Further Studies of the Role of Cyclic β-Glucans in Symbiosis. An ndvC Mutant of Bradyrhizobium japonicum Synthesizes Cyclodecakis-(1→3)-β-Glucosyl1

The cyclic β-(1→3),β-(1→6)-d-glucan synthesis locus of Bradyrhizobium japonicum is composed of at least two genes, ndvB and ndvC. Mutation in either gene affects glucan synthesis, as well as the ability of the bacterium to establish a successful symbiotic interaction with the legume host soybean (Glycine max). B. japonicum strain AB-14 (ndvB::Tn5) does not synthesize β-glucans, and strain AB-1 (ndvC::Tn5) synthesizes a cyclic β-glucan lacking β-(1→6)-glycosidic bonds. We determined that the structure of the glucan synthesized by strain AB-1 is cyclodecakis-(1→3)-β-d-glucosyl, a cyclic β-(1→3)-linked decasaccharide in which one of the residues is substituted in the 6 position with β-laminaribiose. Cyclodecakis-(1→3)-β-d-glucosyl did not suppress the fungal β-glucan-induced plant defense response in soybean cotyledons and had much lower affinity for the putative membrane receptor protein than cyclic β-(1→3),β-(1→6)-glucans produced by wild-type B. japonicum. This is consistent with the hypothesis presented previously that the wild-type cyclic β-glucans may function as suppressors of a host defense response.

Several Thaumatin-Like Proteins Bind to β-1,3-Glucans1

Pathogenesis-related proteins from intercellular fluid washings of stressed barley (Hordeum vulgare L.) leaves were analyzed to determine their binding to various water-insoluble polysaccharides. Three proteins (19, 16, and 15 kD) bound specifically to several water-insoluble β-1,3-glucans. Binding of the barley proteins to pachyman occurred quickly at 22°C at pH 5.0, even in the presence of 0.5 m NaCl, 0.2 m urea, and 1% (v/v) Triton X-100. Bound barley proteins were released by acidic treatments or by boiling in sodium dodecyl sulfate. Acid-released barley proteins could bind again specifically and singly to pachyman. Water-soluble laminarin and carboxymethyl-pachyman competed for the binding of the barley proteins to pachyman. The N-terminal sequence of the 19-kD barley β-1,3-glucan-binding protein showed near identity to the barley seed protein BP-R and high homology to other thaumatin-like (TL) permatins. The 16-kD barley protein was also homologous to TL proteins, whereas the 15-kD barley protein N-terminal sequence was identical to the pathogenesis-related Hv-1 TL protein. Antifungal barley protein BP-R and corn (Zea mays) zeamatin were isolated by binding to pachyman. Two extracellular proteins from stressed pea (Pisum sativum L.) also bound to pachyman and were homologous to TL proteins.

Identification of Inositol 1,3,4-Trisphosphate 5-Kinase and Inositol 1,3,4,5-Tetrakisphosphate 6-Kinase in Immature Soybean Seeds

In extracts of immature soybean (Glycine max [L.] Merr.) seeds inositol tetrakisphosphate was formed from [3H]inositol 1,3,4-trisphosphate but not from [3H]inositol 1,4,5-trisphosphate. Inositol 1,3,4-trisphosphate kinase was purified to a specific activity of 3.55 min−1 mg−1 by polyethylenimine clarification and anion-exchange chromatography. The partially purified enzyme converted [3H]inositol 1,3,4-trisphosphate to inositol 1,3,4,5-tetrakisphosphate as the major product and inositol 1,3,4,6- and/or 1,2,3,4-tetrakisphosphate as the minor product. Subsequent experiments revealed a separate inositol 1,3,4,5-tetrakisphosphate 6-kinase activity, which could link these enzymes to inositol hexakisphosphate synthesis via the previously reported inositol 1,3,4,5,6-pentakisphosphate 2-kinase. The apparent Km values for inositol 1,3,4-trisphosphate kinase were 200 ± 0 nm for inositol 1,3,4-trisphosphate and 171 ± 4 μm for ATP, and the reaction was not reversible. The kinetics were such that no activity could be detected using unlabeled inositol 1,3,4-trisphosphate and [γ-32P]ATP, which suggested that other kinases may have been observed when less purified fractions were incubated with radiolabeled ATP. Inositol 1,3,4-trisphosphate kinase was nonspecifically inhibited more than 80% by various inositol polyphosphates at a concentration of 100 μm.

