Rapid propidium monoazide cPCR assay for exclusive quantification of viable Salmonella spp. cells.

Combination of pretreatment with propidium monoazide by competitive polymerase chain reaction (cPCR) was evaluated to enumerate the viability of Salmonella spp. The results showed that PMA treatment was effective in preventing the cPCR detection of target sequences from non- viable cells. In less than 5 hrs, this method generated a signal from viable but nonculturable (VBNC) Salmonella spp. The standard culture method gave approximately 1-2 log(10)cfu ml(-1) less as compared to the PMA-cPCR results. These results provided evidence to support the VBNC state, whereas, the viable cells failed to be cultured by SCM. The proposed method did not detect DNA from dead Salmonella spp. but recognizes the infectious potential of the VBNC state and is thereby, able to assess the effect of control strategies and provide trustworthy data for risk assessment.

Effect of humic substances on Cu(II) solubility in kaolin-sand soil.

The type and amount of organic matter present in industrially contaminated soils will influence the risk they pose. Previous studies have shown the importance of humic and fulvic acids (FAs) (important components of soil organic matter) in increasing the solubility of toxic metals but were not carried out using toxic metal levels and the pH range typical of industrially contaminated soils. This study investigated the influence of three humic substances (HSs: humates, fulvates and humins) on the solubility of copper(II) ions in kaolinitic soil spiked with Cu at levels representative of industrially contaminated soil. Humates, fulvates and humin were extracted from Irish moss peat, and controlled pH batch leaching tests were conducted on an artificial kaolin-sand soil that was spiked with each. Further leaching tests were conducted on soil spiked with each HS and copper nitrate. Dissolved organic contents were determined by titration and total and free aqueous copper concentrations in the leachate were measured using AAS and ion selective electrode (ISE) potentiometry respectively (dissolved complexed copper levels were determined by difference). It was found that humates and fulvates are partially sorbed by the soil, probably by chemisorption on positively charged gibbsite (Al-hydroxide) sites in the kaolinite. The addition of 340 mg/kg Cu(II) ions did not significantly affect the amount of humate or fulvate sorbed. Dissolved humates and fulvates form soluble complexes with copper over the pH range 3-11. However, in the presence of kaolinite, soluble copper humates and fulvates are unable to compete with the kaolinite for Cu ions at pH 6-7. Above pH 8, humate and fulvate complexes are the only forms of dissolved Cu. Humin is largely insoluble and has little effect on Cu mobility between pH 2 and 12. The implication of this study is that measurement of total soil organic content and water leaching tests should be a standard part of contaminated site investigation.

Leaching behaviour of a chromium smelter waste heap.

This paper reports the results of geochemical sampling and modelling of leachates from a chromite ore processing residue (C.O.P.R.) pile under rainwater infiltration. The waste pile is located in the north of England and consists of 800,000 m3 of waste. The pH of fresh leachate is similar to that of a solution in equilibrium with portlandite Ca(OH)2, which is a major constituent of the waste. The in-gassing of CO2(g) causes the pH of the leachates to drop along the drainage ditch and calcite precipitation to occur. The extent of in-gassing is dependent upon the flow rate within the drainage ditch. The dissolution of solid solutions containing residual chromate is likely to control chromate concentrations within the leachate.

Heterologous biopharmaceutical protein expression in Streptomyces.

The commercial production of human proteins in recombinant microorganisms for therapeutic use is well established. Systems have been developed to exploit the natural ability of certain bacteria to secrete properly folded, bioactive proteins into the extracellular medium. The streptomycetes are a relatively well-characterized group of nonpathogenic filamentous bacteria that have the capacity to secrete large amounts of protein. In particular, Streptomyces lividans has the ability to secrete human proteins at a commercially viable level, thanks to relatively well-established plasmid-based expression system, a high-biomass fermentation process and a low level of endogenous protease activity.

Expression and characterization of soluble human erythropoietin receptor made in Streptomyces lividans 66.

A gene encoding the extracellular domain of the human erythropoietin receptor (EPO-R) was constructed using oligonucleotides, with a view to maintaining preferred codon usage for the Streptomycetes. The gene was subcloned into a multicopy Streptomyces-Escherichia coli shuttle vector, pCAN46 (derived from pIJ680), containing a strong constitutive promoter from the S. fradiae aph gene, a signal peptide coding region derived from the protease B gene of S. griseus, and a transcription terminator sequence also derived from the S. fradiae aph gene. Extracellular expression of authentic EPO-R by S. lividans was demonstrated using SDS-PAGE and Western blot analysis, followed by direct amino terminal sequencing of the purified product. Specific binding of S. lividans-expressed EPO-R to recombinant human glycosylated EPO was demonstrated using BIAcore (surface plasmon resonance) analysis and native gel shift assays.

Troponin-T and glyceraldehyde-3-phosphate dehydrogenase share a common antigenic determinant.

A 37 kDa protein in extracts of bovine aorta and equine platelets was observed on SDS-polyacrylamide gel electrophoretograms to react with polyclonal and monoclonal antibodies to rabbit skeletal troponin-T (TnT) by immunoblotting. Following purification by precipitation at pH 4.6 and several ion-exchange chromatographic steps, it has been identified as glyceraldehyde-3-phosphate dehydrogenase (G3PD) by amino acid analyses and NH2-terminal sequencing. By ELISA, the anti-troponin-T monoclonal antibody reacted with rabbit skeletal G3PD appreciably but 120-fold less specifically than with TnT. A cyanogen bromide fragment (CB2) of TnT (residues 71-151) reacted with the monoclonal antibody nearly as well as intact TnT. This cross-reactivity between G3PD and TnT can be ascribed to a weak homology in the amino acid sequences of the two proteins between residues 72-80 of TnT and residues 157-165 of G3PD. Other regions of limited sequence similarity in the two proteins are also present. We conclude that the identification of diffuse cytoplasmic indirect immunofluorescent staining observed with a monoclonal anti-TnT antibody in chicken gizzard muscle is probably attributable to cross-reactivity with G3PD.

