Antioxidant,anticancer, apoptosis properties and chemical composition of black truffle Terfezia claveryi

Desert truffles are seasonal and important edible fungi that grow wild in many countries around the world. Truffles are natural food sources that have significant compositions. In this work, the antioxidant, chemical composition, anticancer, and antiangiogenesis properties of the Terfezia claveryi truffle were investigated. Solvent extractions of the T. claveryi were evaluated for antioxidant activities using (DPPH, FRAP and ABTS methods). The extracts cytotoxicity on the cancer cell lines (HT29, MCF-7, PC3 and U-87 MG) was determined by MTT assay, while the anti-angiogenic efficacy was tested using ex-vivo assay. All extracts showed moderate anticancer activities against all cancer cells (p < 0.05). The hexane extract inhibited the brain cell line (U-87 MG) with an IC₅₀ of 50 μg/ml and significantly promoted cell apoptosis through the mitochondrial pathway and DNA fragmentation p < 0.001. The ethanol extract demonstrated potent antioxidants; DPPH, FRAP, and ABTS with an IC₅₀ value of 52, 48.5 and 64.7 μg/ml, respectively. In addition, the hexane and ethyl acetate extract significantly (p < 0.001) inhibited the sprouting of microvessels by 100% and 81.2%, at 100 μg/ml, respectively. The GC analysis of the most active extract (hexane) showed the presence of several potent phytochemicals such as stigmasterol, beta-Sitosterol, squalene, lupeol, octadecadienoic acid, and oleic acid.     

Conjugates of COL-1 monoclonal antibody and β-D-galactosidase can specifically kill tumor cells by generation of 5-fluorouridine from the prodrug β-D-galactosyl-5-fluorouridine

5′-O-β-d-galactosyl-5-fluorouridine is a prodrug that can be converted by the enzyme β-d-galactosidase to the potent antineoplastic drug 5-fluorouridine. The prodrug is more than 100x less toxic than the drug to bone marrow cells in Balb/c mice. The ratio of the IC₅₀ of the prodrug to that of the drug determined on a variety of tumor cell lines in vitro ranged from 500∶1–1000∶1. An antibody-enzyme conjugate (AEC) was synthesized and purified. Maleimide-substituted COL-1 anti-CEA monoclonal antibody was linked to free thiol groups of β-d-galactosidase. The conjugate was purified by size exclusion and ion exchange chromatography. It retained full immunoreactivity and enzyme activity. After binding to antigen-positive tumor cells, the conjugate was able to activate the prodrug and specifically kill the cells. We are continuing to investigate this model for its potential use in antibody-directed enzyme prodrug therapy (ADEPT).     

Antifungal activity of novel indole derivative from endophytic bacteria Pantoea ananatis 4G-9 against Mycosphaerella musicola

An endophytic bacterium isolated from banana G-9 (AAA genotype) leaves exhibited strong antagonistic activity against Mycosphaerella musicola. The isolate was identified as Pantoea ananatis 4G-9 by 16S rRNA sequence analysis. Secondary metabolite obtained from P. ananatis 4G-9 was found to have antifungal activity. The active compound was purified from crude extract using column chromatography. Purity of the active compound was assessed using high-performance liquid chromatography. Spectral analysis of compound using infrared, mass spectrometry and nuclear magnetic resonance indicated that the compound structure is an indole derivative. The compound showed strong and dose-dependent antifungal activity against M. musicola. This is the first report on P. ananatis isolated as an endophyte from banana leaves and its antifungal activity against M. musicola.     

Mode switching of Inositol 1,4,5-trisphosphate receptor channel shapes the Spatiotemporal scales of Ca<Superscript>2+</Superscript> signals

The inositol 1,4,5-trisphosphate (InsP₃) receptor (InsP₃R) channel is crucial for the generation and modulation of highly specific intracellular Ca²⁺ signals performing numerous functions in animal cells. However, the single channel behavior during Ca²⁺ signals of different spatiotemporal scales is not well understood. To elucidate the correlation between the gating dynamics of single InsP₃Rs and spatiotemporal Ca²⁺ patterns, we simulate a cluster of InsP₃Rs under varying ligand concentrations and extract comprehensive gating statistics of all channels during events of different sizes and durations. Our results show that channels gating predominantly in the low activity mode with negligible occupancy of intermediate and high modes leads to single channel Ca²⁺ release event blips. Increasing occupancies of intermediate and high modes results in events with increasing size. When the channel has more than 50% probability of gating in the intermediate and high modes, the cluster generates very large puffs that would most likely result in global Ca²⁺ signals. The size, duration and frequency of Ca²⁺ signals all increase linearly with the total probability of channel gating in the intermediate and high modes. To our knowledge, this is the first study that quantitatively relates the modal characteristics of InsP₃R to the shaping of different spatiotemporal scales of Ca²⁺ signals.     

