Evaluation of alpha-D-mannopyranoside glycolipid micelles-lectin interactions by surface plasmon resonance method

It is established that achieving higher binding affinities in carbohydrate-protein interactions requires multivalent presentations of the sugar ligands at the receptor binding site. Several inhibition, calorimetric, mass balance, and other studies have reiterated the beneficial effects of molecular level clustering of the sugar ligands for tight binding to the receptors. We have undertaken an effort to study the multivalent effects involving larger assemblies, represented by micelles, and their lectin interactions. The micelles were constituted with monomer bearing one- or two-sugar moieties at the monomolecular level and with varying the distances between the sugar moieties. Micellar aggregation studies and dynamic light scattering (DLS) studies afforded details of the aggregation numbers and the hydrodynamic diameters of various glycolipid (GL) micelles. The GL micelles were used as analytes of surface plasmon resonance (SPR) experiments on a lectin concanavalin A (Con A)-immobilized surface. SPR studies of the micelle-lectin interactions demonstrate that the ligand-receptor binding can be fit into the bivalent analyte model of interaction. Furthermore, micelles formed from two-sugar containing GLs are able to elicit favorable kinetic association rate constants in comparison to the micelles constituted with one-sugar containing GLs. The kinetic rate constants across the micelles and the effect of the sugar valencies in the GLs are discussed.     

A short survey on protein blocks

Protein structures are classically described in terms of secondary structures. Even if the regular secondary structures have relevant physical meaning, their recognition from atomic coordinates has some important limitations such as uncertainties in the assignment of boundaries of helical and β-strand regions. Further, on an average about 50% of all residues are assigned to an irregular state, i.e., the coil. Thus different research teams have focused on abstracting conformation of protein backbone in the localized short stretches. Using different geometric measures, local stretches in protein structures are clustered in a chosen number of states. A prototype representative of the local structures in each cluster is generally defined. These libraries of local structures prototypes are named as "structural alphabets". We have developed a structural alphabet, named Protein Blocks, not only to approximate the protein structure, but also to predict them from sequence. Since its development, we and other teams have explored numerous new research fields using this structural alphabet. We review here some of the most interesting applications.     

Cationic amino acid transporters and Salmonella Typhimurium ArgT collectively regulate arginine availability towards intracellular Salmonella growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.     

Functional diversity of human protein kinase splice variants marks significant expansion of human kinome

Background

The low pH environment of the human stomach is lethal for most microorganisms; but not Escherichia coli, which can tolerate extreme acid stress. Acid resistance in E. coli is hierarchically controlled by numerous regulators among which are small noncoding RNAs (sncRNA).

Results

In this study, we individually deleted seventy-nine sncRNA genes from the E. coli K12-MG1655 chromosome, and established a single-sncRNA gene knockout library. By systematically screening the sncRNA mutant library, we show that the sncRNA GcvB is a novel regulator of acid resistance in E. coli. We demonstrate that GcvB enhances the ability of E. coli to survive low pH by upregulating the levels of the alternate sigma factor RpoS.

Conclusion

GcvB positively regulates acid resistance by affecting RpoS expression. These data advance our understanding of the sncRNA regulatory network involved in modulating acid resistance in E. coli.     

Annual Rainfall and Seasonality Predict Pan-tropical Patterns of Liana Density and Basal Area

We test the hypotheses proposed by Gentry and Schnitzer that liana density and basal area in tropical forests vary negatively with mean annual precipitation (MAP) and positively with seasonality. Previous studies correlating liana abundance with these climatic variables have produced conflicting results, warranting a new analysis of drivers of liana abundance based on a different dataset. We compiled a pan-tropical dataset containing 28,953 lianas (≥2.5 cm diam.) from studies conducted at 13 Neotropical and 11 Paleotropical dry to wet lowland tropical forests. The ranges in MAP and dry season length (DSL) (number of months with mean rainfall <100 mm) represented by these datasets were 860–7250 mm/yr and 0–7 mo, respectively. Pan-tropically, liana density and basal area decreased significantly with increasing annual rainfall and increased with increasing DSL, supporting the hypotheses of Gentry and Schnitzer. Our results suggest that much of the variation in liana density and basal area in the tropics can be accounted for by the relatively simple metrics of MAP and DSL.     

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-α) secretion by macrophages was essential for robust SHH activation, as TNF-α^−/− macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-α or blockade of TNF-α receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions.     

