Enhanced Antitumor Immunity Contributes to the Radio-Sensitization of Ehrlich Ascites Tumor by the Glycolytic Inhibitor 2-Deoxy-D-Glucose in Mice

Two-deoxy-D-glucose (2-DG), an inhibitor of glycolysis differentially enhances the radiation and chemotherapeutic drug induced cell death in cancer cells in vitro, while the local tumor control (tumor regression) following systemic administration of 2-DG and focal irradiation of the tumor results in both complete (cure) and partial response in a fraction of the tumor bearing mice. In the present studies, we investigated the effects of systemically administered 2-DG and focal irradiation of the tumor on the immune system in Ehrlich ascites tumor (EAT) bearing Strain “A” mice. Markers of different immune cells were analyzed by immune-flow cytometry and secretary cytokines by ELISA, besides monitoring tumor growth. Increase in the expression of innate (NK and monocytes) and adaptive CD4⁺cells, and a decrease in B cells (CD19) have been observed after the combined treatment, suggestive of activation of anti-tumor immune response. Interestingly, immature dendritic cells were found to be down regulated, while their functional markers CD86 and MHC II were up regulated in the remaining dendritic cells following the combination treatment. Similarly, decrease in the CD4⁺ naïve cells with concomitant increase in activated CD4⁺ cells corroborated the immune activation. Further, a shift from Th2 and Th17 to Th1 besides a decrease in inflammatory cytokines was also observed in the animals showing complete response (cure; tumor free survival). This shift was also complimented by respective antibody class switching followed by the combined treatment. The immune activation or alteration in the homeostasis favoring antitumor immune response may be due to depletion in T regulatory cells (CD4⁺CD25⁺FoxP3⁺). Altogether, these results suggest that early differential immune activation is responsible for the heterogenous response to the combined treatment. Taken together, these studies for the first time provided insight into the additional mechanisms underlying radio-sensitization by 2-DG in vivo by unraveling its potential as an immune-modulator besides direct effects on the tumor.     

Threonine-insensitive Homoserine Dehydrogenase from Soybean: GENOMIC ORGANIZATION, KINETIC MECHANISM, AND IN VIVO ACTIVITY*

Aspartate kinase (AK) and homoserine dehydrogenase (HSD) function as key regulatory enzymes at branch points in the aspartate amino acid pathway and are feedback-inhibited by threonine. In plants the biochemical features of AK and bifunctional AK-HSD enzymes have been characterized, but the molecular properties of the monofunctional HSD remain unexamined. To investigate the role of HSD, we have cloned the cDNA and gene encoding the monofunctional HSD (GmHSD) from soybean. Using heterologously expressed and purified GmHSD, initial velocity and product inhibition studies support an ordered bi bi kinetic mechanism in which nicotinamide cofactor binds first and leaves last in the reaction sequence. Threonine inhibition of GmHSD occurs at concentrations (K_i = 160–240 mm) more than 1000-fold above physiological levels. This is in contrast to the two AK-HSD isoforms in soybean that are sensitive to threonine inhibition (K_i∼150 μm). In addition, GmHSD is not inhibited by other aspartate-derived amino acids. The ratio of threonine-resistant to threonine-sensitive HSD activity in soybean tissues varies and likely reflects different demands for amino acid biosynthesis. This is the first cloning and detailed biochemical characterization of a monofunctional feedback-insensitive HSD from any plant. Threonine-resistant HSD offers a useful biotechnology tool for manipulating the aspartate amino acid pathway to increase threonine and methionine production in plants for improved nutritional content.     

Discovery of TAK-960: An orally available small molecule inhibitor of polo-like kinase 1 (PLK1)

Using structure-based drug design, we identified and optimized a novel series of pyrimidodiazepinone PLK1 inhibitors resulting in the selection of the development candidate TAK-960. TAK-960 is currently undergoing Phase I evaluation in adult patients with advanced solid malignancies.     

Sulfhydryl oxidation of reduced insulin in dilute solution

The reduction of insulin by tri-n-butylphosphine followed by air oxidation in dilute solution at pH 9.1 yields A- and B-chain disulfides. A(S-S)2 and B(S-S) have been purified on SP-Sephadex C-25 using a linear gradient of sodium chloride from 0.1 to 0.45 M in 0.5 M acetic acid containing 7 M urea. The overall yield of A(S-S)2 was 70%; and B(S-S), 60%. The A(S-S)2 and B(S-S) had the expected amino acid composition and N-terminal amino acid. The kinetics of reduction and reoxidation of insulin disulfide bonds are discussed.     

