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Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides.

Most virulence (vir) genes of Agrobacterium tumefaciens that are required for the formation of crown gall tumors are expressed in response to such plant signal molecules as acetosyringone and lignin precursors. The phenolic signals are transduced through a receptor VirA protein in the inner membrane of the bacterial cell. The expression of these genes triggers the transfer of a specific DNA segment, called transferred DNA (T-DNA), from the Ti plasmid to plant cells, and its integration into their nuclear DNA. We show here that a group of aldoses (L-arabinose, D-xylose, D-lyxose, D-glucose, D-mannose, D-idose, D-galactose, and D-talose) can markedly enhance acetosyringone-dependent expression of vir genes when the concentration of acetosyringone is limited (10 microM) but does not enhance the expression of noninducible genes. Likewise, a 2-deoxy-D-glucose, a nonmetabolized sugar, is also effective. When a deletion was introduced into the virA gene in the region encoding the periplasmic portion of the VirA protein, enhancement by glucose disappeared, but vir expression was induced by acetosyringone in this mutant. These results suggest that these sugars directly enhance a signaling process initiated by phenolic inducers that results in an increase in expression of the vir genes.

Journal Article↗

Isolectins I-A and I-B of Griffonia (Bandeiraea) simplicifolia. Crystal structure of metal-free GS I-B(4) and molecular basis for metal binding and monosaccharide specificity.

Seeds from the African legume shrub Griffonia simplicifolia contain several lectins. Among them the tetrameric lectin GS I-B(4) has strict specificity for terminal alpha Gal residues, whereas the closely related lectin GS I-A(4) can also bind to alpha GalNAc. These two lectins are commonly used as markers in histology or for research in xenotransplantation. To elucidate the basis for the fine difference in specificity, the amino acid sequences of both lectins have been determined and show 89% identity. The crystal structure of GS I-B(4), determined at 2.5-A resolution, reveals a new quaternary structure that has never been observed in other legume lectins. An unexpected loss of both Ca(2+) and Mn(2+) ions, which are necessary for carbohydrate binding in legume lectins, may be related to a particular amino acid sequence Pro-Glu-Pro in the metal binding loop. Comparison with demetallized concanavalin A reveals a different process for the loss of metal ions and for the subsequent loss of carbohydrate binding activity. The GS I-A x alpha GalNAc and GS I-B x alpha Gal complexes were constructed using homology modeling and docking approaches. The unusual presence of an aromatic amino acid at position 47 (Tyr in I-A and Trp in I-B) explains the strong preference for alpha-anomeric sugars in both isolectins. Alteration at one amino acid position, Ala(106) in I-A versus Glu(106) in I-B, is the basis for the observed specificities toward alpha GalNAc and alpha Gal.

Amino Acid Sequence↗

Gastrointestinal morphology and absorption of monosaccharides in fowls conditioned to different types and levels of dietary fibre.

To test a possible influence of dietary fibre on intestinal sugar uptake, rates of absorption of 10 mM-D-[U-14C] glucose and 10 mM-D-[U-14C] xylose were measured in either jejunum or (distal) caecum, by in vivo lumen perfusion, in immature female fowls preconditioned to a standard diet containing (g/kg) either 0, 100, 200 or 400 added dried grass, 200 powdered cellulose, or 200 grass with a polysaccharidase enzyme supplement. When birds were killed after perfusion, dimensions of (unperfused) parts of their alimentary tracts were determined, and recoveries of 14C radioactivity in some body tissues were compared with measured activities absorbed. On average, absorption of glucose was 1.9 and 1.2 times faster than xylose in jejunum and caecum respectively, although these differences varied with dietary treatment and order of perfusion. Increasing grass in the diet caused significant changes in xylose absorption rate in both jejunum and caecum, but only when it was perfused before glucose. With any one sugar and intestinal segment, mean rates of absorption were correlated positively with corresponding mean rates of fluid loss from perfusate. Although their influence on sugar absorption was not well defined, the dietary fibre treatments had more pronounced effects on gross dimensions of parts of the alimentary tract and, hence, potentially on total rates of absorption. Compared with the basal diet, addition of 100, 200 or 400 g grass/kg or 200 g cellulose/kg caused significant increases in small intestine length while 200 g grass/kg with supplementary enzyme did not, and combined caecal length increased with the 400 g grass/kg and the supplementary enzyme treatments. Absorbed 14C activity was recovered in plasma after jejunal perfusions but not caecal ones, whereas it was recovered in liver and in the flushed perfused segment after both types of perfusion. Since there was overlap in absorption rates between jejunum and caecum, this result suggests that the liver may be able to distinguish and treat differently compounds absorbed in the two regions.

