Binding free energy calculations of galectin-3-ligand interactions

Galectins show remarkable binding specificity towards beta-galactosides. A recently developed method for calculating binding free energies between a protein and its substrates has been used to evaluate the binding specificity of galectin-3. Five disaccharides and a tetrasaccharide were used as the substrates. The calculated binding free energies agree quite well with the experimental data and the ranking of binding affinities is well reproduced. For all the six protein-ligand complexes it was observed that electrostatic interactions oppose binding whereas the non-polar contributions drive complex formation. The observed binding specificity of galectin-3 for galactosides rather than glucosides is discussed in light of our results.     

Growth of beta-amyloid(1-40) protofibrils by monomer elongation and lateral association. Characterization of distinct products by light scattering and atomic force microscopy

Amyloid plaques in brain tissue are a hallmark of Alzheimer's disease. Primary components of these plaques are 40- and 42-residue peptides, denoted A beta(1-40) and A beta(1-42), that are derived by proteolysis of cellular amyloid precursor protein. Synthetic A beta(1-40) and A beta(1-42) form amyloid fibrils in vitro that share many features with the amyloid in plaques. Soluble intermediates in A beta fibrillogenesis, termed protofibrils, have been identified previously, and here we describe the in vitro formation and isolation of A beta(1-40) protofibrils by size exclusion chromatography. In some experiments, the A beta(1-40) was radiomethylated to better quantify various A beta species. Mechanistic studies clarified two separate modes of protofibril growth, elongation by monomer deposition and protofibril-protofibril association, that could be resolved by varying the NaCl concentration. Small isolated protofibrils in dilute Tris-HCl buffers were directed along the elongation pathway by addition of A beta(1-40) monomer or along the association pathway by addition of NaCl. Multi-angle light scattering analysis revealed that protofibrils with initial molecular masses M(w) of (7-30) x 10(3) kDa grew to M(w) values of up to 250 x 10(3) kDa by these two growth processes. However, the mass per unit length of the associated protofibrils was about 2-3 times that of the elongated protofibrils. Rate constants for further elongation by monomer deposition with the elongated, associated, and initial protofibril pools were identical when equal number concentrations of original protofibrils were compared, indicating that the original number of protofibril ends had not been altered by the elongation or association processes. Atomic force microscopy revealed heterogeneous initial protofibrils that became more rodlike following the elongation reaction. Our data indicate that protofibril elongation in the absence of NaCl results from monomer deposition only at the ends of protofibrils and proceeds without an increase in protofibril diameter. In contrast, protofibril association occurs in the absence of monomer when NaCl is introduced, but this association involves lateral interactions that result in a relatively disordered fibril structure.     

Echinocyte shapes: bending, stretching, and shear determine spicule shape and spacing

We study the shapes of human red blood cells using continuum mechanics. In particular, we model the crenated, echinocytic shapes and show how they may arise from a competition between the bending energy of the plasma membrane and the stretching/shear elastic energies of the membrane skeleton. In contrast to earlier work, we calculate spicule shapes exactly by solving the equations of continuum mechanics subject to appropriate boundary conditions. A simple scaling analysis of this competition reveals an elastic length Lambda(el), which sets the length scale for the spicules and is, thus, related to the number of spicules experimentally observed on the fully developed echinocyte.     

Tom34 unlike Tom20 does not interact with the leader sequences of mitochondrial precursor proteins

Tom20 and Tom34 are mammalian liver proteins previously identified by others to be components of the mitochondrial import translocation apparatus. It has been shown that Tom20 interacts with the leader sequence of nuclear coded matrix space precursor proteins. Here we show with recombinantly expressed Tom proteins that Tom34 binds the mature portion of the precursor and not the leader. Both these Tom proteins inhibited the import of newly translated precursor of aldehyde dehydrogenase in an in vitro assay. Only Tom20 inhibited the import of a fusion protein of the leader of aldehyde dehydrogenase attached to dihydrofolate reductase. Antibodies against Tom20 coprecipitated both the precursor of aldehyde dehydrogenase (pALDH) and of dihydrofolate reductase (pA-DHFR). Antibodies against Tom34 interacted only when the mature portion of aldehyde dehydrogenase was present. Similar import inhibition patterns were found when other precursor and chimeric constructs we investigated. When Tom34-green fluorescence protein was transfected to HeLa cells it was observed that Tom34 was found through out the cell. It is concluded from our observation that Tom34 is a cytosolic protein, whose role appeared to be to interact with mature portion of some preproteins and may keep them in an unfolded, import compatible state.     