Comprehensive proteomic analysis of human cervical-vaginal fluid

Cervical-vaginal fluid (CVF) is a potential rich source of biomarkers for enhancing our understanding of human parturition and pathologic conditions affecting pregnancy. In this study, we performed a comprehensive survey of the CVF proteome in pregnancy utilizing multidimensional liquid chromatography (2D-LC) coupled with mass spectrometry and gel-electrophoresis-based protein separation and identification. In total, 150 unique proteins were identified using multiple protein identification algorithms. Metabolism (32%) and immune response-related (22%) proteins are the major functional categories represented in the CVF proteome. A comparison of the CVF, serum, and amniotic fluid proteomes showed that 77 proteins are unique to CVF, while 56 and 17 CVF proteins also occur in serum and amniotic fluid, respectively. This data set provides a foundation for evaluation of these proteins as potential CVF biomarkers for noninvasive diagnosis of pregnancy-related disorders.     

Novel and Efficient Transfer Hydrogenolysis of Protected Peptides Using Recyclable Polymer-Supported Hydrogen Donor

Palladium catalyzed transfer hydrogenolysis of protected peptides using a recyclable polymer-supported formate as hydrogen donor affords pure hydrogenolyzed products without the need for any chromatographic purification steps and provides a facile method for the clean and efficient removal of some of the commonly used protecting groups in peptide synthesis.     

Method for Label-Free Quantitative Proteomics for <Emphasis Type="Italic">Sorghum bicolor</Emphasis> L. Moench

Sorghum (Sorghum bicolor L. Moench) is a rapidly emerging high biomass feedstock for bioethanol and lignocellulosic biomass production. The robust varietal germplasm of sorghum and its completed genome sequence provide the necessary genetic and molecular tools to study and engineer the biotic/abiotic stress tolerance. Traditional proteomics approaches for outlining the sorghum proteome have many limitations like, demand for high protein amounts, reproducibility and identification of only few differential proteins. In this study, we report a gel-free, quantitative proteomic method for in-depth coverage of the sorghum proteome. This novel method combining phenol extraction and methanol chloroform precipitation gives high total protein yields for both mature sorghum root and leaf tissues. We demonstrate successful application of this method in comparing proteomes of contrasting cultivars of sorghum, at two different phenological stages. Protein identification and relative quantification analyses were performed by a label-free liquid chromatography tandem mass spectrometry (LC/MS-MS) analyses. Several unique proteins were identified respectively from sorghum tissues, specifically 271 from leaf and 774 from root tissues, with 193 proteins common in both tissues. Using gene ontology analysis, the differential proteins identified were finely corroborated with their leaf/root tissue specific functions. This method of protein extraction and analysis would contribute substantially to generate in-depth differential protein data in sorghum as well as related species. It would also increase the repertoire of methods uniquely suited for gel-free plant proteomics that are increasingly being developed for studying abiotic and biotic stress responses.     

Diesel exhaust particle extracts and associated polycyclic aromatic hydrocarbons inhibit Cox-2-dependent prostaglandin synthesis in murine macrophages and fibroblasts

Diesel exhaust particles (DEP) and their organic constituents modulate the immune system and exacerbate allergic airway inflammation. We investigated the role of DEP extract and associated polycyclic aromatic hydrocarbons (PAHs) on prostaglandin synthesis in endotoxin-activated murine macrophages and in mitogen-stimulated fibroblasts. In both macrophages and fibroblasts, DEP extract, phenanthrene, anthracene, phenanthrenequinone, and beta-napthoflavone inhibit prostaglandin production from endogenous arachidonic acid in response to ligand stimulation. However, DEP extract and PAHs do not block ligand induction of cyclooxygenase-2 (COX-2) protein, either in mitogen-stimulated fibroblasts or endotoxin-treated macrophages. Release of total arachidonic acid and total lipid products is not reduced by DEP or PAHs following ligand stimulation of macrophages or fibroblasts. DEP extract and the PAHs inhibit the activity of purified COX-2 enzyme in vitro but do not inhibit COX-1 activity. Thus, DEP and PAHs do not affect ligand-induced COX-2 gene expression, phospholipase activation, or arachidonic acid release in macrophages and fibroblasts but exert their inhibitory effect on prostaglandin production by preferentially blocking COX-2 enzyme activity.