Galactose-Specific Fimbrial Adhesin of Enteroaggregative <Emphasis Type="Italic">Escherichia coli</Emphasis>: A Possible Aggregative Factor

A galactose-specific adhesin was isolated from the fimbriae of an enteroaggregative Escherichia coli (EAEC) strain. The adhesin was found to be a high molecular weight aggregate of the 18-kDa monomer. The dimeric (36 kDa) and tetrameric (76 kDa) forms appeared in sodium dodecyl sulphate polyacrylamide gel electrophoresis when a higher concentration of the adhesin was used. The IgG_AD (IgG against adhesin) obtained from the immune sera raised in rabbits against purified adhesin could detect all three forms of the adhesin even from the crude fimbrial preparation. The IgG_AD failed to recognize the adhesin in the presence of galactose, thereby suggesting the antibody-binding site and the sugar-binding site on the adhesin might be same or overlapping. Furthermore, the IgG_AD could localize the adhesin exclusively on the fimbriae as observed in immunogold electron microscopy. The aggregative adherence of the bacteria to HEp-2 cells was reduced to 70% in the presence of the IgG_AD. A glycoprotein (34 kDa) present in the membrane fraction of HEp-2 cells interacted with the purified adhesin in a galactose-specific manner. The IgG_AD could recognize the adhesin from the crude fimbrial preparation of 9 out of 10 clinical isolates of EAEC strains but failed to identify any protein from the crude fimbrial preparation of Salmonella typhimurium (fim +ve as well as fim −ve strain), Vibrio cholerae (WO7) or Escherichia coli DH5α.     

Improvement of pea biomass and seed productivity by simultaneous increase of phloem and embryo loading with amino acids

The development of sink organs such as fruits and seeds strongly depends on the amount of nitrogen that is moved within the phloem from photosynthetic‐active source leaves to the reproductive sinks. In many plant species nitrogen is transported as amino acids. In pea (Pisum sativum L.), source to sink partitioning of amino acids requires at least two active transport events mediated by plasma membrane‐localized proteins, and these are: (i) amino acid phloem loading; and (ii) import of amino acids into the seed cotyledons via epidermal transfer cells. As each of these transport steps might potentially be limiting to efficient nitrogen delivery to the pea embryo, we manipulated both simultaneously. Additional copies of the pea amino acid permease PsAAP1 were introduced into the pea genome and expression of the transporter was targeted to the sieve element‐companion cell complexes of the leaf phloem and to the epidermis of the seed cotyledons. The transgenic pea plants showed increased phloem loading and embryo loading of amino acids resulting in improved long distance transport of nitrogen, sink development and seed protein accumulation. Analyses of root and leaf tissues further revealed that genetic manipulation positively affected root nitrogen uptake, as well as primary source and sink metabolism. Overall, the results suggest that amino acid phloem loading exerts regulatory control over pea biomass production and seed yield, and that import of amino acids into the cotyledons limits seed protein levels.     

Docking and free energy simulations to predict conformational domains involved in hCG-LH receptor interactions using recombinant antibodies

Single chain fragment variables (ScFvs) have been extensively employed in studying the protein-protein interactions. ScFvs derived from phage display libraries have an additional advantage of being generated against a native antigen, circumventing loss of information on conformational epitopes. In the present study, an attempt has been made to elucidate human chorionic gonadotropin (hCG)-luteinizing hormone (LH) receptor interactions by using a neutral and two inhibitory ScFvs against hCG. The objective was to dock a computationally derived model of these ScFvs onto the crystal structure of hCG and understand the differential roles of the mapped epitopes in hCG-LH receptor interactions. An anti-hCG ScFv, whose epitope was mapped previously using biochemical tools, served as the positive control for assessing the quality of docking analysis. To evaluate the role of specific side chains at the hCG-ScFv interface, binding free energy as well as residue interaction energies of complexes in solution were calculated using molecular mechanics Poisson-Boltzmann/surface area method after performing the molecular dynamic simulations on the selected hCG-ScFv models and validated using biochemical and SPR analysis. The robustness of these calculations was demonstrated by comparing the theoretically determined binding energies with the experimentally obtained kinetic parameters for hCG-ScFv complexes. Superimposition of hCG-ScFv model onto a model of hCG complexed with the 51-266 residues of LH receptor revealed importance of the residues previously thought to be unimportant for hormone binding and response. This analysis provides an alternate tool for understanding the structure-function analysis of ligand-receptor interactions.     

