Evaluation of health risks from a buried mass of diesel fuel before and after bioremediation

Plans are being formulated for in situ bioremediation of a subsurface plume of diesel fuel No. 2 that resulted from an accidental fuel release. Raoult's law and the aqueous solubilities of the toxic components were used to estimate organic contaminant concentrations in leachate from the untreated fuel mass. Carcinogenic risks and noncarcinogenic hazard indices were calculated for undiluted leachate. An 80% decrease in hydrocarbon mass and increases in the average molecular weights of the component fractions were assumed to result from the treatment. Sample calculations are provided to show how to evaluate results of analyses for petroleum hydrocarbons after bioremediation.     

Factor VIII: structure, function and analysis

Factor VIII is an important blood coagulation protein whose genetic deficiency leads to the serious bleeding disorder, classic haemophilia (haemophilia A).Here we review the structure, function and analysis of this protein for diagnostic and therapeutic applications. Because factor VIII is tightly associated with von Willebrand factor some recent work on the latter is also considered so as to clarify the relationship between them.     

First chemical synthesis of a scorpion alpha-toxin affecting sodium channels: the Aah I toxin of Androctonus australis hector.

Aah I is a 63-residue alpha-toxin isolated from the venom of the Buthidae scorpion Androctonus australis hector, which is considered to be the most dangerous species. We report here the first chemical synthesis of Aah I by the solid-phase method, using a Fmoc strategy. The synthetic toxin I (sAah I) was renatured in DMSO-Tris buffer, purified and subjected to thorough analysis and comparison with the natural toxin. The sAah I showed physico-chemical (CD spectrum, molecular mass, HPLC elution), biochemical (amino-acid composition, sequence), immunochemical and pharmacological properties similar to those of the natural toxin. The synthetic toxin was recognized by a conformation-dependent monoclonal anti-Aah I antibody, with an IC50 value close to that for the natural toxin. Following intracerebroventricular injection, the synthetic and the natural toxins were similarly lethal to mice. In voltage-clamp experiments, Na(v) 1.2 sodium channel inactivation was inhibited by the application of sAah I or of the natural toxin in a similar way. This work describes a simple protocol for the chemical synthesis of a scorpion alpha-toxin, making it possible to produce structural analogues in time.     

Tyr115 is the key residue for determining agonist selectivity in the V1a vasopressin receptor.

Using a three-dimensional model of G protein-coupled receptors (GPCR), we have previously succeeded in docking the neurohypophysial hormone arginine-vasopressin (AVP) into the V1a receptor. According to this model, the hormone is completely embedded in the transmembrane part of the receptor. Only the side chain of the Arg residue at position 8 projects outside the transmembrane core of the receptor and possibly interacts with a Tyr residue located in the first extracellular loop at position 115. Residue 8 varies in the two natural neurohypophysial hormones, AVP and oxytocin (OT); similarly, different residues are present at position 115 in the different members of the AVP/OT receptor family. Here we show that Arg8 is crucial for high affinity binding of AVP to the rat V1a receptor. Moreover, when Tyr115 is replaced by an Asp and a Phe, the amino acids naturally occurring in the V2 and in the OT receptor subtypes, the agonist selectivity of the V1a receptor switches accordingly. Our results indicate that the interaction between peptide residue 8 and the receptor residue at position 115 is not only crucial for agonist high affinity binding but also for receptor selectivity.     

Regulation of O-antigen chain length is required for Shigella flexneri virulence

It is shown that Shigella flexneri maintains genetic control over the modal chain length of the O-antigen polysaccharide chains of its lipopolysaccharide (LPS) molecules because such a distribution is required for virulence. The effect of altering O-antigen chain length on S. flexneri virulence was investigated by inserting a kanamycin (Km)-resistance cassette into the rol gene (controlling the modal O-antigen chain length distribution), and into the rfbD gene, whose product is needed for synthesis of dTDP-rhamnose (the precursor of rhamnose in the O-antigen). The mutations had the expected effect on LPS structure. The rol ::Km mutation was impaired in the ability to elicit keratoconjunctivitis, as determined by the Serény test. The rol ::Km and rfbD ::Km mutations prevented plaque formation on HeLa cells, but neither mutation affected the ability of S. flexneri to invade and replicate in HeLa cells. Microscopy of bacteria-infected HeLa cells stained with fluorescein isothiocyanate (FITC)-phalloidin demonstrated that both the rol ::Km and rfbD ::Km mutants were defective in F-actin tail formation: the latter mutant showed distorted F-actin tails. Plasma-membrane protrusions were occasionally observed. Investigation of the location of IcsA (required for F-actin tail formation) on the cell surface by immunofluorescence and immunogold electron microscopy showed that while most rol mutant bacteria produced little or no cell-surface IcsA, 10% resembled the parental bacterial cell (which had IcsA at one cell pole; the rfbD mutant had IcsA located over its entire cell surface although it was more concentrated at one end of the cell). That the O-antigen chains of the rol ::Km mutant did not mask the IcsA protein was demonstrated by using the endorhamnosidase activity of Sf6c phage to digest the O-antigen chains, and comparing untreated and Sf6c-treated cells by immunofluorescence with anti-IcsA serum.     

