Galactosyltransferase of Neurospora.

An enzyme, galactosyltransferase, able to catalyze the formation of galactose polymers was detected in cell-free extracts of a wild type strain of Neurospora crassa. Enzyme activity was found in both the supernatant and the particle fractions after centrifugation at 100,000 X g. The enzyme assayed in the 100,000 X g supernatant showed a 4fold difference in specific activity as compared to that found in the particle fraction.     

Affinity chromatography of soluble galactosyltransferases using an easily synthesized N-acetylglucosamine-agarose bead

Summary Agarose derivatized withp-nitrophenyl-N-acetyl--d-glucosamine was used for affinity chromatography for soluble galactosyltransferase from various sources. Moreover, this adsorbant acts as an acceptor for galactose when incubated with prepurified galactosyltransferase.This work was supported by a grant from the USPHS (grant No. 1 PO1 CA 14294-2).     

Galactosyltransferase of Neurospora

Summary An enzyme, galactosyltransferase, able to catalyze the formation of galactose polymers was detected in cell-free extracts of a wild type strain of Neurospora crassa. Enzyme activity was found in both the supernatant and the particle fractions after centrifugation at 100,000 xg. The enzyme assayed in the 100,000 xg supernatant showed a 4fold difference in specific activity as compared to that found in the particle fraction.Acknowledgment. This work was supported by a grant from the Research Corporation.     

TGFβ-induced protein mediates lymphatic endothelial cell adhesion to the extracellular matrix under low oxygen conditions

TGFbeta-induced protein (TGFBI) is an extracellular protein that mediates cell adhesion to collagen, laminin and fibronectin through its interaction with different beta integrins. We had previously reported that hypoxia-induced TGFBI mRNA expression in lymphatic endothelial cells (LEC). Here, we demonstrate that TGFBI can contribute to hypoxia-induced increases in LEC adhesion to the ECM. We show that while there are no changes in alpha1, alpha4, alphav, beta1, beta2, beta3, alpha5beta1, alphavbeta3, alphavbeta5 integrin expression on the LEC surface after hypoxia exposure, there exists an accumulation of TGFBI adaptor protein in LEC supernatants. We also demonstrate that hypoxia driven TGBFI expression is dependent on TGFbeta production by LEC. Furthermore, we show that TGFBI mediated LEC adhesion and migration through the ECM by its binding to the beta3 integrin. The identification of the specific mechanisms regulating LEC-ECM interactions may help us design new therapeutic applications for diseases in which lymphatic vessel function is compromised.     

Evolution of the arginase fold and functional diversity

Novel structural superfamilies can be identified among the large number of protein structures deposited in the Protein Data Bank based on conservation of fold in addition to conservation of amino acid sequence. Since sequence diverges more rapidly than fold in protein Evolution, proteins with little or no significant sequence identity are occasionally observed to adopt similar folds, thereby reflecting unanticipated evolutionary relationships. Here, we review the unique alpha/beta fold first observed in the manganese metalloenzyme rat liver arginase, consisting of a parallel eight-stranded beta-sheet surrounded by several helices, and its evolutionary relationship with the zinc-requiring and/or iron-requiring histone deacetylases and acetylpolyamine amidohydrolases. Structural comparisons reveal key features of the core alpha/beta fold that contribute to the divergent metal ion specificity and stoichiometry required for the chemical and biological functions of these enzymes.     

Enzymatic in vitro synthesis of I-branches of mammalian polylactosamines: generation of scaffolds for multiple selectin-binding saccharide determinants

Polylactosamines are covalent monosaccharide assemblies of the animal kingdom and some bacteria, and are characterized by backbones of interlinked N-acetyllactosamine units (Galbeta1-4GlcNAc, LacNAc). The mammalian LacNAc arrays are linear (blood group i-type) and branched (blood group I-type), and are linked to the core elements of glycolipids as well as O- and N-glycans of glycoproteins and keratan sulfate proteoglycans. Generation of I-branches to linear i-type polylactosamines is initiated by two kinds of beta6GlcNAc transferases. One type of the enzymes transfers to Glc-NAcbeta 1-3Galbeta 1-OR of growing i-chains at the peridistal (underlined) Gal; these enzymes are called dIGnT (d for 'distally acting'). The other enzymes transfer to internal Gal units of preformed i-chains; they are called cIGnT (c for 'centrally acting'). Purified natural and recombinant enzymes of both types have been described. The structures of I-type polylactosamines result from a collaboration of dIGnTs, cIGnTs, beta4GalTs and the i-chain-elongating iGnTs. At present, the interplay of these enzymes in vivo is poorly understood. By contrast, enzyme-assisted in vitro synthesis of branched polylactosamines representing distinct LacNAc arrays that are multiply capped by a variety of decorations is possible. Some of the synthetic polylactosamines reduce the lymphocyte-endothelium adhesion in a tissue-specific mode, raising the possibility of achieving local immunosuppression in the future. Useful applications of multiply decorated I-type polylactosamines may also be found in prevention of mammalian gamete adhesion and in inhibition of bacterial and viral adhesion to host tissues.     

