Synthesis of a tetrasaccharide unit of the capsular polysaccharide of Streptococcus pneumoniae type 9V

In the synthesis of 8-methoxycarbonyloctyl O-(alpha-D-galactopyranosyl)-(1----3)-O-(2-acetamido-2-deoxy-beta-D- mannopyranosyl)-(1----4)-O-(beta-D-glucopyranosyl)-(1----4)-alpha-D- glucopyranoside, which represents a component of the capsular polysaccharide of Streptococcus pneumoniae type 9V, the key step was the coupling of alpha-D-Galp-(1----3)-beta-D-ManpNAc-(1----4)-D-Glc as glycosyl donor with 8-ethoxy-carbonyloctyl 6-O-acetyl-2,3-di-O-benzyl-alpha-D-glucopyranoside as glycosyl acceptor by use of the imidate method. Only the beta-imidate of the trisaccharide could be employed in this glycosidation reaction to give stereoselectively the tetrasaccharide in high yield. The alpha-imidate of the trisaccharide led to hydrolysis of the imidate group.     

Membrane transport proteins: implications of sequence comparisons.

Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.     

tRNA topography during translocation: steady-state and kinetic fluorescence energy-transfer studies

The distances between the anticodon loops of fluorescent tRNAPhe bound to the E site and to either the A or the P site of poly(U)-programmed Escherichia coli ribosomes were measured by fluorescence energy transfer. Donor and acceptor molecules were wybutine and proflavin, respectively, both located 3' to the anticodon of tRNAPhe. The anticodon loops were found to be separated by 42 +/- 10 A (A to E site) and 34 +/- 8 A (P to E site). The latter distance is much larger than the one measured between the anticodon loops of A and P site bound tRNAs [24 +/- 4 A; Paulsen, H., Robertson, J. M., & Wintermeyer, W. (1983) J. Mol. Biol. 167, 411-426], rendering unlikely simultaneous codon-anticodon interaction in the P and E sites. In kinetic stopped-flow measurements, the energy transfer between the anticodon loops of the tRNA molecules was followed during translocation. The transfer efficiency decreases in three steps with apparent rate constants on the order of 1, 0.1, and 0.01 s-1. The fast step is ascribed to the simultaneous displacement of the deacylated tRNAPhe out of the P site and of the N-AcPhe-tRNAPhe from the A site to the P site. The distance between the anticodon loops does not change appreciably during this reaction. A significant separation of the two tRNAs occurs during the intermediate and the slow steps. The latter most likely represents a rearrangement of the posttranslocation complex containing both tRNA molecules.(ABSTRACT TRUNCATED AT 250 WORDS)     

An interspecies comparison of normal levels of glycosylated hemoglobin and glycosylated albumin

Aminophenylboronic acid affinity chromatography was used to measure glycosylated hemoglobin and glycosylated albumin levels in a variety of species. The highest glycosylated hemoglobin levels were found in man, the lowest in the chicken and the pig. The highest glycosylated albumin levels were found in avian species, the lowest in the mouse and the rat. A simple kinetic model was used to analyze the rates of formation of glycosylated hemoglobin and albumin in the various species. Rates of glycosylated albumin formation were very similar across the species while rates of glycosylated hemoglobin formation were quite different, presumably reflecting wide differences in erythrocyte permeability to glucose among the species.     

Mode of high temperature injury to wheat during grain development

High temperature stress adversely affects wheat growth in many important production regions, but the mode of injury is unclear. Wheat ( Triticum aestivum L. cv. Newton) was grown under controlled conditions to determine the relative magnitude and sequences of responses of source and sink processes to high temperature stress during grain development. Regimes of 25°C day/15°C night, 30°C day/20°C night, and 35°C day/25°C night from 5 days after anthesis to maturity differentially affected source and sink processes. High temperatures accelerated the normal decline in viable leaf blade area and photosynthetic activities per unit leaf area. Electron transport, as measured by Hill reaction activity, declined earlier and faster than other photosynthetic processes at the optimum temperature of 25/15 °C and at elevated temperatures. Changes in RUBP carboxylase activities were similar in direction but smaller in magnitude than changes in photosynthesic rate. Increased protease activity during senscence was markedly accentuated by high temperature stress. Specific protease activity increased 4-fold at 25/15 °C and 28-fold at 35/25 °C from 0 to 21 days after initiation of temperature treatments. Grain-filling rate decreased from the lowest to the highest temperature, but the change was smaller than the decrease in grain-filling duration at the same temperatures. We concluded that a major effect of high temperature is acceleration of senescence, including cessation of vegetative and reproductive growth, deterioration of photosynthetic activities, and degradation of proteinaceous constituents.     