Pleiotropic roles of a ribosomal protein in Dictyostelium discoideum

The cell cycle phase at starvation influences post-starvation differentiation and morphogenesis in Dictyostelium discoideum. We found that when expressed in Saccharomyces cerevisiae, a D. discoideum cDNA that encodes the ribosomal protein S4 (DdS4) rescues mutations in the cell cycle genes cdc24, cdc42 and bem1. The products of these genes affect morphogenesis in yeast via a coordinated moulding of the cytoskeleton during bud site selection. D. discoideum cells that over- or under-expressed DdS4 did not show detectable changes in protein synthesis but displayed similar developmental aberrations whose intensity was graded with the extent of over- or under-expression. This suggested that DdS4 might influence morphogenesis via a stoichiometric effect--specifically, by taking part in a multimeric complex similar to the one involving Cdc24p, Cdc42p and Bem1p in yeast. In support of the hypothesis, the S. cerevisiae proteins Cdc24p, Cdc42p and Bem1p as well as their D. discoideum cognates could be co-precipitated with antibodies to DdS4. Computational analysis and mutational studies explained these findings: a C-terminal domain of DdS4 is the functional equivalent of an SH3 domain in the yeast scaffold protein Bem1p that is central to constructing the bud site selection complex. Thus in addition to being part of the ribosome, DdS4 has a second function, also as part of a multi-protein complex. We speculate that the existence of the second role can act as a safeguard against perturbations to ribosome function caused by spontaneous variations in DdS4 levels.     

Propyl-2-(8-(3,4-Difluorobenzyl)-2′,5′-Dioxo-8-Azaspiro[Bicyclo[3.2.1] Octane-3,4′-Imidazolidine]-1′-yl) Acetate Induces Apoptosis in Human Leukemia Cells through Mitochondrial Pathway following Cell Cycle Arrest

Background

Due to the functional defects in apoptosis signaling molecules or deficient activation of apoptosis pathways, leukemia has become an aggressive disease with poor prognosis. Although the majority of leukemia patients initially respond to chemotherapy, relapse is still the leading cause of death. Hence targeting apoptosis pathway would be a promising strategy for the improved treatment of leukemia. Hydantoin derivatives possess a wide range of important biological and pharmacological properties including anticancer properties. Here we investigated the antileukemic activity and mechanism of action of one of the potent azaspiro hydantoin derivative, (ASHD).

Materials and Methods

To investigate the antileukemic efficacy of ASHD, we have used MTT assay, cell cycle analysis by FACS, tritiated thymidine incorporation assay, Annexin V staining, JC1 staining and western blot analysis.

Results

Results showed that ASHD was approximately 3-fold more potent than the parent compounds in inducing cytotoxicity. Tritiated thymidine assay in conjunction with cell cycle analysis suggests that ASHD inhibited the growth of leukemic cells. The limited effect of ASHD on cell viability of normal cells indicated that it may be specifically directed to cancer cells. Translocation of phosphatidyl serine, activation of caspase 3, caspase 9, PARP, alteration in the ratio of BCL2/BAD protein expression as well as the loss of mitochondrial membrane potential suggests activation of the intrinsic pathway of apoptosis.

Conclusion

These results could facilitate the future development of novel hydantoin derivatives as chemotherapeutic agents for leukemia.     

Investigating proline puckering states in diproline segments in proteins

In the current study, the puckering states of the Proline ring occurring in diproline segments (^LPro‐^LPro) in proteins has been investigated with a segregation made on the basis of cis and trans states for the Pro‐Pro peptide bond and the conformational states for the diproline segment to investigate the effects of conformation of the diproline segment on the corresponding puckering state of the Proline ring in the segment if any. The value of the endocyclic ring torsional angles of the pyrrolidine ring has been used for calculating and visualizing various puckering states using a proposed new sign convention (+/?) nomenclature. The results have been compared to that obtained in a previous study on peptides from this group. In this study, quite interestingly, the Planar (G) conformation that was present in 14.3% of the cases in peptides, appears to be nearly a rare conformation in the case of proteins (1.9%). The present study indicates that the (C^γ‐exo/C^γ‐exo), (C^γ‐exo/Twisted C^γ‐exo‐C^β‐endo) and (Twisted C^γ‐endo‐C^β‐exo/Twisted C^γ‐endo‐C^β‐exo) categories are the most preferred combinations. For Proline rings in proteins, the states C^γ‐exo, Twisted C^γ‐exo‐C^β‐endo and Twisted C^γ‐endo‐C^β‐exo are the most preferred states. Within diproline segments, the pyrrolidine ring conformations do not show a strong co‐relation to the backbone conformation in which they are observed. It is likely that five‐membered rings have a considerable plasticity of structure and are readily deformed to accommodate a variety of energetically preferred backbone conformations. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 605–610, 2013.