Trigonal crystals of porin from Escherichia coli

Trigonal crystals of the integral membrane protein porin from Escherichia coli have been grown and characterized. They belong to space group P321 with unit cell constants a = b = LL8.4, c = 52.7 A, alpha = beta = 90 degrees, gamma = 120 degrees. The crystals grow as well-defined hexagonal prisms to a size of 0.25 mm in all dimensions, and diffract to 2.7 A. The molecular symmetry coincides with 3-fold crystallographic symmetry, giving two trimers per unit cell (1 monomer/asymmetric unit). This corresponds to VM = 2.9 A3/Da. Native X-ray data to 3.0 A resolution have been collected on a FAST area detector and a search for heavy atom derivatives is underway.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Structure of a Bimodular Botulinum Neurotoxin Complex Provides Insights into Its Oral Toxicity

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and cause the fatal disease botulism, a flaccid paralysis of the muscle. BoNTs are released together with several auxiliary proteins as progenitor toxin complexes (PTCs) to become highly potent oral poisons. Here, we report the structure of a ∼760 kDa 14-subunit large PTC of serotype A (L-PTC/A) and reveal insight into its absorption mechanism. Using a combination of X-ray crystallography, electron microscopy, and functional studies, we found that L-PTC/A consists of two structurally and functionally independent sub-complexes. A hetero-dimeric 290 kDa complex protects BoNT, while a hetero-dodecameric 470 kDa complex facilitates its absorption in the harsh environment of the gastrointestinal tract. BoNT absorption is mediated by nine glycan-binding sites on the dodecameric sub-complex that forms multivalent interactions with carbohydrate receptors on intestinal epithelial cells. We identified monosaccharides that blocked oral BoNT intoxication in mice, which suggests a new strategy for the development of preventive countermeasures for BoNTs based on carbohydrate receptor mimicry.     

Effect of Metalaxyl on the incidence and severity of Pearl millet downy mildew disease in Northern Nigeria

Pearl millet downy mildew (DM) incidence, severity and yield losses of two pearl millet varieties (local and improved) due to the disease were determined in the field. Significant differences in the disease incidence and severity were recorded in the plots sown with metalaxyl-treated seeds and those sown with non-treated seeds, indicating the efficacy of the fungicide on the fungus. Yield losses due to non-treatment of seeds with metalaxyl was 40.88 and 45.39% in a local variety and 43.00 and 18.60% in an improved variety in the 2000 and 2001 cropping seasons respectively. Significant differences between plots sown with metalaxyl-treated and those sown with non-treated seeds were obtained for other yield components such as 1000-grains weight, panicle length and weight.     

Effect of temperature on the saccharide composition obtained after alpha-amylolysis of starch

The hydrolysis of starch to low-molecular-weight products (normally characterised by their dextrose equivalent (DE), which is directly related to the number-average molecular mass) was studied at different temperatures. Amylopectin potato starch, lacking amylose, was selected because of its low tendency towards retrogradation at lower temperatures. Bacillus licheniformis alpha-amylase was added to 10% [w/w] gelatinised starch solutions. The hydrolysis experiments were done at 50, 70, and 90 degrees C. Samples were taken at defined DE values and these were analysed with respect to their saccharide composition. At the same DE the oligosaccharide composition depended on the hydrolysis temperature. This implies that at the same net number of bonds hydrolysed by the enzyme, the saccharide composition was different. The hydrolysis temperature also influenced the initial overall molecular-weight distribution. Higher temperatures led to a more homogenous molecular weight distribution. Similar effects were observed for alpha-amylases from other microbial sources such as Bacillus amyloliquefaciens and Bacillus stearothermophilus. Varying the pH (5.1, 6.2, and 7.6) at 70 degrees C did not significantly influence the saccharide composition obtained during B. licheniformis alpha-amylase hydrolysis. The underlying mechanisms for B. licheniformis alpha-amylase were studied using pure linear oligosaccharides, ranging from maltotriose to maltoheptaose as substrates. Activation energies for the hydrolysis of individual oligosaccharides were calculated from Arrhenius plots at 60, 70, 80, and 90 degrees C. Oligosaccharides with a degree of polymerisation exceeding that of the substrate could be detected. The contribution of these oligosaccharides increased as the degree of polymerisation of the substrate decreased and the temperature of hydrolysis increased. The product specificity decreased with increasing temperature of hydrolysis, which led to a more equal distribution between the possible products formed. Calculations with the subsite map as determined for the closely related alpha-amylase from B. amyloliquefaciens reconfirmed this finding of a decreased substrate specificity with increased temperature of hydrolysis. Copyright 1999 John Wiley & Sons, Inc.     

The HCC-domain of botulinum neurotoxins A and B exhibits a singular ganglioside binding site displaying serotype specific carbohydrate interaction

Tetanus and botulinum neurotoxins selectively invade neurons following binding to complex gangliosides. Recent biochemical experiments demonstrate that two ganglioside binding sites within the tetanus neurotoxin HC-fragment, originally identified in crystallographic studies to bind lactose or sialic acid, are required for productive binding to target cells. Here, we determine by mass spectroscopy studies that the HC-fragment of botulinum neurotoxins A and B bind only one molecule of ganglioside GT1b. Mutations made in the presumed ganglioside binding site of botulinum neurotoxin A and B abolished the formation of these HC-fragment/ganglioside complexes, and drastically diminished binding to neuronal membranes and isolated GT1b. Furthermore, correspondingly mutated full-length neurotoxins exhibit significantly reduced neurotoxicity, thus identifying a single ganglioside binding site within the carboxyl-terminal half of the HC-fragment of botulinum neurotoxins A and B. These binding cavities are defined by the conserved peptide motif H...SXWY...G. The roles of tyrosine and histidine in botulinum neurotoxins A and B in ganglioside binding differ from those in the analogous tetanus neurotoxin lactose site. Hence, these findings provide valuable information for the rational design of potent botulinum neurotoxin binding inhibitors.