Comparison of Transmitter Amino Acid Levels in Rat Globus Pallidus and Neostriatum During Hypoglycemia or After Treatment with Methionine Sulfoximine or γ-Vinyl γ-Aminobutyric Acid

The levels of amino acids in globus pallidus, a structure heavily innervated with gamma-aminobutyric acid (GABA)-ergic terminals but few glutamergic terminals, were compared with the levels in neostriatum, a structure richly innervated with glutamergic terminals but intermediate in GABAergic terminals. The level of glutamate in neostriatum was twice as high as in globus pallidus whereas the level of GABA in globus pallidus was three times higher than in neostriatum. The level of aspartate was similar in both regions whereas the level of glutamine was correlated with the level of glutamate. Methionine sulfoximine, a glutamine synthetase inhibitor, reduced the level of glutamine to 10-20% of control in both structures. This reduction was accompanied by the largest decrease in the level of glutamate in neostriatum, indicating that transmitter glutamate turns over more rapidly than other glutamate pools. Likewise, insulin decreased the levels of glutamate and glutamine more in neostriatum than in globus pallidus. gamma-Vinyl GABA increased the level of GABA in globus pallidus more than in neostriatum although the percent increase was largest in neostriatum. Treatment with gamma-vinyl GABA was accompanied by a large reduction in the level of GABA, indicating that a substantial proportion of the glutamine pool is linked to GABA metabolism.     

Proton-dependent multidrug efflux systems.

Multidrug efflux systems display the ability to transport a variety of structurally unrelated drugs from a cell and consequently are capable of conferring resistance to a diverse range of chemotherapeutic agents. This review examines multidrug efflux systems which use the proton motive force to drive drug transport. These proteins are likely to operate as multidrug/proton antiporters and have been identified in both prokaryotes and eukaryotes. Such proton-dependent multidrug efflux proteins belong to three distinct families or superfamilies of transport proteins: the major facilitator superfamily (MFS), the small multidrug resistance (SMR) family, and the resistance/ nodulation/cell division (RND) family. The MFS consists of symporters, antiporters, and uniporters with either 12 or 14 transmembrane-spanning segments (TMS), and we show that within the MFS, three separate families include various multidrug/proton antiport proteins. The SMR family consists of proteins with four TMS, and the multidrug efflux proteins within this family are the smallest known secondary transporters. The RND family consists of 12-TMS transport proteins and includes a number of multidrug efflux proteins with particularly broad substrate specificity. In gram-negative bacteria, some multidrug efflux systems require two auxiliary constituents, which might enable drug transport to occur across both membranes of the cell envelope. These auxiliary constituents belong to the membrane fusion protein and the outer membrane factor families, respectively. This review examines in detail each of the characterized proton-linked multidrug efflux systems. The molecular basis of the broad substrate specificity of these transporters is discussed. The surprisingly wide distribution of multidrug efflux systems and their multiplicity in single organisms, with Escherichia coli, for instance, possessing at least nine proton-dependent multidrug efflux systems with overlapping specificities, is examined. We also discuss whether the normal physiological role of the multidrug efflux systems is to protect the cell from toxic compounds or whether they fulfil primary functions unrelated to drug resistance and only efflux multiple drugs fortuitously or opportunistically.     

The transient receptor potential protein (Trp), a putative store-operated Ca2+ channel essential for phosphoinositide-mediated photoreception, forms a signaling complex with NorpA, InaC and InaD.

The transient receptor potential protein (Trp) is a putative capacitative Ca2+ entry channel present in fly photoreceptors, which use the inositol 1,4,5-trisphosphate (InsP3) signaling pathway for phototransduction. By immunoprecipitation studies, we find that Trp is associated into a multiprotein complex with the norpA-encoded phospholipase C, an eye-specific protein kinase C (InaC) and with the InaD protein (InaD). InaD is a putative substrate of InaC and contains two PDZ repeats, putative protein-protein interaction domains. These proteins are present in the photoreceptor membrane at about equimolar ratios. The Trp homolog analyzed here is isolated together with NorpA, InaC and InaD from blowfly (Calliphora) photoreceptors. Compared to Drosophila Trp, the Calliphora Trp homolog displays 77% amino acid identity. The highest sequence conservation is found in the region that contains the putative transmembrane domains S1-S6 (91% amino acid identity). As investigated by immunogold labeling with specific antibodies directed against Trp and InaD, the Trp signaling complex is located in the microvillar membranes of the photoreceptor cells. The spatial distribution of the signaling complex argues against a direct conformational coupling of Trp to an InsP3 receptor supposed to be present in the membrane of internal photoreceptor Ca2+ stores. It is suggested that the organization of signal transducing proteins into a multiprotein complex provides the structural basis for an efficient and fast activation and regulation of Ca2+ entry through the Trp channel.     

Trimerization and crystallization of reconstituted light-harvesting chlorophyll a/b complex.

The major light-harvesting complex (LHCII) of photosystem II, the most abundant chlorophyll-containing complex in higher plants, is organized in trimers. In this paper we show that the trimerization of LHCII occurs spontaneously and is dependent on the presence of lipids. LHCII monomers were reconstituted from the purified apoprotein (LHCP), overexpressed in Escherichia coli, and pigments, purified from chloroplast membranes. These synthetic LHCII monomers trimerize in vitro in the presence of a lipid fraction isolated from pea thylakoids. The reconstituted LHCII trimers are very similar to native LHCII trimers in that they are stable in the presence of mild detergents and can be isolated by partially denaturing gel electrophoresis or by centrifugation in sucrose density gradients. Moreover, both native and reconstituted LHCII trimers exhibit signals in circular dichroism in the visible range that are not seen in native or reconstituted LHCII monomers, indicating that trimer formation either establishes additional pigment-pigment interactions or alters pre-existing interactions. Reconstituted LHCII trimers readily form two-dimensional crystals that appear to be identical to crystals of the native complex.