Synthesis, in vitro evaluation, and in vivo metabolism of fluor/quencher compounds containing IRDye 800CW and Black Hole Quencher-3 (BHQ-3)

Protease-cleavable peptides containing a suitable fluor/quencher (Fl/Q) pair are optically dark until cleaved by their target protease, generating fluorescence. This approach has been used with many Fl/Q pairs, but little has been reported with IRDye 800CW, a popular near-infrared (NIR) fluor. We explored the use of the azo-bond-containing Black Hole Quencher 3 (BHQ-3) as a quencher for IRDye 800CW and found that IRDye 800CW/BHQ-3 is a suitable Fl/Q pair, despite the lack of proper spectral overlap for fluorescence resonance energy transfer (FRET) applications. Cleavage of IRDye 800CW-PLGLK(BHQ-3)AR-NH(2) (8) and its D-arginine (Darg) analogue (9) by matrix metalloproteinases (MMPs) in vitro yielded the expected cleavage fragments. In vivo, extensive metabolism was found. Significant decomposition of a "non-cleavable" control IRDye 800CW-(1,13-diamino-4,7,10-trioxatridecane)-BHQ-3 (10) was evident in plasma of normal mice by 3 min post injection. The major metabolite showed a m/z and UV/vis spectrum consistent with azo bond cleavage in the BHQ-3 moiety. Preparation of an authentic standard of this metabolite (11) confirmed the assignment. Although the IRDye 800CW/BHQ-3 constructs showed efficient contact quenching prior to enzymatic cleavage, BHQ-3 should be used with caution in vivo, due to instability of its azo bond.     

Biocontrol efficacy of protoplast fusants between Bacillus thuringiensis and Bacillus subtilis against Spodoptera litura Fabr.

Bacillus thuringiensis (Bt) is a microbial pesticide widely used to control crop pests. Its strains have good biocontrol activity against crop insect pest, but lack some desirable characteristics that are found in Bacillus subtilis. An attempt has been made to combine those desirable characteristics; we used a highly effective biocontrol strain of B. thuringiensis in protoplast fusions with a strain of B. subtilis. The fusants were identified through cell culture and stained with crystal violet. The Bt and B. subtilis protoplasts were induced to fuse by PEG 6000. The fusants were produced almost 95% mortality in first instar larvae of Spodoptera litura. The lethal doses (The LC₅₀ and LC₉₀) for mortality of S. litura values were significantly in lower level in the fusant-treated larvae, when compared with Bt and B. subtilis individual treatment. The consumption and digestion of S. litura significantly decreased after treatment with fusant. Also the approximate digestibility of S. litura increased significantly.     

A database for human Y chromosome protein data

The human Y chromosome is the sex determining chromosome. The number of proteins associated with this chromosome is 196 and 107 of the 196 proteins have yet not been characterised. Here, we describe the analysis of these 107 proteins by computing various physicochemical properties using sequence and predicted structural data to elucidate molecular function. We present the derived data in the form a form a database made freely available for download, review, refinement and update.

Availability

http://puratham.googlepages.com/ or http://puratham.googlepages.com/ftpconnection     

Enhanced structure prediction of gene products containing class III adenylyl cyclase domains

Domain finding algorithms are useful to understand overall domain architecture and to propose biological function to gene products. Automated methods of applying these tools to large-scale genome studies often employ stringent thresholds to recognize sequence domains. The realization of additional domains can be tedious involving manual intervention but can lead to better understanding of overall biological function. We propose a multi-step approach for the further examination of unassigned linker regions that exploits properties such as the conservation of domain architectures of homologous proteins to propose connections. Improved structure prediction is possible starting from initial domain architectures, obtained from simple 'domain finding' techniques, by concentrating on connecting unassigned regions. 254 unassigned regions have been examined in 114 gene products that potentially contain at least one class III adenylyl cyclase domain for a pilot study. Reliable structure prediction was possible for nearly 80% of unassigned regions. New connections were recognized that assign putative structure and function to these regions by indirect searches (26%). Several others (34%) could be associated with three-dimensional models that might pertain to novel folds and new functions with enough structural content and evolutionary conservation. The presence of additional domains will provide further clues to the overall function of the gene products and their recruitment in particular biochemical pathways.     

