Purification and properties of brain alkaline phosphatase

Alkaline phosphatase from sheep brain has been purified to homogeneity. The method includes butanol extraction, fractional ethanol precipitation, ion-exchange chromatography on DEAE-cellulose, and on DEAE-Sephadex followed by Sephadex G-200 filtration. By these steps, the enzyme is purified 22,920-fold with 15% recovery. The homogeneous enzyme is shown to be a sialoglycoprotein in nature. Neuraminidase treatment reduces the electrophoretic mobility of the enzyme. The enzyme shows pyridoxal phosphate phosphatase activity along with p-nitrophenylphosphate phosphatase activity. Both these compounds behave as mutual alternate competitive substrates. The general properties of the enzyme are described

Sweetening of Glutamine Metabolism in Cancer Cells by Rho GTPases Through Convergence of Multiple Oncogenic Signaling Pathways

Comment on: Lukey MJ, Greene KS, Erickson JW, et al. The oncogenic transcription factor c-Jun regulates glutaminase expression and sensitizes cells to glutaminase-targeted therapy. Nat Commun 2016;7:11321

Fluorescence studies on the interaction of some ligands with carcinoscorpin, the sialic acid specific lectin, from the horseshoe crab, Carcinoscorpius rotundacauda

The binding affinities of some ligands towards the sialic acid-specific lectin carcinoscorpin, from hemolymph of the horseshoe crab Carcinoscorpius rotundacauda have been determined by protein fluorescence quenching in presence of ligands. Among the ligands studied, the disaccharide O-(N-acetylneuraminyl)-(2→6)-2-acetamido-2-deoxy-D-galactitol has the highest Ka (l.15 × 106 M−1) for carcinoscorpin. Studies on the effect of pH on Ka values of disaccharide suggests the possible involvement of amino acid residues having pKa values around 6.0 and 9.0 in the binding activity of carcinoscorpin. There were distinct changes in the accessibility of the fluorescent tryptophan residues of carcinoscorpin by ligand-binding as checked through potassium iodide quenching

NRH:quinone Oxidoreductase 2 (NqO2) and Glutaminase (GLS) Both Play a Role in Large Extracellular Vesicles (LEV) Formation in Preclinical LNCaP-C4-2B Prostate Cancer Model of Progressive Metastasis

In the course of studies aimed at the role of oxidative stress in the development of metastatic potential in the LNCaP-C4-2B prostate cancer progression model system, we found a relative decrease in the level of expression of the cytoplasmic nicotinamide riboside: quinone oxidoreductase (NQO2) and an increase in the oxidative stress in C4-2B cells compared to that in LNCaP or its derivatives C4 and C4-2. It was also found that C4-2B cells specifically shed large extracellular vesicles (LEVs) suggesting that these LEVs and their cargo could participate in the establishment of the osseous metastases. The level of expression of caveolin-1 increased as the system progresses from LNCaP to C4-2B. Since NQO2 RNA levels were not changed in LNCaP, C4, C4-2, and C4-2B, we tested an altered cellular distribution hypothesis of NQO2 being compartmentalized in the membrane fractions of C4-2B cells which are rich in lipid rafts and caveolae. This was confirmed when the detergent resistant membrane fractions were probed on immunoblots. Moreover, when the LEVs were analyzed for membrane associated caveolin-1 as possible cargo, we noticed that the enzyme NQO2 was also a component of the cargo along with caveolin-1 as seen in double immunofluorescence studies. Molecular modeling studies showed that a caveolin-1 accessible site is present in NQO2. Specific interaction between NQO2 and caveolin-1 was confirmed using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Interestingly, whole cell lysate and mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B showed an increasing expression of glutaminase (GLS, kidney type). The extrusion of LEVs appears to be a specific property of the bone metastatic C4-2B cells and this process could be inhibited by a GLS specific inhibitor BPTES, suggesting the critical role of a functioning glutamine metabolism. Our results indicate that a high level of expression of caveolin-1 in C4-2B cells contributes to an interaction between caveolin-1 and NQO2 and to their packaging as cargo in the shed LEVs. These results suggest an important role of membrane associated oxidoreductases in the establishment of osseous metastases in prostate cancer

Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes That Have Cysteine S-Conjugate Beta-Lyase Activity and Can Transaminate L-Selenomethionine

Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-L-selenocysteine (MSC) and L-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites

Microvesicles and exosomes in pulmonary hypertension

Pulmonary hypertension is a serious disorder with a high morbidity and mortality rate. The juxtaposition of endothelial cells and smooth muscle cells maintains vascular homeostasis. Vascular injury results in endothelial dysfunction, leading to impaired vascular relaxation, cell proliferation, and altered immune and metabolic states. In addition, injury induces pulmonary arterial endothelium and other cells to release increased levels of extracellular vesicles, including exosomes and microparticles that may be involved in enhancing the proliferation of apoptosis-resistant smooth muscle cells. These extracellular vesicles carry proteins, lipids, RNA, miRNA, chemokines cytokines and modulate immune function, inflammation, embryogenesis, regenerative processes, and serve as intercellular messengers. Importantly, mesenchymal stem cells-derived extracellular vesicles exert inhibitory effects on inflammation and restore homeostasis. This article reviews the pathophysiological role of extracellular vesicles in pulmonary hypertension

ISCHAEMIC GANGRENE OF LEG DUE TO RING CONSTRICTION: A CASE REPORT

An infant with bilateralcongenital ringconstrictions in the legs presenting with ischaemic gangrene of one leg is reported. The ischaemiawas precipitated by greenstick fracture, the constriction ring producing a 'tourniquet effect ' on the swollen tissues. Prophylactic release of deep annular constrictions is advised to prevent complications

High Levels of Glutaminase II Pathway Enzymes in Normal and Cancerous Prostate Suggest a Role in \u27Glutamine Addiction\u27

Abstract: Many tumors readily convert l-glutamine to α-ketoglutarate. This conversion is almost invariably described as involving deamidation of l-glutamine to l-glutamate followed by a transaminase (or dehydrogenase) reaction. However, mammalian tissues possess another pathway for conversion of l-glutamine to α-ketoglutarate, namely the glutaminase II pathway: l-Glutamine is transaminated to α-ketoglutaramate, which is then deamidated to α-ketoglutarate by ω-amidase. Here we show that glutamine transaminase and ω-amidase specific activities are high in normal rat prostate. Immunohistochemical analyses revealed that glutamine transaminase K (GTK) and ω-amidase are present in normal and cancerous human prostate and that expression of these enzymes increases in parallel with aggressiveness of the cancer cells. Our findings suggest that the glutaminase II pathway is important in providing anaplerotic carbon to the tricarboxylic acid (TCA) cycle, closing the methionine salvage pathway, and in the provision of citrate carbon in normal and cancerous prostate. Finally, our data also suggest that selective inhibitors of GTK and/or ω-amidase may be clinically important for treatment of prostate cancer. In conclusion, the demonstration of a prominent glutaminase II pathway in prostate cancer cells and increased expression of the pathway with increasing aggressiveness of tumor cells provides a new perspective on \u27glutamine addiction\u27 in cancers