Regulation of human intestinal intraepithelial lymphocyte cytolytic function by biliary glycoprotein (CD66a).

Human small intestinal intraepithelial lymphocytes (iIEL) are a unique population of CD8alphabeta+ TCR-alphabeta+ but CD28- T lymphocytes that may function in intestinal epithelial cell immunosurveillance. In an attempt to define novel cell surface molecules involved in iIEL function, we raised several mAbs against activated iIELs derived from the small intestine that recognized an Ag on activated, but not resting, iIELs. Using expression cloning and binding studies with Fc fusion proteins and transfectants, the cognate Ag of these mAbs was identified as the N domain of biliary glycoprotein (CD66a), a carcinoembryonic Ag-related molecule that contains an immune receptor tyrosine-based inhibitory motif. Functionally, these mAbs inhibited the anti-CD3-directed and lymphokine-activated killer activity of the P815 cell line by iIELs derived from the human small intestine. These studies indicate that the expression of biliary glycoprotein on activated human iIELs and, potentially, other mucosal T lymphocytes is involved in the down-regulation of cytolytic function.     

Studies of a myosin-cleaving protease from dystrophic hamster heart

A myosin-cleaving protease was purified to homogeneity from the cardiac myofibrils of dystrophic hamsters. The biochemical properties of the enzyme was studied with both [³H] acetyl-casein and purified myosin as substrates. Steadily increasing levels of the enzyme correlated to the development of cardiomyopathy.     

The Genomic Blueprint of Salmonella enterica subspecies enterica serovar Typhi P-stx-12

Salmonella enterica subspecies enterica serovar Typhi is a rod-shaped, Gram-negative, facultatively anaerobic bacterium. It belongs to the family Enterobacteriaceae in the class Gammaproteobacteria, and has the capability of residing in the human gallbladder by forming a biofilm and hence causing the person to become a typhoid carrier. Here we present the complete genome of Salmonella enterica subspecies enterica serotype Typhi strain P-stx-12, which was isolated from a chronic carrier in Varanasi, India. The complete genome comprises a 4,768,352 bp chromosome with a total of 98 RNA genes, 4,691 protein-coding genes and a 181,431 bp plasmid. Genome analysis revealed that the organism is closely related to Salmonella enterica serovar Typhi strain Ty2 and Salmonella enterica serovar Typhi strain CT18, although their genome structure is slightly different.     

Synthetic,Structural, and Conformational Studies of Methylated Ring-Expanded Nucleosides Containing the Ihidazo[4, 5-e][1, 4]Diazepine Ring System

Abstract

Syntheses of 4- and 7-methyl 4, 5, 7, 8-tetrahydro-6H-3-(β-ribofuranosyl)imidazo[4, 5-e] [1, 4]diazepine-5, 8-dione, 3 and 1, respectively, are reported. Single-crystal X-ray diffraction analysis of the aglycon of 3 aided in confirming the site of methylation in 3, and that of 4 in elucidating the solid state conformation of 4. Solution conformations of 3 and 4, along with their parent nucleoside 1 and the latter's 1-glycosyl regioisomer 2, were investigated by NOE and CD measurements.     

The Synthesis of Novel Regioisomeric Ring-Expanded Xanthine Nucleosides Containing The 5:7-Fused Imidazo[4,5-e][1,2,4]Triazepine Ring System

Abstract

The syntheses of novel regioisomeric ring-expanded purine nucleosides containing the imidazo[4,5-e][1,2,4]triazepine nucleus are reported. The glycosylation of the heterocycle 3,4,6,7-tetrahydroimidazo[4,5-e][1,2,4]triazepine-5,8-dione (2a) by the stannic chloride procedure gave nucleosides 3 and 4, with the sugar moiety attached at the 7-and 3-positions of the heterocycle, respectively. On the other hand, the mercuric cyanide procedure for glycosylation of 2a yielded nucleosides 4 and 5, with the sugar attached at the 1-position in the latter. In either procedure, 4 was the minor isomer and was obtained only in trace amounts. While debenzoylation of 3 and 5 provided the respective parent nucleosides 8 and 10, that of 4 resulted in ring-opening to produce 9. Attempted enzymic glycosylation of 2a with purine nucleoside phosphorylase failed to yield any nucleoside product.     

