Investigation of the role of the disulphide bond in the activity and structure of staphylococcal enterotoxin C1

The goal of this study was to Investigate the role of the disulphide bond of staphylococcal enterotoxin C1 (SEC1) in the structure and activity of the toxin. Mutants unable to form a disulphide bond were generated by substituting alanine or serine for cysteine at positions 93 and/or 110. Although we did not directly investigate the residues between the disulphide linkage, tryptic lability showed that significant native structure in the cystine loop is preserved in the absence of covalent bonding between residues 93 and 110. Since no correlation was observed between the behaviour of these mutants with regard to toxin stability, emesis and T cell proliferation, we conclude that SEC1 -induced emesis and T cell proliferation are dependent on separate regions of the molecule. The disulphide bond itself is not an absolute requirement for either activity. However, conformation within or adjacent to the loop is important for emesis. Although mutants with alanine substitutions were not emetic, those with serine substitutions retained this activity, suggesting that the disulphide linkage stabilizes a crucial conformation but can be replaced by residues which hydrogen bond.     

TheEscherichia coli mannitol permease as a model for transport via the bacterial phosphotransferase system

The bacterial phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) consists of several proteins whose primary functions are to transport and phosphorylate their substrates. The complexity of the PTS undoubtedly reflects its additional roles in chemotaxis to PTS substrates and in regulation of other metabolic processes in the cell. The PTS permeases (Enzymes II) are the membrane-associated proteins of the PTS that sequentially recognize, transport, and phosphorylate their specific substrates in separate steps, and theEscherichia coli mannitol permease is one of the best studied of these proteins. It consists of two cytoplasmic domains (EIIA and EIIB) involved in mannitol phosphorylation and an integral membrane domain (EIIC) which is sufficient to bind mannitol, but which transports mannitol at a rate that is dependent on phosphorylation of the EIIA and EIIB domains. Recent results show that several residues in a hydrophilic, 85-residue segment of the EIIC domain are important for the binding, transport, and phosphorylation of mannitol. This segment may be at least partially exposed to the cytoplasm of the cell. A model is proposed in which this region of the EIIC domain is crucial in coupling phosphorylation of the EIIB domain to transport through the EIIC domain of the mannitol permease.     

Transport and transformation of hexavalent chromium through soils and into ground water

A detailed characterization of the underlying and adjacent soils of a chrome‐plating shop was performed to provide information on the extent of soil and aquifer contamination at the site and on the potential for off‐site migration and environmental impact. Intact, moist cores were obtained from more than 40 different locations, resulting in more than 200 discrete samples for total metal analysis, selective extraction tests, and adsorption‐reduction experiments, to assess the chemical speciation and distribution of chromium on the contaminated soils and its leaching potential. Surface analytical techniques were also used to determine chemical speciation and to further elucidate mineral fractions responsible for retention of the chromium on the soils and sediments. Adsorption and reduction capacities of the saturated aquifer sediments were variable and low, while the unsaturated soils’ reduction capacities were much greater and were correlated with depth (decreasing capacity with increasing depth). The soils’ adsorption and reduction capacities were eventually overwhelmed, however, and permitted the passage of Cr(VI) into the underlying ground water. Adsorption capacity differences were primarily related to clay content and pH, and less so to the presence of amorphous iron oxide coatings on matrix minerals as operationally defined by the selective extraction methods used in the study. Reduction of Cr(VI) to Cr(III) and subsequent precipitation as (Fe, Cr)(OH)₃ is proposed as the primary attenuation mechanism in the unsaturated soils immediately beneath the shop, based on extraction and surface analyses results.     

Cloning and characterization of the gene encoding an esterase from Spirulina platensis

The gene encoding a 23 kDA serine esterase from the cyanobacterium Spirulina platensis has been identified, cloned, characterized and expressed in Escherichia coli. The primary structure of the esterase deduced from the DNA sequence displayed 32% sequence identity with the carboxylesterase (esterase II) encoded by estB of Pseudomonas fluorescens; the highest degree of homology is found in a stretch of 11 identical or highly conserved amino acid residues corresponding to the GXSXG consensus motif found in the catalytic site of many serine proteases, lipases and esterases.     

