The C-type lectin fold as an evolutionary solution for massive sequence variation

Only few instances are known of protein folds that tolerate massive sequence variation for the sake of binding diversity. The most extensively characterized is the immunoglobulin fold. We now add to this the C-type lectin (CLec) fold, as found in the major tropism determinant (Mtd), a retroelement-encoded receptor-binding protein of Bordetella bacteriophage. Variation in Mtd, with its approximately 10(13) possible sequences, enables phage adaptation to Bordetella spp. Mtd is an intertwined, pyramid-shaped trimer, with variable residues organized by its CLec fold into discrete receptor-binding sites. The CLec fold provides a highly static scaffold for combinatorial display of variable residues, probably reflecting a different evolutionary solution for balancing diversity against stability from that in the immunoglobulin fold. Mtd variants are biased toward the receptor pertactin, and there is evidence that the CLec fold is used broadly for sequence variation by related retroelements.     

A topological model of biofeedback based on catecholamine interactions

Background  

The present paper describes a topological model of biofeedback. This model incorporates input from a sensory organ and a transduction phase mediated through catecholamine production in the feedback path. The transduction phase comprises both conservative and dissipative systems, from which the appropriate output is combined in a closed loop.     

Characterization of the calcium-binding sites of Listeria monocytogenes InlB

The Listeria monocytogenes protein InlB promotes invasion of mammalian cells through activation of the receptor tyrosine kinase Met. The InlB N-cap, a approximately 40 residue part of the domain that binds Met, was previously observed to bind two calcium ions in a novel and unusually exposed manner. Because subsequent work raised questions about the existence of these calcium-binding sites, we assayed calcium binding in solution to the InlB N-cap. We show that calcium ions are bound with dissociation constants in the low micromolar range at the two identified sites, and that the sites interact with one another. We demonstrate that the calcium ions are not required for structure, and also find that they have no appreciable effect on Met activation or intracellular invasion. Therefore, our results indicate that the sites are fortuitous in InlB, but also suggest that the simple architecture of the sites may be adaptable for protein engineering purposes.     

Preparation and in vitro evaluation of polystyrene-coated diltiazem-resin complex by oil-in-water emulsion solvent evaporation method

The purpose of this study was to examine the suitability of polystyrene-coated (PS-coated) microcapsules of drug-resin complex for achieving prolonged release of diltiazem-HCl, a highly water-soluble drug, in simulated gastric and intestinal fluid. The drug was bound to Indion 254, a cation-exchange resin, and the resulting resinate was microencapsulated with PS using an oil-in-water emulsion-solvent evaporation method. The effect of various formulation parameters on the characteristics of the microcapsules was studied. Mean diameter and encapsulation efficiency of the microcapsules rose with an increase in the concentration of emulsion stabilizer and the coat/core ratio, while the same characteristics tended to decrease with an increase in the volume of the organic disperse phase. The desorption of drug from the uncoated resinate was quite rapid and independent of the pH of the dissolution media. On the other hand, the drug release from the microcapsules was prolonged for different periods of time depending on the formulation parameters and was also found to be independent of the pH of the dissolution media. Both the encapsulation efficiency and the retardation of drug release were found to be dependent on the uniformity of coating, which in turn was influenced by the formulation parameters. Kinetic studies revealed that the desorption of drug from the resinate obeyed the typical particle diffusion process, whereas the drug release from the microencapsulated resinate followed the diffusion-controlled model in accordance with the Higuchi equation. PS appeared to be a suitable polymer to provide prolonged release of diltiazem independent of the pH of the dissolution media.     

Comparative effect of Ocimum sanctum, Commiphora mukul, folic acid and ramipril on lipid peroxidation in experimentally-induced hyperlipidemia

Treatment with C. mukul and O. sanctum, showed a significant decrease in cholesterol and triglyceride levels respectively. O. sanctum also significantly increased serum HDL-cholesterol compared to control. Serum MDA levels were significantly reduced in all the treated groups compared to control suggesting that each of the drugs under study were effective in their free radical scavenging action. Erythrocyte SOD activity was increased in all the treatment groups with C. mukul showing the maximum effect followed by O. sanctum, folic acid and ramipril. The erythrocyte CAT activity was significantly increased in all the drug treated groups with maximum increase seen in O. sanctum and ramipril treated groups, whereas lesser effects were observed with C. mukul and folic acid groups. Thus, the indigenous drugs, C. mukul and O. sanctum had beneficial effect on hypercholesterolemic rabbit model, both in terms of lipid profile as well as antioxidant potential. Ocimum sanctum was found to be the most promising of all the drugs. Moreover, it could be hypothesized that these plant products along with folic acid and ramipril can be explored for synergistic effect for treatment for hypercholesterolemic conditions.     