Evidence for the extranuclear localization of thymosins in thymus.

A new radioimmunoassay has been developed for thymosin beta 4 by generating rabbit polyclonal antibodies against the synthetic N-terminal peptide fragment 1-15 coupled to KLH. The synthetic analogue [Tyr12]-thymosin beta 4 (1-15) was used as tracer. This radioimmunoassay, with a useful range of 10-1000 pmoles, showed cross-reactivity with the second homologous beta-thymosin of man and rat (thymosin beta 10) but not of calf (thymosin beta 9). This radioimmunoassay, together with an improved radioimmunoassay for the N-terminus of parathymosin alpha, was employed for the measurement of the levels of thymosin beta 4 and parathymosin alpha in nuclear and extranuclear extracts of calf thymus. The bulk of these polypeptides was found in the extranuclear material whereas only traces were observed in the nuclear environment, which indicates the extranuclear localisation of alpha- and beta-thymosins.     

(—)-Epigallocatechin-3-gallate interferes with mast cell adhesiveness, migration and its potential to recruit monocytes

Mast cells are multipotent effector cells of the immune system. They are able to induce and enhance angiogenesis via multiple pathways. (-)-Epigallocatechin-3-gallate (EGCG), a major component of green tea and a putative chemopreventive agent, was reported to inhibit tumor invasion and angiogenesis, processes that are essential for tumor growth and metastasis. Using the human mast cell line HMC-1 and commercial cDNA macroarrays, we evaluated the effect of EGCG on the expression of angiogenesis-related genes. Our data show that among other effects, EGCG treatment reduces expression of two integrins (alpha5 and beta3) and a chemokine (MCP1), resulting in a lower adhesion of mast cells associated with a decreased potential to produce signals eliciting monocyte recruitment. These effects on gene expression levels are functionally validated by showing inhibitory effects in adhesion, aggregation, migration and recruitment assays.     

Isolation and partial characterization of a phytotoxic glycoprotein from culture filtrates ofRhynchosporium secalis

Summary Rhynchosporium secalis (Oud.) Davis produces a phytotoxic compound with a mol. wt of 275×10³ which is able to induce chlorotic symptoms in both susceptible and resistant barley leaves. Collectively, the data suggest that the toxin is a glycoprotein. Mild base treatment, by elimination, indicates that threonine and serine are involved in o-glycosidic linkages with the carbohydrate moiety. Sugar residues occur in the molecule in the ratio of mannose, rhammnose, galactose, glucosamine 13.6111.Acknowledgments. We acknowledge the assistance of Mr M. McNeil and P. Albersheim in doing the sugar analyses shown in this report. Financial support for this project was provided by a BARD grant 1-31-79, the French government, and the Montana Ag. Expt. Station.     

Modulation of C2 and C3 gene expression of human peripheral blood monocytes by interleukin 1 beta, interferon gamma, tumor necrosis factor alpha and lipopolysaccharide.

The effect of interleukin 1 beta (IL-1 beta), interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF alpha) and lipopolysaccharide (LPS) on the expression of the C2 and C3 genes in human adherent monocytes was studied. Stimulation of monocytes with IFN-gamma increased both C2 and C3 mRNA. IL-1 beta also increased C2 mRNA level, whereas C3 gene expression was not enhanced. TNF alpha failed to increase either C2 or C3 mRNA. LPS increased C2 mRNA, but suppressed C3 gene expression. These results suggest that C2 and C3 production by monocytes is regulated by IL-1 beta and IFN-gamma in the local tissues.     

Biological indicators of cadmium exposure and toxicity

The increasing environmental and occupational exposure of populations to cadmium creates the need for biological indicators of cadmium exposure and toxicity. The advantages and disadvantages of monitoring blood cadmium, urinary, fecal, hair, and tissue cadmium, serum creatinine, beta 2-microglobulin, alpha 1-antitrypsin and other proteins, and urinary amino acids, enzymes, total proteins, glucose, beta 2-microglobulin, retinol-binding protein, lysozyme, and metallothionein are discussed. It is concluded that urinary cadmium, metallothionein and beta 2-microglobulin may be used together to assess cadmium exposure and toxicity.     