Novel Vibrio cholerae O139 genes involved in lipopolysaccharide biosynthesis.

The sequence of part of the rfb region of Vibrio cholerae serogroup O139 and the physical map of a 35-kb region of the O139 chromosome have been determined. The O139 rfb region presented contains a number of open reading frames which show similarities to other rfb and capsular biosynthesis genes found in members of the Enterobacteriaceae family and in V. cholerae O1. The cloned and sequenced region can complement the defects in O139 antigen biosynthesis in transposon insertions within the O139 rfb cluster. Linkage is demonstrated among IS1358 of V. cholerae O139, the rfb region, and the recently reported otnA and otnB genes (E. M. Bik, A. E. Bunschoten, R. D. Gouw, and F. R. Mooi, EMBO J. 14:209-216, 1995). In addition, the whole of this region has been linked to the rfaD gene. Furthermore, determination of the sequence flanking IS1358 has revealed homology to other rfb-like genes. The exact site of insertion with respect to rfaD is defined for the novel DNAs of both the Bengal and the Argentinian O139 isolates.     

Inducible nitric oxide synthase and guinea-pig ileitis induced by adjuvant

We sought to establish a model of inflammatory bowel disease by augmenting the activity of the local immune system with Freund's complete adjuvant, and to determine if inducible nitric oxide synthase (iNOS) expression and peroxynitrite formation accompanied the inflammatory condition. In anaesthetized guinea-pigs, a loop of distal ileum received intraluminal 50% ethanol followed by Freund's complete adjuvant. Control animals were sham operated. When the animals were killed 7 or 14 days later, loop lavage fluid was examined for nitrite and PGE(2) levels; mucosal levels of granulocyte and macrophages were estimated by myeloperoxidase (MPO) and N-acetyl-D-glucosaminidase (NAG) activity, respectively. Cellular localization if iNOS and peroxynitrite formation were determined by immunohistochemistry with polyclonal antibodies directed against peptide epitopes of mouse iNOS and nitrotyrosine, respectfully. Adjuvant administration resulted in a persistent ileitis, featuring gut thickening, crypt hyperplasia, villus tip swelling and disruption, and cellular infiltration. Lavage levels of PGE(2) and nitrite were markedly elevated by adjuvant treatment. Immunoreactive iNOS and nitrotyrosine bordered on detectability in normal animals but were markedly evident with adjuvant treatment at day 7 and particularly day 14. Immunohistochemistry suggested that enteric neurons and epithelia were major sites of iNOS activity and peroxynitrite formation. We conclude that local administration of adjuvant establishes a chronic ileitis. Inducible nitric oxide synthase may contribute to the inflammatory process.     

A common-immunogenic Vibrio outer membrane protein

Abstract The presence of antibodies in rabbit antisera to cell envelope proteins of Vibrio cholerae has been examined using a two-dimensional system, in which the cell envelopes are electrophoresed in sodium dodecyl sulfate (SDS) in polyacrylamide gels in the first dimension, and in agarose containing antibodies in the second. The results show that a 25-kDa protein is markedly immunogenic and appears to be common to Vibrio strains; it is not present in a number of other organisms. This 25-kDa protein is an outer membrane protein as judged by sucrose gradient centrifugation and it is accessible to iodination by lactoperoxidase, suggesting that it is exposed on the cell surface.     

A method for gene enrichment based on the avidin-biotin interaction. Application to the Drosophila ribosomal RNA genes.

A method of enriching, from the total DNA of an organism, for long DNA strands carrying a particular gene is described. The purified RNA corresponding to the gene is covalently attached to biotin via a cytochrome c bridge. This modified RNA is hybridized to the total DNA. Those DNA strands which hybridize are separated from all the other DNA, using the avidin-biotin interaction, by one of two methods. Avidin is covalently attached to submicroscopic polymer spheres; the complexes of avidin spheres with the DNA: RNA-biotin hybrids band in CsCl at a much lower buoyant density than does free DNA. Alternatively, the DNA:RNA-biotin hybrids are isolated by affinity chromatography on an avidin-solid support column. These methods have been used to prepare long single strands of Drosophila ribosomal DNA (rDNA) in high yield and 42 to 80% pure.     

Analysis of the promoter-distal region of the tra operon of the F sex factor of Escherichia coli K-12 encoded by EcoRI restriction fragments f17, f19, and f2.

The promoter-distal region of the tra operon of the F sex factor Escherichia coli K-12 was analyzed, using the chimeric plasmid pRS31, which contains the F EcoRI restriction fragments f17, f19, and f2 cloned into the EcoRI site of pSC101. A series of deletion plasmids of pRS31, extending increasing distances from a site in f17 through f19 and ending in f2, were isolated. These plasmids were examined by heteroduplex analysis with the parent DNA, and a restriction map of this region of DNA was constructed. A series of Tn5 insertion derivatives of pRS31 were also isolated and mapped, using both heteroduplex analysis and restriction mapping. Both the insertion and deletion mutants were tested in minicells for the synthesis of radioactively labeled proteins. This allowed the identification of the individual gene products and mapping of the genes. The result is a saturated physical map of this region of DNA from fragment f17 through to the IS3 insertion sequence near the promoter-distal end of f2.