Evaluation of toxic potential of captan: Induction of hsp70 and tissue damage in transgenic Drosophila melanogaster (hsp70-lacZ) Bg9

The study investigated the working hypothesis that a widely used fungicide captan exerts toxic effects on nontarget organisms. Transgenic Drosophila melanogaster (hsp70-lacZ) was used as a model by assaying stress gene expression as an endpoint for cytotoxicity and also to evaluate whether stress gene expression is sufficient enough to protect and to prevent tissue damage against toxic insult of the chemical. The study was further extended to understand the effect of the pesticide on development, life cycle, and reproduction of the organism and finally to evaluate a concentration of the chemical to be nontoxic to the organism. The study showed that (i) captan causes cytotoxicity at and above 0.015 ppm; (ii) at 0.0015 ppm captan, absence of hsp70 expression in the exposed organism was evaluated as the concentration referred to as no observed adverse effect level (NOAEL) for Drosophila; (iii) emergence pattern of flies was affected only at the highest concentration of captan by 4 days, while hatching and survivorship were unaffected even at this concentration; (iv) reproductive performance was significantly affected only at 125.0 and 1250.0 ppm captan, while in the lower dietary concentrations no such deleterious effects were observed; (v) at 1250.0 ppm, hsp70 failed to protect the cells from toxicant assault after 48 h exposure, thus leading to tissue damage as revealed by Trypan Blue staining. The present study shows the cytotoxic potential of captan and further reveals the application of stress genes in determining NOAEL and its expression as bioindicator of exposure to environmental contaminants.     

Structural and interactional homology of clinically potential trypsin inhibitors: molecular modelling of cucurbitaceae family peptides using the X-ray structure of MCTI-II

Several trypsin inhibitor peptides (with 28-32 amino acid residues) belonging to the Cucurbitaceae (LA-1, LA-2, MCTI-I, CMTI-I, CMTI-III, CMTI-IV), characterized by a distinctive tertiary fold with three conserved disulphide bonds and with mostly arginine at their active centre, were modelled using the high-resolution X-ray structure of a homologous inhibitor, MCTI-II, isolated from bitter gourd. All the inhibitors were modelled in both their native and complexed state with the trypsin molecule, keeping the active site the same as was observed in the trypsin-MCTI-II complex, by homology modelling using the InsightII program. The minimized energy profile supported the binding constants (binding behaviour) of the inhibitor-trypsin complexes in the solution state. A difference accessible surface area (DASA) study of the trypsin with and without inhibitors revealed the subsites of trypsin where the inhibitors bind. It revealed that the role of mutation of these peptides through evolution is to modulate their inhibitory function depending on the biological need rather than changing the overall structural folding characteristics which are highly conserved. The minor changes of amino acids in the non-conserved regions do not influence significantly the basic conformational and interactional sequences at the trypsin binding subsites during complex formation.     

Selective activation of antitumor activity of macrophages by the delivery of muramyl dipeptide using a novel polynucleotide-based carrier recognized by scavenger receptors

We have shown that muramyl dipeptide (MDP) conjugated to a 10-mer polyguanylic acid (PolyG) is specifically internalized by macrophages through scavenger receptor (SCR)-mediated endocytosis. Macrophages activated by PolyG-MDP displayed about 20-fold higher cytotoxic activity against nonmacrophage tumor cells compared to that elicited by free MDP. The PolyG-MDP was found to trigger the secretion of higher levels of interleukin-6, interleukin-1alpha, TNF-alpha, and nitric oxide in comparison to free MDP. Addition of antibodies directed against IL-6 and TNF-alpha to macrophage culture completely abrogated the tumoricidal response of PolyG-MDP, indicating that these two cytokines are primarily responsible for bioefficacy. This general approach of PolyG as a vehicle may find wide application in the delivery of genes and antisense oligonucleotides to macrophages.     

Virulence genes in environmental strains of Vibrio cholerae

The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression. The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibrio cholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups. In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as well as toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V. cholerae cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental V. cholerae strains. Sequencing of a part of the tcpA gene carried by an environmental strain showed 97.7% homology to the tcpA gene of the classical biotype of V. cholerae O1. Strains carrying the tcpA gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili. Strains carrying ctxAB genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of V. cholerae, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of V. cholerae.