The structure of Cryptococcus neoformans galactoxylomannan contains β-d-glucuronic acid

The structure of galactoxylomannan, a capsular polysaccharide from the opportunistic yeast Cryptococcus neoformans, was re-examined by NMR spectroscopy and GC-MS. The residue that is 3-linked to the side chain galactose and was previously assigned as β-d-xylose [Vaishnav, V. V.; Bacon, B. E.; O’Neill, M.; Cherniak, R. Carbohydr. Res.1998, 306, 315-330] was determined to be β-d-glucuronic acid. A revised structure for this polymer is presented, along with a proposal that this compound be termed glucuronoxylomannogalactan (GXMGal).     

Genomic organization and mapping of the human and mouse neuronal β2-nicotinic acetylcholine receptor genes

As a first step in determining whether there are polymorphisms in the nicotinic acetylcholine receptor (nAChR) genes that are associated with nicotine addiction, we isolated genomic clones of the β2-nAChR genes from human and mouse BAC libraries. Although cDNA sequences were available for the human gene, only the promoter sequence had been reported for the mouse gene. We determined the genomic structures by sequencing 12 kb of the human gene and over 7 kb of the mouse gene. While the sizes of exons in the mouse and human genes are the same, the introns differ in size. Both promoters have a high GC content (60–80%) proximal to the AUG and share a neural-restrictive silencer element (NRSE), but overall sequence identity is only 72%. Using a 6-Mb YAC contig of Chr 1, we mapped the human β2-nAChR gene, CHRNB2, to 1q21.3 with the order of markers cen, FLG, IVL, LOR, CHRNB2, tel. The mouse gene, Acrb2, had previously been mapped to Chr 3 in a region orthologous to human Chr 1. We refined mapping of the mouse gene and other markers on a radiation hybrid panel of Chr 3 and found the order cen, Acrb2, Lor, Iv1, Flg, tel. Our results indicate that this cluster of markers on human Chr 1 is inverted with respect to its orientation on the chromosome compared with markers in the orthologous region of mouse Chr 3. Received: 26 January 1999 / Accepted: 10 May 1999     

Crystal structure of α/β-galactoside α2,3-sialyltransferase from a luminous marine bacterium, Photobacterium phosphoreum

α/β-Galactoside α2,3-sialyltransferase produced by Photobacterium phosphoreum JT-ISH-467 is a unique enzyme that catalyzes the transfer of N-acetylneuraminic acid residue from cytidine monophosphate N-acetylneuraminic acid to acceptor carbohydrate groups. The enzyme recognizes both mono- and di-saccharides as acceptor substrates, and can transfer Neu5Ac to both α-galactoside and β-galactoside, efficiently. To elucidate the structural basis for the broad acceptor substrate specificity, we determined the crystal structure of the α2,3-sialyltransferase in complex with CMP. The overall structure belongs to the glycosyltransferase-B structural group. We could model a reasonable active conformation structure based on the crystal structure. The predicted structure suggested that the broad substrate specificity could be attributed to the wider entrance of the acceptor substrate binding site.     

Genomic organization of sheep TRDJ segments and their expression in the δ-chain repertoire in thymus

cDNA sequences obtained from polymerase chain reaction products of reverse-transcribed RNA from sheep thymus showed the presence of a large number of members of the TRDV1 gene family. Some are TRDV1 genes also found in peripheral blood lymphocytes, while four genes had not been described so far. The cDNA sequences also showed extensive junctional diversity and a preferential usage of the three TRDJ elements. We characterized the genomic organization of the sheep TRDJ locus and detected a correlation between the nonrandom usage of TRDJ elements during development and their chromosomal order.     

Extracellular expression and biochemical characterization of α-cyclodextrin glycosyltransferase from Paenibacillus macerans

The cgt gene encoding α-cyclodextrin glycosyltransferase (α-CGTase) from Paenibacillus macerans strain JFB05-01 was expressed in Escherichia coli as a C-terminal His-tagged protein. After 90 h of induction, the activity of α-CGTase in the culture medium reached 22.5 U/mL, which was approximately 42-fold higher than that from the parent strain. The recombinant α-CGTase was purified to homogeneity through either nickel affinity chromatography or a combination of ion-exchange and hydrophobic interaction chromatography. Then, the purified enzyme was characterized in detail with respect to its cyclization activity. It is a monomer in solution. Its optimum reaction temperature is 45 °C, and half-lives are approximately 8 h at 40 °C, 1.25 h at 45 °C and 0.5 h at 50 °C. The recombinant α-CGTase has an optimum pH of 5.5 with broad pH stability between pH 6 and 9.5. It is activated by Ca²⁺, Ba²⁺, and Zn²⁺ in a concentration-dependent manner, while it is dramatically inhibited by Hg²⁺. The kinetics of the α-CGTase-catalyzed cyclization reaction could be fairly well described by the Hill equation.     

Binding and Cleavage of E. coli HUβ by the E. coli Lon Protease

The Escherichia coli Lon protease degrades the E. coli DNA-binding protein HUβ, but not the related protein HUα. Here we show that the Lon protease binds to both HUβ and HUα, but selectively degrades only HUβ in the presence of ATP. Mass spectrometry of HUβ peptide fragments revealed that region K18-G22 is the preferred cleavage site, followed in preference by L36-K37. The preferred cleavage site was further refined to A20-A21 by constructing and testing mutant proteins; Lon degraded HUβ-A20Q and HUβ-A20D more slowly than HUβ. We used optical tweezers to measure the rupture force between HU proteins and Lon; HUα, HUβ, and HUβ-A20D can bind to Lon, and in the presence of ATP, the rupture force between each of these proteins and Lon became weaker. Our results support a mechanism of Lon protease cleavage of HU proteins in at least three stages: binding of Lon with the HU protein (HUβ, HUα, or HUβ-A20D); hydrolysis of ATP by Lon to provide energy to loosen the binding to the HU protein and to allow an induced-fit conformational change; and specific cleavage of only HUβ.     

Seed rain and seed bank of Chinese yew (Taxus chinensis var. mairei) population in Tianmu Mountain☆

Seed rain and seed bank of a Chinese yew (Taxus chinensis var. mairei) population in Tianmu Mountain were researched in 2008 and 2009. The seed rain lasted from 16th–23th of October to 5th–14th of December, and the heaviest seed falling period was from 2nd to18th of November. The intensity of seed rain showed a great inter-annual variation, with a good harvest in 2008. The fallen seeds were composed of 49.9% proportion of immature seed, 33.8% proportion of chewed seed and 16.3% proportion of mature seed. The analysis on the soil seed bank under mother forest showed that the number of intact seeds was 122.75 ± 108.08 grain/m² in October, 279.25 ± 210.73 grain/m² in December 2008, and 166.5 ± 165.34 grain/m² in October, 322.5 ± 275.73 grain/m² in December 2009. The increased number of seed was 156.5 ± 222.723 grain/m² in 2008 and 156 ± 275grain/m² in 2009, which showed a significant variation. Large number of intact seeds added into soil seed bank after seed rain each year. The number of intact seeds in soil seed bank decreased 112.75 ± 47.74 grain/m² from December 2008 to October 2009. Large number of intact seeds lost from seed rot and seed predation by animals. The number of seeds in soil bank under bamboo forest was much lower than that of mother tree forest, and the increased number of seeds was 0.63 ± 1.60 grain/m² in 2008 and 2.88 ± 1.86 grain/m² in 2009. The number of seedling was 0.73 ± 1.10 trees/m² in mother tree forest and 0.09 ± 0.35 trees/m² in bamboo forest. Seedling survival ratio was 0.37% in mother tree forest and 10.23% in bamboo forest. The micro-habitat in bamboo forest was fit for seed germination. Birds transported seeds to bamboo forest, and had an important effect on the regeneration of Chinese yew.     

Xanthonolignoids from Kielmeyera and Caraipa species—13C NMR spectroscopy of xanthones

Kielcorin and the cadensins A and B, isolated respectively from Kielmeyera coriacea and Caraipa densiflora (Guttiferae), were shown to be xanthonolignoids. The structure of (5S,6S)-6(or 5)-hydroxymethyl-5(or 6)-(4″-hydroxy-3″-methoxyphenyl)-2,3:3′,4′-(2′-methoxyxanthono)-1,4-dioxane was proposed for kielcorin by analysis of high resolution MS and PMR spectra. The carbon shifts of xanthone were assigned and used in the ¹³C NMR spectral confirmation of the proposed structure.     

Monochoria vaginalis var. angustifolia，a new variety of the Pontederiaceae from Thailand

A new variety of Monochoria C. Presl from Thailand, M. vaginalis （N. L. Burman）Kunth var.angustifolia G. X. Wang, is described. This variety can be distinguished from the typical one, M. vaginalis var. vaginalis, by having mature leaves narrowly lanceolat     

A 3D Structure Model of Integrin α4β1 Complex: I. Construction of a Homology Model of β1 and Ligand Binding Analysis

It is well established that integrin α4β1 binds to the vascular cell adhesion molecule (VCAM) and fibronectin and plays an important role in signal transduction. Blocking the binding of VCAM to α4β1 is thought to be a way of controlling a number of disease processes. To better understand how various inhibitors might block the interaction of VCAM and fibronectin with α4β1, we began constructing a structure model for the integrin α4β1 complex. As the first step, we have built a homology model of the β1 subunit based on the I domain of the integrin CD11B subunit. The model, including a bound Mg²⁺ ion, was optimized through a specially designed relaxation scheme involving restrained minimization and dynamics steps. The native ligand VCAM and two highly active small molecules (TBC772 and TBC3486) shown to inhibit binding of CS-1 and VCAM to α4β1 were docked into the active site of the refined model. Results from the binding analysis fit well with a pharmacophore model that was independently derived from active analog studies. A critical examination of residues in the binding site and analysis of docked ligands that are both potent and selective led to the proposal of a mechanism for β1/β7 ligand binding selectivity.