Non-specific interactions of histones with serum components and contractile proteins.

Strong, apparently non-specific interactions are often observed between antisera and proteins in the assay of biological systems by methods employing antibodies. We have documented the interaction between immunoglobulin-enzyme complexes and nuclear histone proteins. This binding has been demonstrated for all the histones derived from bovine thymus. The interaction between histones and the immunoglobulin-enzyme complexes has been shown to be inhibited by both whole serum and individual serum components as well as a number of the major contractile proteins.

Excimer fluorescence of equine platelet tropomyosin labeled with N-(1-pyrenyl)iodoacetamide.

Tropomyosin from equine platelets was reacted with N-(1-pyrenyl)iodoacetamide, a sulfhydryl-specific fluorescent reagent, to give an average extent of incorporation of 1.12 pyrene (Py) groups per platelet tropomyosin (P-TM) chain. The predominant site of reaction on P-TM was the penultimate COOH-terminal residue, Cys-246. The high proportion of the total emission that is due to pyrene ecximers and the pretransition observed in thermal denaturation of Py-P-TM point to a rather loose structure for the COOH-terminal amino acid residues of P-TM. The label on Cys-246 also reports on end-to-end overlap interactions that occur between two different tropomyosin molecules. Additions to a Py-P-TM solution at low ionic strength of unlabeled P-TM, rabbit cardiac tropomyosin (C-TM), or a carboxypeptidase A treated, nonpolymerizable derivative of C-TM all reduce the extent of excimer fluorescence from the sample. Addition of salt greatly reduces the effects of the unlabeled TM species on the Py-P-TM emission spectrum. Circular dichroism measurements indicate Py-P-TM still to be greater than 95% helical. However, analysis of excimer fluorescence levels in samples that contained a constant protein concentration but different mole ratios of labeled to unlabeled P-TM suggests that the bulky pyrene group may diminish the tendency of Py-P-TM to polymerize in an end-to-end manner.

Redistribution of membrane-bound and cytosolic action in rabbit polymorphonuclear leucocytes during phagocytosis.

In the analysis of highly purified surface membrane from both resting and phagocytosing neutrophils an increase in the surface membrane associated actin has been demonstrated. This change at the cell periphery is associated with a coincident increase in the F-actin content of the cells following stimulation of the cells by exposure to opsonized Oil Red O droplets. The actin which is newly associated with the surface membrane of the phagocytosing cells was more susceptible to removal by detergent than the membrane-associated actin in resting cells and it was also noted that the F-actin associated with phagosomes was readily disrupted by detergent. A redistribution of the surface membrane glycoprotein 5'-nucleotidase was observed during phagocytosis, but no change in distribution of a 125I-labelled Lens culinaris lectin was observed during the entire phagocytic process.

Preparation of a highly purified surface membrane fraction from rabbit polymorphonuclear leucocytes by high-voltage free-flow electrophoresis.

A surface membrane fraction of high purity and good yield has been prepared from homogenates of rabbit peritoneal polymorphonuclear leucocytes, using a preliminary sorbitol density gradient sedimentation followed by preparative high voltage electrophoresis in a thin flowing buffer film. Enrichment values for the plasma membrane marker enzyme 5'-nucleotidase and 125I-labelled Lens culinaris lectin, after the latter had been applied at the whole cell level, were 18-fold and 6-fold, respectively. Contamination of the surface membrane fraction by other organelles was negligible and approximately 1 mg of surface membrane protein can be obtained from 2 . 10(9) leucocytes. A triacylglycerol-rich, protein-poor fraction that lacks any definable structure in electron microscopy separates discretely from the surface membrane vesicles during electrophoresis. It is considered that this may be a contaminant not previously recognized as present in membrane fractions prepared by more conventional procedures.

Identification of a troponin-I like protein in platelet preparations as histone H2B.

A tropomyosin-binding protein (app. Mr 17000) was detected in equine platelet preparations by a gel overlay technique. Its isolation, amino acid and partial sequence analyses have shown it to be histone H2B. As with a similar protein from pig platelet preparations [der Terrossian et al. (1983) FEBS Lett. 152, 202-206], it inhibits Mg2+-dependent actomyosin S1 ATPase. This inhibition is partially reversed in the presence of calmodulin and Ca2+ but is not potentiated, unlike troponin-I, by tropomyosin. This protein, along with the other histones, is almost certainly derived from a low level of contaminating nucleated cells in most platelet preparations.

Redistribution of membrane 5'-nucleotidase in rabbit peritoneal polymorphonuclear leucocytes during phagocytosis.

Using a quantitative phagocytic model involving oil droplet internalisation rabbit polymorphonuclear leucocytes display selective segregation of membrane constituents during the phagocytic event. A resting cell surface membrane and fractions representing vesicle membranes and uninvolved surface membranes from the active cells have been purified by density gradient sedimentation and free flow electrophoresis. The specific activity of 5'-nucleotidase, a major neutrophil surface membrane glycoprotein, was 3-fold higher in the uninvolved membrane of phagocytosing cells than in the resting cell membrane. The activity in the vesicle membranes was substantially depleted. In contrast Lens culinaris receptors showed no redistribution during phagocytosis: the two surface domains showing essentially the same enrichment with respect to homogenate as the resting cell surface membrane.