Alpha-lactalbumin affects the acceptor specificity of Lymnaea stagnalis albumen gland UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalactosaminyltransferase: synthesis of GalNAc beta 1-->4Glc.

The N,N'-diacetyllactosediamine (lacdiNAc) pathway of complex-type oligosaccharide synthesis is controlled by a UDP-GalNAc:GlcNAc beta-R beta 1-->4-N-acetylgalac-tesaminyltransferase (beta 4-GalNAcT) that acts analogously to the common UDP-Gal:GlcNAc beta-R beta 1-->4-galactosyltransferase (beta 4-GalT). LacdiNAc-based chains particularly occur in invertebrates and cognate beta 4-GalNAcTs have been identified in the snail Lymnaea stagnalis, in two schistosomal species, and in several lepldopteran insect cell lines. Because of the similarity in reactions catalyzed by both enzymes, we investigated whether L. stagnalis albumen gland beta 4-GalNAcT would share with mammalian beta 4-GalT the property of interacting with alpha-lactalbumin (alpha-LA), a protein that only occurs in the lactating mammary gland, to form a complex in which the specificity of the enzyme is changed. It was found that, under conditions where beta 4-GalT forms the lactose synthase complex with alpha-LA, the snail beta 4-GalNAcT was induced by this protein to act on Glc with a > 100-fold increased efficiency, resulting in the formation of the lactose analog GalNAc beta 1-->4Glc. This forms the second example of a glycosyltransferase, the specificity of which can be altered by a modifier protein. So far, however, no protein fraction could be isolated from L. stagnalis that could likewise interact with the beta 4-GalNAcT. Neither had lysozyme c, a protein that is homologous to alpha-LA, an effect on the specificity of the enzyme. These results raise the question of how the capability to interact with alpha-LA has been conserved in the snail enzyme during evolution without any apparent selective pressure. They also suggest that snail beta 4-GalNAcT and mammalian beta 4-GalT show similarity at a molecular level and allows the identification of the beta 4-GalNAcT as a candidate member of the beta 4-GalT family.

Human immunodeficiency virus tat gene transfer to the murine central nervous system using a replication-defective herpes simplex virus vector stimulates transforming growth factor beta 1 gene expression.

The high incidence of neurological disorders in patients afflicted with acquired immunodeficiency syndrome (AIDS) may result from human immunodeficiency virus type 1 (HIV-1) induction of chemotactic signals and cytokines within the brain by virus-encoded gene products. Transforming growth factor beta1 (TGF-beta1) is an immunomodulator and potent chemotactic molecule present at elevated levels in HIV-1-infected patients, and its expression may thus be induced by viral trans-activating proteins such as Tat. In this report, a replication-defective herpes simplex virus (HSV)-1 tat gene transfer vector, dSTat, was used to transiently express HIV-1 Tat in glial cells in culture and following intracerebral inoculation in mouse brain in order to directly determine whether Tat can increase TGF-beta1 mRNA expression. dSTat infection of Vero cells transiently transfected by a panel of HIV-1 long terminal repeat deletion mutants linked to the bacterial chloramphenicol acetyltransferase reporter gene demonstrated that vector-expressed Tat activated the long terminal repeat in a trans-activation response element-dependent fashion independent of the HSV-mediated induction of the HIV-1 enhancer, or NF-kappaB domain. Northern blot analysis of human astrocytic glial U87-MG cells transfected by dSTat vector DNA resulted in a substantial increase in steady-state levels of TGF-beta1 mRNA. Furthermore, intracerebral inoculation of dSTat followed by Northern blot analysis of whole mouse brain RNA revealed an increase in levels of TGF-beta1 mRNA similar to that observed in cultured glial cells transfected by dSTat DNA. These results provided direct in vivo evidence for the involvement of HIV-1 Tat in activation of TGF-beta1 gene expression in brain. Tat-mediated stimulation of TGF-beta1 expression suggests a novel pathway by which HIV-1 may alter the expression of cytokines in the central nervous system, potentially contributing to the development of AIDS-associated neurological disease.