The Synthesis,Structure, and Conformation of 2′-Deoxy Analogues of „Fat“ Xanthine Nucleosides,Containing the Imidazo[4,5-e][1,4]Diazepine Ring System

Abstract

The syntheses of 1-(2-deoxy-β-D-erythro-pentofuranosyl)-4,5,7,8-tetrahydro-6H-imidazo[4,5-e] [1,4]diazepine-5,8-dione (9β), its 3-glycosyl regioisomer (16β), and their respective α anomers (9α and 16α), are reported. Conformational and configurational studies, employing ¹H NMR NOE and CD spectroscopy, are described. The single-crystal X-ray structural analysis of 9β is presented. The attempted enzymic glycosylation of the heterocyclic base 6 with a bacterial purine nucleoside phosphorylase was not successful.     

Heterogeneous behavior of metalloproteins toward metal ion binding and selectivity: insights from molecular dynamics studies

About one-third of the existing proteins require metal ions as cofactors for their catalytic activities and structural complexities. While many of them bind only to a specific metal, others bind to multiple (different) metal ions. However, the exact mechanism of their metal preference has not been deduced to clarity. In this study, we used molecular dynamics (MD) simulations to investigate whether a cognate metal (bound to the structure) can be replaced with other similar metal ions. We have chosen seven different proteins (phospholipase A₂, sucrose phosphatase, pyrazinamidase, cysteine dioxygenase (CDO), plastocyanin, monoclonal anti-CD4 antibody Q425, and synaptotagmin 1 C₂B domain) bound to seven different divalent metal ions (Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Cu²⁺, Ba²⁺, and Sr²⁺, respectively). In total, 49 MD simulations each of 50 ns were performed and each trajectory was analyzed independently. Results demonstrate that in some cases, cognate metal ions can be exchanged with similar metal ions. On the contrary, some proteins show binding affinity specifically to their cognate metal ions. Surprisingly, two proteins CDO and plastocyanin which are known to bind Fe²⁺ and Cu²⁺, respectively, do not exhibit binding affinity to any metal ion. Furthermore, the study reveals that in some cases, the active site topology remains rigid even without cognate metals, whereas, some require them for their active site stability. Thus, it will be interesting to experimentally verify the accuracy of these observations obtained computationally. Moreover, the study can help in designing novel active sites for proteins to sequester metal ions particularly of toxic nature.     

Development of a Recombinant Escherichia coli Strain for Overproduction of the Plant Pigment Anthocyanin

Anthocyanins are water-soluble colored pigments found in terrestrial plants and are responsible for the red, blue, and purple coloration of many flowers and fruits. In addition to the plethora of health benefits associated with anthocyanins (cardioprotective, anti-inflammatory, antioxidant, and antiaging properties), these compounds have attracted widespread attention due to their promising potential as natural food colorants. Previously, we reported the biotransformation of anthocyanin, specifically cyanidin 3-O-glucoside (C3G), from the substrate (+)-catechin in Escherichia coli. In the present work, we set out to systematically improve C3G titers by enhancing substrate and precursor availability, balancing gene expression level, and optimizing cultivation and induction parameters. We first identified E. coli transporter proteins that are responsible for the uptake of catechin and secretion of C3G. We then improved the expression of the heterologous pathway enzymes anthocyanidin synthase (ANS) and 3-O-glycosyltransferase (3GT) using a bicistronic expression cassette. Next, we augmented the intracellular availability of the critical precursor UDP-glucose, which has been known as the rate-limiting precursor to produce glucoside compounds. Further optimization of culture and induction conditions led to a final titer of 350 mg/liter of C3G. We also developed a convenient colorimetric assay for easy screening of C3G overproducers. The work reported here constitutes a promising foundation to develop a cost-effective process for large-scale production of plant-derived anthocyanin from recombinant microorganisms.