Generation of novel radiolabeled opiates through site-selective iodination

Tritiated opioid radioligands have proven valuable in exploring opioid binding sites. However, tritium has many limitations. Its low specific activity and limited counting efficiency makes it difficult to examine low abundant, high affinity sites and its disposal is problematic due to the need to use organic scintillants and its relatively long half-life. To overcome these issues, we have synthesized both unlabeled and carrier-free radioiodinated iodobenzoyl derivatives of 6β-naltrexamine (¹²⁵I-BNtxA, 18), 6β-naloxamine (¹²⁵I-BNalA, 19) and 6β-oxymorphamine (¹²⁵I-BOxyA, 20) with specific activities of 2100 Ci/mmol. To optimize the utility of the radioligand, we designed a synthesis in which the radiolabel is incorporated in the last synthetic step, which required the selective iodination of the benzoyl moiety without incorporation into the phenolic A ring. Competition studies demonstrated high affinity of the unlabelled compounds for opioid receptors in transfected cell lines, as did the direct binding of the ¹²⁵I-ligands to the opioid receptors. The radioligand displayed very high sensitivity, enabling a marked reduction in tissue, as well as excellent signal/noise characteristics. These new ¹²⁵I-radioligands should prove valuable in future studies of opioid binding sites.     

Exosomes mediate LTB4 release during neutrophil chemotaxis

Leukotriene B₄ (LTB₄) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB₄ and its synthesizing enzymes localize to intracellular multivesicular bodies, which, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in an LTB₄ receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB₄ release. Our findings establish that the exosomal pool of LTB₄ acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments.     

Lung and alveolar wall elastic and hysteretic behavior in rats: effects of in vivo elastase treatment.

We investigated the relationship between the microscopic elastic and hysteretic behavior of the alveolar walls and the macroscopic mechanical properties of the whole lung in an in vivo elastase-treated rat model of emphysema. We measured the input impedance of isolated lungs at three levels of transpulmonary pressure (Ptp) and used a linear model to estimate the dynamic elastance and hysteresivity of the lungs. The elastance of the normal lungs increased steeply with Ptp, whereas this dependence diminished in the treated lungs. Hysteresivity decreased significantly with Ptp in the normal lungs, but this dependence disappeared in the treated lungs. To investigate the microscopic origins of these changes, the alveolar walls were immunofluorescently labeled in small tissue strips. By using a fluorescent microscope, the lengths and angular orientations of individual alveolar walls were followed during cyclic uniaxial stretching of the tissue strips. The microstrains (relative change in segment length) and changes in angle of the alveolar walls showed considerable heterogeneity, which was interpreted in terms of a network model. In the normal strips, the alveolar walls showed larger angular changes compared with the treated tissue, whereas the alveolar walls of the treated tissue tended to be more extensible. Hysteresis in the average angle change was also larger in the treated tissue than in the normal tissue. We conclude that the decreased Ptp dependence of elastance and the constant hysteresivity in the treated lungs are related to microstructural remodeling and network phenomena at the level of the alveolar walls.     

Effect of Ocimum sanctum fixed oil on vascular permeability and leucocytes migration

Ocimum sanctum fixed oil significantly inhibited the rise in protein concentration and dye leakage in peritoneal fluid in experimentally induced peritoneal inflammation in mice. In carrageenan-induced pleurisy in rats, the fixed oil showed significant inhibition of leucocytes migration in the pleural exudate. The results suggest that the fixed oil can inhibit enhancement of the vascular/capillary permability and leucocyte migration following inflammatory stimulus.     