Radical catalysis of B12 enzymes: structure,mechanism, inactivation,and reactivation of diol and glycerol dehydratases

Enzymatic radical catalysis is defined as a mechanism of catalysis by which enzymes catalyze chemically difficult reactions by utilizing the high reactivity of free radicals. Adenosylcobalamin (coenzyme B12) serves as a cofactor for enzymatic radical reactions. The recent structural analysis of adenosylcobalamin-dependent diol dehydratase revealed that the substrate 1,2-propanediol and an essential potassium ion are located inside a (beta/alpha)8 barrel. Two hydroxyl groups of the substrate coordinate directly to the potassium ion which binds to the negatively charged inner part of the cavity. Cobalamin bound in the base-on mode covers the cavity to isolate the active site from solvent. Based on the three-dimensional structure and theoretical calculations, a new mechanism for diol dehydratase is proposed in which the potassium ion plays a direct role in the catalysis. The mechanisms for generation of a catalytic radical by homolysis of the coenzyme Co-C bond and for protection of radical intermediates from undesired side reactions during catalysis are discussed based on the structure. The reactivating factors for diol and glycerol dehydratases have been identified. These factors are a new type of molecular chaperone which participate in reactivation of the inactivated holoenzymes by mediating ATP-dependent exchange of the modified coenzyme for free intact coenzyme.     

Regulated expression and neural functions of human natural killer-1 (HNK-1) carbohydrate

Human natural killer-1 (HNK-1) carbohydrate, comprising a unique trisaccharide HSO₃-3GlcAβ1-3Galβ1-4GlcNAc, shows well-regulated expression and unique functions in the nervous system. Recent studies have revealed sophisticated and complicated expression mechanisms for HNK-1 glycan. Activities of biosynthetic enzymes are controlled through the formation of enzyme-complexes and regulation of subcellular localization. Functional aspects of HNK-1 carbohydrate were examined by overexpression, knockdown, and knockout studies of these enzymes. HNK-1 is involved in several neural functions such as synaptic plasticity, learning and memory, and the underlying molecular mechanisms have been illustrated upon identification of the target carrier glycoproteins of HNK-1 such as the glutamate receptor subunit GluA2 or tenascin-R. In this review, we describe recent findings about HNK-1 carbohydrate that provide further insights into the mechanism of its expression and function in the nervous system.     

Histone methylation and ubiquitination with their cross-talk and roles in gene expression and stability

Methylation of lysine residues of histones is associated with functionally distinct regions of chromatin, and, therefore, is an important epigenetic mark. Over the past few years, several enzymes that catalyze this covalent modification on different lysine residues of histones have been discovered. Intriguingly, histone lysine methylation has also been shown to be cross-regulated by histone ubiquitination or the enzymes that catalyze this modification. These covalent modifications and their cross-talks play important roles in regulation of gene expression, heterochromatin formation, genome stability, and cancer. Thus, there has been a very rapid progress within past several years towards elucidating the molecular basis of histone lysine methylation and ubiquitination, and their aberrations in human diseases. Here, we discuss these covalent modifications with their cross-regulation and roles in controlling gene expression and stability. Received 24 September 2008; received after revision 21 November 2008; accepted 28 November 2008     

Defensive steroids from a carrion beetle (Silpha americana)

The defensive anal effluent discharged by Silpha americana in response to disturbance contains a mixture of steroids stemming from a glandular annex of the rectum. The compounds have been characterized as 15 beta-hydroxyprogesterone (1, principal component), 5 beta-pregnan-15 beta-ol-3,20-dione (2), 5 beta-pregnan-3 alpha, 15 beta-diol-20-one (3), 5 beta-pregnan-7 beta, 15 beta-diol-3,20-dione (4), 5 beta-pregnan-3 alpha, 7 beta, 15 beta-triol-20-one (5), 5 beta-pregnan-16 alpha-ol-3,20-dione (6), and 5 beta-pregnan-3 alpha, 16 alpha-diol-20-one (7), none previously found in insects. Bioassays with jumping spiders showed compounds 1 and 6 to be feeding deterrents at the 1 microgram level.     

The origin,diversity, and structure function relationships of insect luciferases

Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. Whereas the oxidative mechanism which leads to light emission is similar for most luciferases, these enzymes and their substrates are evolutionarily unrelated. Among all bioluminescent groups, insects constitute one of the most diverse in terms of biochemistry. In the fungus-gnats (Mycetophilidae: Diptera), for example, bioluminescence is generated by two biochemically distinct systems. Despite the diversity, investigations on insect luciferases and biochemistry have been conducted mostly with fireflies. The luciferases from the related phengodid beetles, which can produce green to red bioluminescence using the same chemistry as firefly luciferases, have been recently investigated. Beetle luciferases originated from ancestral acyl-CoA ligases. Present data suggest that conserved motifs among this class of ligases are involved in substrate adenylation. The three-dimensional structure of firefly luciferase was recently solved and mutagenesis studies have been performed identifying putative residues involved in luciferin binding and bioluminescence color determination in several beetle luciferases. The knowledge gained through these studies is helping in the development of useful reporter gene tools for biotechnological and biomedical purposes. Received 4 March 2002; received after revision 13 May 2002; accepted 21 May 2002