Evolution of regulatory genes and patterns: Relationships to evolutionary rates and to metabolic functions

In an effort to understand the forces shaping evolution of regulatory genes and patterns, we have compared data on interspecific differences in enzyme expression patterns among the rapidly evolving Hawaiian picture-winged Drosophila to similar data on the more conservative virilis species group. Divergence of regulatory patterns is significantly more common in the former group, but cause and effect are difficult to discern. Random fixation of regulatory variants in small populations and/or during speciation may be somewhat more likely than divergence driven by selection. Within the picture-winged group, we also have compared enzymes that fulfill different metabolic roles. There are highly significant differences between individual enzymes, but no obvious correlations to functional categories. Correspondence to: W.J. Dickinson     

ANTIVIRAL ACTIVITY OF CULTURED BLUE-GREEN ALGAE (CYANOPHYTA)1

Lipophilic and hydrophilic extracts from approximately 600 strains of cultured cyanophytes, representing some 300 species, were examined for antiviral activity against three pathogenic viruses. Approximately 10% of the cultures produced substances that caused significant reduction in cytopathic effect normally associated with viral infection. The screening program identified the order Chroococcales as commonly producing antiviral agents.     

Widespread formation of intracellular calcium carbonates by the bloom-forming cyanobacterium Microcystis

The formation of intracellular amorphous calcium carbonates (iACC) has been recently observed in a few cultured strains of Microcystis, a potentially toxic bloom-forming cyanobacterium found worldwide in freshwater ecosystems. If iACC-forming Microcystis are abundant within blooms, they may represent a significant amount of particulate Ca. Here, we investigate the significance of iACC biomineralization by Microcystis. First, the presence of iACC-forming Microcystis cells has been detected in several eutrophic lakes, indicating that this phenomenon occurs under environmental conditions. Second, some genotypic (presence/absence of ccyA, a marker gene of iACC biomineralization) and phenotypic (presence/absence of iACC) diversity have been detected within a collection of strains isolated from one single lake. This illustrates that this trait is frequent but also variable within Microcystis even at a single locality. Finally, one-third of publicly available genomes of Microcystis were shown to contain the ccyA gene, revealing a wide geographic and phylogenetic distribution within the genus. Overall, the present work shows that the formation of iACC by Microcystis is common under environmental conditions. While its biological function remains undetermined, this process should be further considered regarding the biology of Microcystis and implications on the Ca geochemical cycle in freshwater environments.     

A differential proteomics study of cerebrospinal fluid from individuals with Niemann-Pick disease,Type C1

Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity.     

COMPARATIVE ONTOGENY OF A WILD CUCURBIT AND ITS DERIVED CULTIVAR

Most previous studies of evolutionary modification of form in plants have focused primarily on individual organs or flowers. Few have investigated the role of evolutionary changes in timing or position at the level of whole plant ontogeny. This study compares ontogenies of the primary shoots of two subspecies of Cucurbita argyrosperma, one a cultivar and the other its wild progenitor. Differences in flowering times between these subspecies suggested that the cultivar may have evolved from the wild subspecies via heterochronic processes leading to paedomorphosis. Analyses showed that both subspecies are similar in vegetative architecture and rates of leaf production. Earlier flowering in the cultivar, both in terms of position and absolute time, appears to have arisen through progenesis. Initial observations of leaf blade morphology led to the hypothesis that paedomorphosis and gigantism also may have been involved in the evolution of leaf blade shape in the cultivar: all leaves of the cultivar are larger and visually similar in shape to early leaves of the wild subspecies. However, quantitative analysis revealed that leaves of the cultivar are neither geometrically, nor solely allometrically larger versions of early leaves of the progenitor. Leaf shape in the cultivar exhibits novel features as well as effects of allometry shared with the progenitor, hence a simple hypothesis of paedomorphic evolution of leaf shape is not supported.