GOPET: A tool for automated predictions of Gene Ontology terms

Background  

Vast progress in sequencing projects has called for annotation on a large scale. A Number of methods have been developed to address this challenging task. These methods, however, either apply to specific subsets, or their predictions are not formalised, or they do not provide precise confidence values for their predictions.     

Evaluation of antipyretic activity of leaf extracts of Mallotus peltatus (Geist) Muell. arg. var acuminatus: a folk medicine.

A study was carried out to evaluate the anti-pyretic potential of the methanol extract of Mallotus peltatus (Geist) Muell. Arg. var acuminatus leaf, a folk medicine of Onge tribes of Bay Islands, on normal body temperature and yeast-induced pyrexia in Wister albino rats. The leaf extract at oral doses of 100, 200 and 300 mg kg(-1), p.o., showed significant reduction in normal body temperature and yeast-provoked elevated temperature in a dose-dependent manner and the anti-pyretic effect was comparable to that of standard anti-pyretic agent paracetamol (150 mg kg(-1), p.o.). The effect also extended up to 5 hours after the drug administration.     

Biosorption of inorganic and methyl mercury by a biosorbent from Aspergillus niger

A biosorbent prepared by alkaline extraction of Aspergillus niger biomass was evaluated for its potential to remove mercury species – inorganic (Hg²⁺) and methyl mercury (CH₃Hg⁺) – from aqueous solutions. Batch experiments were carried out to determine the pH and time profile of sorption for both species in the pH range 2–7. The Hg²⁺ exhibited more rapid sorption and higher capacity than the CH₃Hg⁺. Further, removal of both mercury species from spiked ground water samples was efficient and not influenced by other ions. Sorption studies with esterified biosorbent indicated loss of binding of both mercury species (>80%), which was regained when the ester groups were removed by alkaline hydrolysis, suggesting the involvement of carboxyl groups in binding. Further, no interconversion of sorbed species occurred on the biomass. The biosorbent was reusable up to six cycles without serious loss of binding capacity. Our results suggest that the biosorbent from Aspergillus niger can be used for removal of mercury and methyl mercury ions from polluted aqueous effluents.     

Anti-diabetic potential of Barleria lupulina extract in rats.

We have undertaken a study to evaluate the anti-hyperglycemic effect of a methanol extract of aerial parts of Barleria lupulina Lindl. (Acanthaceae) in streptozotocin-diabetic rats, based on folkloric reports its use as an anti-diabetic agent. The extract exerted significant (p < 0.05) anti-hyperglycemic efficacy at all levels tested from 4 h after its administration, as compared with the control group, and the effect was also prolonged up to 12 h. The extract at doses of 200 mg kg(-1) body wt. and above exhibited a maximum activity (p < 0.001) at 12 h after administration. The most significant activity (15.35% blood glucose reduction) was observed for the group administered 300 mg kg(-1) body wt. at 12 h after administration, while the standard drug glibenclamide (10 mg/kg(-1) body wt.) showed an 18.80% reduction of blood glucose at the same time interval. Based on our current results, it appears that the methanol extract of aerial parts of Barleria lupulina Lindl. shows a pronounced blood-glucose-lowering potential in streptozotocin hyperglycemic rats, and is thus provided with a pharmacological support of the folklore claims of anti-diabetic activity.     

Dynamics of sialyl Lewis(a) in aqueous solution and prediction of the structure of the sialyl Lewis(a)-SelectinE complex

In this article we investigate all possible three-dimensional structures for sialyl Lewis^a (SLe^a) in aqueous solution and we predict without a priori experimental information its conformation when bound to SelectinE by using a combination of long molecular dynamics (MD) simulations. Based on 10 ns MD studies, three structures differing in glycosidic conformations are proposed for SLe^a in aqueous solution. Based on a 4 ns MD study of the SLe^a-SelectinE complex with initial structures derived from our prediction tools, we find that, fucose and N-acetyl neuraminic acid are in close contact with SelectinE and therefore expect interactions of the protein with these two sugar rings to be significantly more important than in the case of galactose and N-acetyl glucosamine. Our predictions indicate that the N-acetyl glucosamine of SLe^a is positioned primarily in the aqueous phase. In order to be able to interact with SLe^a the side chains of amino acid residues Lys99 and Lys111 in SelectinE appear to undergo large conformational changes when contrasted with the positions of these residues in the X-ray crystal structure. Furthermore, amino acid residues Arg97, Glu98 and Lys99 are acting as a holding arm to position the NeuNAc of SLe^a in the binding pocket.