Role of phenylalanine B10 in plant nonsymbiotic hemoglobins

All plants contain an unusual class of hemoglobins that display bis-histidyl coordination yet are able to bind exogenous ligands such as oxygen. Structurally homologous hexacoordinate hemoglobins (hxHbs) are also found in animals (neuroglobin and cytoglobin) and some cyanobacteria, where they are thought to play a role in free radical scavenging or ligand sensing. The plant hxHbs can be distinguished from the others because they are only weakly hexcacoordinate in the ferrous state, yet no structural mechanism for regulating hexacoordination has been articulated to account for this behavior. Plant hxHbs contain a conserved Phe at position B10 (Phe(B10)), which is near the reversibly coordinated distal His(E7). We have investigated the effects of Phe(B10) mutation on kinetic and equilibrium constants for hexacoordination and exogenous ligand binding in the ferrous and ferric oxidation states. Kinetic and equilibrium constants for hexacoordination and ligand binding along with CO-FTIR spectroscopy, midpoint reduction potentials, and the crystal structures of two key mutant proteins (F40W and F40L) reveal that Phe(B10) is an important regulatory element in hexacoordination. We show that Phe at this position is the only amino acid that facilitates stable oxygen binding to the ferrous Hb and the only one that promotes ligand binding in the ferric oxidation states. This work presents a structural mechanism for regulating reversible intramolecular coordination in plant hxHbs.     

The tumour-suppressor genes NF2/Merlin and Expanded act through Hippo signalling to regulate cell proliferation and apoptosis

Merlin, the protein product of the Neurofibromatosis type-2 gene, acts as a tumour suppressor in mice and humans. Merlin is an adaptor protein with a FERM domain and it is thought to transduce a growth-regulatory signal. However, the pathway through which Merlin acts as a tumour suppressor is poorly understood. Merlin, and its function as a negative regulator of growth, is conserved in Drosophila, where it functions with Expanded, a related FERM domain protein. Here, we show that Drosophila Merlin and Expanded are components of the Hippo signalling pathway, an emerging tumour-suppressor pathway. We find that Merlin and Expanded, similar to other components of the Hippo pathway, are required for proliferation arrest and apoptosis in developing imaginal discs. Our genetic and biochemical data place Merlin and Expanded upstream of Hippo and identify a pathway through which they act as tumour-suppressor genes.     

Functional Glycomic Analysis of Human Milk Glycans Reveals the Presence of Virus Receptors and Embryonic Stem Cell Biomarkers

Human milk contains a large diversity of free glycans beyond lactose, but their functions are not well understood. To explore their functional recognition, here we describe a shotgun glycan microarray prepared from isolated human milk glycans (HMGs), and our studies on their recognition by viruses, antibodies, and glycan-binding proteins (GBPs), including lectins. The total neutral and sialylated HMGs were derivatized with a bifunctional fluorescent tag, separated by multidimensional HPLC, and archived in a tagged glycan library, which was then used to print a shotgun glycan microarray (SGM). This SGM was first interrogated with well defined GBPs and antibodies. These data demonstrated both the utility of the array and provided preliminary structural information (metadata) about this complex glycome. Anti-TRA-1 antibodies that recognize human pluripotent stem cells specifically recognized several HMGs that were then further structurally defined as novel epitopes for these antibodies. Human influenza viruses and Parvovirus Minute Viruses of Mice also specifically recognized several HMGs. For glycan sequencing, we used a novel approach termed metadata-assisted glycan sequencing (MAGS), in which we combine information from analyses of glycans by mass spectrometry with glycan interactions with defined GBPs and antibodies before and after exoglycosidase treatments on the microarray. Together, these results provide novel insights into diverse recognition functions of HMGs and show the utility of the SGM approach and MAGS as resources for defining novel glycan recognition by GBPs, antibodies, and pathogens.     

Tryptamine-gallic acid hybrid prevents non-steroidal anti-inflammatory drug-induced gastropathy: correction of mitochondrial dysfunction and inhibition of apoptosis in gastric mucosal cells

We have investigated the gastroprotective effect of SEGA (3a), a newly synthesized tryptamine-gallic acid hybrid molecule against non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy with mechanistic details. SEGA (3a) prevents indomethacin (NSAID)-induced mitochondrial oxidative stress (MOS) and dysfunctions in gastric mucosal cells, which play a pathogenic role in inducing gastropathy. SEGA (3a) offers this mitoprotective effect by scavenging of mitochondrial superoxide anion (O(2)(·-)) and intramitochondrial free iron released as a result of MOS. SEGA (3a) in vivo blocks indomethacin-mediated MOS, as is evident from the inhibition of indomethacin-induced mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. SEGA (3a) corrects indomethacin-mediated mitochondrial dysfunction in vivo by restoring defective electron transport chain function, collapse of transmembrane potential, and loss of dehydrogenase activity. SEGA (3a) not only corrects mitochondrial dysfunction but also inhibits the activation of the mitochondrial pathway of apoptosis by indomethacin. SEGA (3a) inhibits indomethacin-induced down-regulation of bcl-2 and up-regulation of bax genes in gastric mucosa. SEGA (3a) also inhibits indometacin-induced activation of caspase-9 and caspase-3 in gastric mucosa. Besides the gastroprotective effect against NSAID, SEGA (3a) also expedites the healing of already damaged gastric mucosa. Radiolabeled ((99m)Tc-labeled SEGA (3a)) tracer studies confirm that SEGA (3a) enters into mitochondria of gastric mucosal cell in vivo, and it is quite stable in serum. Thus, SEGA (3a) bears an immense potential to be a novel gastroprotective agent against NSAID-induced gastropathy.