Insights into unbound–bound states of GPR142 receptor in a membrane-aqueous system using molecular dynamics simulations

G protein coupled receptors (GPCRs) are source machinery in signal transduction pathways and being one of the major therapeutic targets play a significant in drug discovery. GPR142, an orphan GPCR, has been implicated in the regulation of insulin, thereby having a crucial role in Type II diabetes management. Deciphering of the structures of orphan, GPCRs (O-GPCRs) offer better prospects for advancements in research in ion translocation and transduction of extracellular signals. As the crystallographic structure of GPR142 is not available in PDB, therefore, threading and ab initio-based approaches were used for 3D modeling of GPR142. Molecular dynamic simulations (900 ns) were performed on the 3D model of GPR142 and complexes of GPR142 with top five hits, obtained through virtual screening, embedded in lipid bilayer with aqueous system using OPLS force field. Compound 1, 3, and 4 may act as scaffolds for designing potential lead agonists for GPR142. The finding of GPR142 MD simulation study provides more comprehensive representation of the functional properties. The concern for Type II diabetes is increasing worldwide and successful treatment of this disease demands novel drugs with better efficacy.     

Cross-linking of the enzymes in the glycosome of Trypanosoma brucei

Glycosomes, the microbody-like organelles containing mainly glycolytic enzymes, were purified from the long slender bloodstream form of Trypanosoma brucei EATRO 110 monomorphic strain by an improved method in which the protozoa were frozen and thawed in 15% glycerol to free, from the plasma membrane, much of the variant surface glycoprotein which used to constitute the major contaminant of our purified glycosomes. The purified glycosomes have 11 major proteins, 6 of which, tentatively identified as phosphofructose kinase, hexokinase, 3-phosphoglycerate kinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and alpha-glycerophosphate dehydrogenase, constitute 87% of the total glycosomal protein. The bifunctional cross-linking reagents dimethyl suberimidate and dimethyl-3,3'-dithiobispropionimidate can penetrate the glycosomal membrane and cause extensive cross-linking of all the major glycosomal proteins. The cross-linked complex, insoluble in 0.1% Triton X-100 plus 0.15 M NaCl, contains all the glycosomal enzyme activities with only partial inactivations. All the enzymes are probably cross-linked into one large complex since they all sediment rapidly to the bottom of a 5-20% (v/v) sucrose density gradient. This successful cross-linking with reagents of span lengths of 11-12 A suggests close proximities among the glycosomal enzymes which may explain the extraordinarily high rate of glycolysis in T. brucei. Whether such a close association represents specific spatial arrangement required for genuine substrate channeling among the enzymes will be verified by future kinetic studies of the cross-linked enzyme complex.     

NADPH Oxidase 4 Induces Cardiac Arrhythmic Phenotype in Zebrafish

Oxidative stress has been implicated in cardiac arrhythmia, although a causal relationship remains undefined. We have recently demonstrated a marked up-regulation of NADPH oxidase isoform 4 (NOX4) in patients with atrial fibrillation, which is accompanied by overproduction of reactive oxygen species (ROS). In this study, we investigated the impact on the cardiac phenotype of NOX4 overexpression in zebrafish. One-cell stage embryos were injected with NOX4 RNA prior to video recording of a GFP-labeled (myl7:GFP zebrafish line) beating heart in real time at 24–31 h post-fertilization. Intriguingly, NOX4 embryos developed cardiac arrhythmia that is characterized by irregular heartbeats. When quantitatively analyzed by an established LQ-1 program, the NOX4 embryos displayed much more variable beat-to-beat intervals (mean S.D. of beat-to-beat intervals was 0.027 s/beat in control embryos versus 0.038 s/beat in NOX4 embryos). Both the phenotype and the increased ROS in NOX4 embryos were attenuated by NOX4 morpholino co-injection, treatments of the embryos with polyethylene glycol-conjugated superoxide dismutase, or NOX4 inhibitors fulvene-5, 6-dimethylamino-fulvene, and proton sponge blue. Injection of NOX4-P437H mutant RNA had no effect on the cardiac phenotype or ROS production. In addition, phosphorylation of calcium/calmodulin-dependent protein kinase II was increased in NOX4 embryos but diminished by polyethylene glycol-conjugated superoxide dismutase, whereas its inhibitor KN93 or AIP abolished the arrhythmic phenotype. Taken together, our data for the first time uncover a novel pathway that underlies the development of cardiac arrhythmia, namely NOX4 activation, subsequent NOX4-specific NADPH-driven ROS production, and redox-sensitive CaMKII activation. These findings may ultimately lead to novel therapeutics targeting cardiac arrhythmia.     

A requirement for lipid rafts in B cell receptor induced Ca(2+) flux

Although the major biochemical events triggered by ligation of the B-cell receptor (BCR) have been well defined [1] [2], little is known about the spatio-temporal organization of BCR signaling components within the cell membrane and the mechanisms by which signaling specificity is achieved. Partitioning of signaling complexes into specialized domains in the plasma membrane may provide a mechanism for channeling specific stimuli into distinct signaling pathways. Here, we report that multiple tyrosine-phosphorylated proteins accumulate transiently upon BCR activation in detergent-insoluble membrane microdomains known as lipid rafts. We found an activation-dependent translocation to the rafts of the BCR itself, as well as phospholipase Cgamma2 (PLCgamma2), an enzyme critical for BCR-induced Ca(2+) flux in B cells. An intact raft structure was required for BCR-induced tyrosine phosphorylation of PLCgamma2 and the induction of Ca(2+) flux. Taken together, these data provide a functional role for lipid rafts in BCR signaling.     

High-Resolution Crystal Structures of Drosophila melanogaster Angiotensin-Converting Enzyme in Complex with Novel Inhibitors and Antihypertensive Drugs

Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE.