Thermal-induced unfolding domains in aldolase identified by amide hydrogen exchange and mass spectrometry.

Amide hydrogen exchange has been measured in short segments of intact rabbit muscle aldolase at temperatures of 14-50 degrees C by the protein fragmentation/mass spectrometry method (Zhang Z, Smith DL, 1993, Protein Sci 2:522-531). Deuterium levels in some segments did not change over the temperature range of the measurements, whereas deuterium levels in other segments increased rapidly with temperature. These results demonstrate that the equilibrium constant for local unfolding, Kunf, of some segments increases with temperature in the low temperature range (14-30 degrees C) of this study. Aldolase begins to lose activity at temperatures above 40 degrees C. In the 40-50 degrees C temperature range, Kunf is greater than 10(-4) in some regions and less than 10(-6) in other regions. This wide range of regional stability in the temperature range where aldolase begins to denature is interpreted in terms of cooperative unfolding/folding domains. Regions of highest stability were located along the hydrophobic subunit binding surface. It is proposed that hydrogen exchange might be used to identify unfolding domains in multidomain proteins whose thermodynamic properties have been determined by differential scanning calorimetry.     

The effect of an interleukin receptor antagonist (IL-1ra) on colonocyte eicosanoid release

We investigated whether an interleukin 1 receptor antagonist (IL-1ra) altered cellular release of prostanoids and leukotrienes in a transformed colonic cell line (CACO-2) in the presence of proinflammatory stimuli. Cellular inflammation was induced by treatment with lipopolysaccharide (LPS) or the cytokine, interleukin 1 beta (IL-1(beta)). In a separate set of experiments, cells were pretreated with IL-1ra prior to exposure to LPS or IL-1(beta). Prostaglandin E(2) and leukotriene B(4) (LTB(4)) levels were quantified by ELISA assays. Both LPS and IL-1(beta) exposure were noted to stimulate cellular PGE(2) release, a response which was significantly inhibited by IL-1ra treatment. Either stimulant when administered alone failed to stimulate release of LTB(4). When administered after IL-1ra pretreatment however, both stimuli caused a significant increase in LTB(4) release. These results suggest that a cytokine receptor antagonist can selectively influence eicosanoid production in this cell line. Furthermore, this study suggests that a IL-1ra may have a future clinical role in the treatment of inflammatory disorders of the colon which are intimately linked to enhanced eicosanoid synthesis.     

Life cycle assessment standards

The newly emerging LCA standards provide an opportunity to review and improve upon the current LCA methodology. As more industrial practitioners enter the arena, the opportunity arises to not only demand environmental improvement from industrial service and product providers but also to fill LCA data gaps. A framework is suggested for improvement in the current LCA framework that focuses on the business relationships of the industrial practitioner. The framework seeks to promote environmental improvement from industrial sectors through the identification of state-of-the-art technologies used throughout a life cycle. Basing LCAs on the best performers in an industry will create a market for a high level of environmental performance, disperse the responsibility of inventory data gathering, and improve upon the advancements already anticipated through the widespread application of LCA.     

A gene product related to Tral is required for the mobilization of Bacteroides mobilizable transposons and plasmids

The antibiotic-resistance transposon Tn4555 from Bacteroides can be transferred between strains by conjugation. The transposon is not self-transmissible and must be mobilized by resident chromosomal tetracy-cline-resistance elements. In the present report, the mechanism of transfer was examined at the genetic level by deletion analysis and nucleotide sequencing of clones that conferred a transmissible phenotype on a non-mobilizable plasmid. The results suggested that the product of mobA_Tn was required for mobilization and it worked in concert with a cis-acting oriT-like sequence. This mechanism was compared with the mobilization system of a cryptic Bacteroides plasmid, pBl143, and the two systems were found to share a common transfer strategy. The mobA gene products from both genetic elements were related and they had limited homology to the broad group of mobilization proteins (relaxases) typified by Tral of RP4. Phylogenetic analysis of MobA and several other mobilization proteins from commensal gastrointestinal tract organisms suggested that they formed a new subgroup of the Tral superfamily. The mobilization regions of both Tn4555 and pBl143 were located on discrete segments of DNA within the parent genetic element. These segments were delineated by regions of secondary structure, suggesting that they could be defined mobilization cassettes.     

The elongation of amylose and amylopectin chains in isolated starch granules

The aim of this work was to investigate the conditions required for amylose synthesis in starch granules. Although the major granule-bound isoform of starch synthase - GBSSI - catalyses the synthesis of amylose in vivo, ¹⁴C from ADP[¹⁴C]glucose was incorporated primarily into a specific subset of amylopectin chains when supplied to starch granules isolated from pea (Pisum sativum L.) embryos and potato (Solanum tuberosum L.) tubers. Incubation of granules with soluble extracts of these organs revealed that the extracts contained compounds that increased the incorporation of ¹⁴C into amylose. These compounds were rendered inactive by treatment of the extracts with α-glucosidase, suggesting that they were malto-oligosaccharides. Consistent with this idea, provision of pure malto-oligosaccharides to isolated granules resulted in a dramatic shift in the pattern of incorporation of ¹⁴C, from amylopectin chains to amylose molecules. Comparison of the pattern of incorporation in granules from wild-type peas and lam mutant peas which lack GBSSI showed that this effect of malto-oligosaccharides was specifically on GBSSI. The significance of these results for understanding of the synthesis of amylose and amylopectin in storage organs is discussed.     

大鼠胼胝体内神经肽Y免疫反应阳性纤维的发育

本实验用免疫组织化学ＡＢＣ法研究了大鼠胼胝体内神经肽Ｙ免疫反应阳性（ＮＰＹ－ＩＲ）纤维的生后发育。结果发现,许多ＮＰＹ－ＩＲ纤维在大鼠出生时便存在于胼胝体内。ＮＰＹ－ＩＲ胼胝体纤维的密度在生后１周内继续逐渐增高,在第２周内达到最高峰。之后,ＮＰＹ－ＩＲ胼胝体纤维的密度逐渐下降,至第３周末时接近成年时的水平,即仅有少量ＮＰＹ－ＩＲ纤维存在于胼胝体内。这些结果提示在大鼠早期生后发育过程中许多ＮＰＹ－ＩＲ胼胝体纤维是暂时性的,其作用可能与大脑皮质的机能发育有关。     

Enzymes of Choline Synthesis in Spinach (Response of Phospho-Base N-Methyltransferase Activities to Light and Salinity)

In spinach (Spinacia oleracea L.), choline is synthesized by the sequential N-methylation of phosphoethanolamine -> phosphomono- -> phosphodi- -> phosphotrimethylethanolamine (i.e. phosphocholine) followed by hydrolysis to release choline. Differential centrifugation of spinach leaf extracts shows that enzymes catalyzing the three N-methylations are cytosolic. These enzymes were assayed in leaf extracts prepared from plants growing under various light/dark periods. Under a diurnal, 8-h light/16-h dark photoperiod, the activity of the enzyme catalyzing the N-methylation of phosphoethanolamine is highest at the end of the light period and lowest following the dark period. Prolonged dark periods (exceeding 16 h) lead to a further reduction in the activity of this enzyme, although activity is restored when plants are reexposed to light. In contrast, the activity of the enzyme(s) catalyzing the N-methylations of phosphomono- and phosphodimethylethanolamine does not undergo comparable changes in response to light/dark treatments. Salt shock of plants with 200 mM NaCl results in a 2-fold increase in all three N-methylation activities relative to nonsalinized controls but only in plants exposed to light. Thus, light is required for the salt-responsive up-regulation of choline synthesis in spinach.