day-old nodules, label also bound adjacent to the peribacteroid membranes. Statistical analysis of the number of gold particles per cell area indicated that in 5-day-old nodules, glyceraldehyde-3-phosphate dehydrogenase was distributed equally between infected, interstitial and cortical cells, but in older nodules the enzyme was more prominent in the interstitial and cortical cells than in infected cells.     

Clickable gold nanoparticles for streamlining capture,enrichment and release of alkyne-labelled proteins

Alkyne-labelled proteins are generated as key intermediates in the chemical probe-based approaches to proteomics analysis. Their efficient and selective detection and isolation is an important problem. We designed and synthesized azide-functionalized gold nanoparticles as new clickable capture reagents to streamline click chemistry-mediated capture, enrichment and release of the alkyne-labelled proteins in one-pot to expedite the post-labelling analysis. Because hydrophobic surface functionalities are known to render gold nanoparticles poorly water-dispersible, hydrophilic PEG linkers with two different lengths were explored to confer colloidal stability to the clickable capture reagents. We demonstrated the ability of the capture reagents to conjugate the alkyne containing proteins at a nanomolar concentration via click chemistry, which can be immediately followed by their enrichment and elution. Furthermore, a bifunctional clickable capture reagent bearing sulforhodamine and azide groups was shown to conveniently attach a fluorophore to the alkyne-labelled protein upon click capture, which facilitated their rapid detection in the gel analysis.     

Structure and assembly of epiglucan, the extracellular (1-->3;1-->6)-beta-glucan produced by the fungus Epicoccum nigrum strain F19

In a previous article [Carbohydr. Res.2001, 331, 163-171] two different structures for the possible modular repeating unit of the extracellular beta-glucan, epiglucan produced by the fungus Epicoccum nigrum strain F19 were proposed. Clarifying which was the more likely one was considered essential before attempts were made to understand how epiglucan was assembled by this fungus. Data from Smith degradation analyses of epiglucan were consistent with the repeating unit of structure I, where single glucosyl residues are attached by (1-->6)-beta-linkages to two out of every three glucosyl residues in the (1-->3)-beta-linked glucan backbone. Repeated Smith degradations of 14C-glucose labelled epiglucan showed that chain elongation occurred from its non-reducing end. Side chain insertion into the growing glucan was followed by analysis of real time incorporation of 13C-glucose into epiglucan by 13C NMR, and 14C-glucose by enzymic digestion of the synthesised 14C-epiglucan. All data obtained were consistent with the view that single (1-->6)-beta-linked glucosyl side residues are inserted simultaneously as the glucan backbone elongates.     

Direct labelling of BAC-DNA by rolling-circle amplification

Efficient amplification and labelling of probes are crucial for successful sequence detection by fluorescent in situ hybridization (FISH). In particular, chromosome painting to visualize chromosome segments or entire chromosomes by FISH requires large amounts of probes derived from extended templates. There are a number of techniques for probe labelling. The most widespread is nick translation, based on the replicational incorporation of modified nucleotides. Here we demonstrate successful rolling-circle amplification (RCA) of very low amounts of long circular template sequences (single bacterial artificial chromosomes (BACs) or pools of BACs). The amplicons were suitable for labelling by nick translation and subsequent FISH. A novel achievement is the use of RCA for simultaneous amplification and labelling of single BACs or BAC pools in a labour- and cost-effective manner.     

The cancer cell secretome: A good source for discovering biomarkers?

Cancer is a leading cause of death. Early detection is usually associated with better clinical outcomes. Recent advances in genomics and proteomics raised hopes that new biomarkers for diagnosis, prognosis or monitoring therapeutic response will soon be discovered. Proteins secreted by cancer cells, referred also as “the cancer cell secretome”, is a promising source for biomarker discovery. In this review we will summarize recent advances in cancer cell secretome analysis, focusing on the five most fatal cancers (lung, breast, prostate, colorectal, and pancreatic). For each cancer type we will describe the proteomic approaches utilized for the identification of novel biomarkers. Despite progress, identification of markers that are superior to those currently used has proven to be a difficult task and very few, if any, newly discovered biomarker has entered the clinic the last 10 years.     

High yield expression and purification of isotopically labelled human endothelin-1 for use in NMR studies

Human endothelin-1 (ET-1) is a potent vasocontractile 21-residue peptide hormone with significant pharmacological importance. An efficient and straightforward expression strategy that enables cost-effective incorporation of stable isotopes is not available thus far. In this report, we describe a cost-effective expression system in Escherichia coli for the production of ET-1 enriched with (15)N and (13)C isotopes. Employing thioredoxin as carrier protein, specific and nearly quantitative cleavage of ET-1 from the fusion was mediated by Factor Xa, and purification to homogeneity (final purity of >95%) was achieved by RP-HPLC. Purified recombinant ET-1 was found to be indistinguishable from the synthetic counterpart as determined by mass spectrometry and NMR spectroscopy. Our expression strategy offers the potential for production of isotopically labeled ET-1 in large (mg) quantities for the purpose of heteronuclear NMR experiments. Moreover, the method devised should be applicable for recombinant expression of small peptides in general.     

Synergistic activity between Bacillus thuringiensis Cry1Ab and Cry1Ac toxins against maize stem borer (Chilo partellus Swinhoe)

Aim: To select a toxin combination for the management of maize stem borer (Chilo partellus) and to understand possible mechanism of synergism among Bacillus thuringiensis Cry1A toxins tested. Methods and Results: Three Cry1A toxins were over expressed in Escherichia coli strain JM105 and used for diet overlay insect bioassay against C. partellus neonate larvae, both alone and in combinations. Probit analysis revealed that the three Cry1A toxins tested have synergistic effect against C. partellus larvae. In vitro binding analysis of fluorescein isothiocyanate (FITC)‐labelled Cry1A toxins to midgut brush border membrane vesicle (BBMV) shows that increase in toxicity is directly correlated to an increase in binding of toxin mix. Conclusions: A high Cry1Ac to Cry1Ab ratio leads to an increase in efficacy of these toxins towards C. partellus larvae and this increase in toxicity comes from an increase in toxin binding. Significance and Impact of the Study: Use of Cry1Ab and Cry1Ac combination could be an effective approach to control C. partellus. Furthermore, we show it first time that possible reason behind increase in toxicity of synergistic Cry1A proteins is an increase in toxin binding.