Perturbing the linker regions of the alpha-subunit of transducin: a new class of constitutively active GTP-binding proteins

The GDP-GTP exchange activity of the retinal G protein, transducin, is markedly accelerated by the photoreceptor rhodopsin in the first step of visual transduction. The x-ray structures for the alpha subunits of transducin (alpha(T)) and other G proteins suggest that the nucleotide-binding (Ras-like) domain and a large helical domain form a "clam shell" that buries the GDP molecule. Thus, receptor-promoted G protein activation may involve "opening the clam shell" to facilitate GDP dissociation. In this study, we have examined whether perturbing the linker regions connecting the Ras-like and helical domains of Galpha subunits gives rise to a more readily exchangeable state. The sole glycine residues in linkers 1 and 2 were individually changed to proline residues within an alpha(T)/alpha(i1) chimera (designated alpha(T)(*)). Both alpha(T)(*) linker mutants showed significant increases in their basal rates of GDP-GTP exchange when compared either to retinal alpha(T) or recombinant alpha(T)(*). The alpha(T)(*) linker mutants were responsive to aluminum fluoride, which binds to alpha-GDP complexes and induces changes in Switch 2. Although both linker mutants were further activated by light-activated rhodopsin together with the betagamma complex, their activation was not influenced by betagamma alone, arguing against the idea that the betagamma complex helps to pry apart the helical and Ras-like domains of Galpha subunits. Once activated, the alpha(T)(*) linker mutants were able to stimulate the cyclic GMP phosphodiesterase. Overall, these findings highlight a new class of activated Galpha mutants that constitutively exchange GDP for GTP and should prove valuable in studying different G protein-signaling systems.     

De‐ubiquitinases on the move: an emerging field in plant biology

A balance between the synthesis and degradation of active proteins governs diverse cellular processes in plants, spanning from cell‐cycle progression and circadian rhythm to the outcome of several hormone signalling pathways. Ubiquitin‐mediated post‐translational modification determines the degradative fate of the target proteins, thereby altering the output of cellular processes. An equally important, and perhaps under‐appreciated, aspect of this pathway is the antagonistic process of de‐ubiquitination. De‐ubiquitinases (DUBs), a group of processing enzymes, play an important role in maintaining cellular ubiquitin homeostasis by hydrolyzing ubiquitin poly‐proteins and free poly‐ubiquitin chains into mono‐ubiquitin. Further, DUBs rescue the cellular proteins from 26S proteasome‐mediated degradation to their active form by cleaving the poly‐ubiquitin chain from the target protein. Any perturbation in DUB activity is likely to affect proteostasis and downstream cellular processes. This review illustrates recent findings on the biological significance and mechanisms of action of the DUBs in Arabidopsis thaliana, with an emphasis on ubiquitin‐specific proteases (UBPs), the largest family among the DUBs. We focus on the putative roles of various protein–protein interaction interfaces in DUBs and their generalized function in ubiquitin recycling, along with their pre‐eminent role in plant development.     

Saccharide-RNA recognition in a complex formed between neomycin B and an RNA aptamer.

BACKGROUND: Aminoglycoside antibiotics can target RNA folds with micromolar affinity and inhibit biological processes ranging from protein biosynthesis to ribozyme action and viral replication. Specific features of aminoglycoside antibiotic-RNA recognition have been probed using chemical, biochemical, spectroscopic and computational approaches on both natural RNA targets and RNA aptamers identified through in vitro selection. Our previous studies on tobramycin-RNA aptamer complexes are extended to neomycin B bound to its selected RNA aptamer with 100 nM affinity. RESULTS: The neamine moiety (rings I and II) of neomycin B is sandwiched between the major groove floor of a 'zippered-up' G.U mismatch aligned segment and a looped-out purine base that flaps over the bound antibiotic. Specific intermolecular hydrogen bonds are observed between the charged amines of neomycin B and base mismatch edges and backbone phosphates. These interactions anchor 2-deoxystreptamine ring I and pyranose ring II within the RNA-binding pocket. CONCLUSIONS: The RNA aptamer complexes with tobramycin and neomycin B utilize common architectural principles to generate RNA-binding pockets for the bound aminoglycoside antibiotics. In each case, the 2-deoxystreptamine ring I and an attached pyranose ring are encapsulated within the major groove binding pocket, which is lined with mismatch pairs. The bound antibiotic within the pocket is capped over by a looped-out base and anchored in place through intermolecular hydrogen bonds involving charged amine groups of the antibiotic.