Animals↗

Active transport into the human erythrocyte; evidence from comparative kinetics and competition among monosaccharides.

1. Applicability of the previously postulated active transport system for conveying glucose into the human red cell was tested in connection with a number of related substances, comprising 6-carbon aldoses and ketoses, 5-carbon aldoses, and 5- or 6-carbon polyhydric alcohols. 2. The alcohols did not perceptibly penetrate the cells; all the sugars penetrated, the rates differing, but all of the same order of magnitude. 3. All the aldoses penetrated according to the pattern previously reported for glucose, in that the rate of penetration was not directly related to the gradient, but subject to some limiting factor. 4. The ketoses penetrated approximately according to the pattern of passive diffusion. 5. When present in equal concentrations, any aldose prevented or greatly delayed the entrance of a ketose, while the ketose did not perceptibly alter the rate of aldose uptake. Within each class, similar inhibitory relations were observed. 6. Penetration of all the sugars showed a high temperature coefficient and was inhibited by the mercuric ion or p-chloromercuribenzoate; certain of the sugars showed a residual degree of penetration not thus inhibitable. 7. Penetration of the ketoses was selectively inhibited by barbital. 8. These observations are interpreted in terms of simple equilibria between the various sugars and a hypothetical carrier molecule in the membrane, with which the sugars form a complex during their passage through the membrane. Comparisons between the sugars in relation to their reactions with the carrier system are indicated.

Biological Transport, Active↗

Glycosphingolipids in insects. Chemical structures of ceramide monosaccharide, disaccharide, and trisaccharide from pupae of Calliphora vicina (Insecta: Diptera).

The presence of glycosphingolipids in the pupae of the blowfly, Calliphora vicina, was established. The thin layer chromatographic pattern of the total neutral glycolipids revealed the presence of more than 13 components, the major one being ceramide monohexoside. By the use of high performance liquid chromatography, the three simplest components were isolated and their chemical structures determined: Glc(beta 1-1)Cer, Man(beta 1-4)-Glc(beta 1-1)Cer [with minor component Gal(beta 1-4)Glc(beta 1-1)Cer] and GlcNAc(beta 1-3)Man(beta 1-4)Glc(beta 1-1)-Cer. The ceramide composition of the parent insect glycosphingolipids is dominated by the 20:0 fatty acid, arachidic acid, and the sphingoid tetradecasphing-4-enine.

Animals↗

Generalized N-acetylneuraminic acid storage disease: quantitation and identification of the monosaccharide accumulating in brain and other tissues.

Brain and other tissues from a patient with extensive neonatal ascites and clinical symptoms suggestive of a severe neurovisceral storage disorder were examined following autopsy for the accumulation of oligosaccharides. This carbohydrate analysis revealed the presence of large amounts (3--21) mumol/g fresh weight) of sialic acid in brain, liver, and kidney tissue as the major abnormality. Exhaustive characterization of the accumulating material by gel filtration, gas-liquid chromatography, thin-layer chromatography, and GLC-mass spectrometry positively identified the saccharide as free N-acetylneuraminic acid. Based on the accumulation of only free N-acetylneuraminic acid in the tissue of this patient, and normal activities of lysosomal enzymes involved in the catabolism of cellular glycoproteins, this storage disorder appears to result from a previously unreported defect in glycoconjugate metabolism.

Brain↗