A resuscitation/selection system for rapid determination of Salmonella in foods.

A resuscitation medium was developed consisting of a trypticase soy broth base supplemented with 0.5% yeast extract, 0.25% sodium pyruvate, 0.01% sodium thioglycollate, and 0.1% chicken fat. After a resuscitation period of 4 h, the medium was made selective by addition of either sodium thiosulfate, bile salts and iodine, or sodium selenite and L-cystine. The now selective medium was incubated for 16 h. The presence or absence of Salmonella was determined by the Salmonella-Tek antibody-based detection kit. The present system was compared with a method of the Bacteriological Analytical Manual (BAM) for naturally contaminated foods. Nineteen egg products were screened; 3/19 were positive using the BAM method, 9/19 were positive using the present system. Seventeen chicken samples were assayed; 10/17 were positive using the BAM method; 13/17 were positive using the present system. Of 8 pepper samples, 4/8 were positive using the BAM method; 6/8 were positive using the present system. Of 8 spice samples, 6/8 were positive using the BAM method, 7/8 were positive using the present system. Of 6 onion products sampled, 5/6 were positive using the BAM method; 6/6 were positive using the present system.

Some studies on the preservation of indometacin suspensions intended for ophthalmic use.

Suspensions of indometacin (1% w/v), buffered to pH 5.6, may be satisfactorily preserved by 0.002% w/v phenylmercuric nitrate in the presence of hydroxypropylmethylcellulose (0.5% w/v), despite 90% of the preservative being adsorbed to the indometacin powder. Polyvinyl alcohol (1.4%) could be used as an alternative suspending agent.

Purification and biochemical characterization of SELB, a translation factor involved in selenoprotein synthesis.

The product of the selB gene from Escherichia coli is required for co-translational insertion of selenocysteine into protein. To make the SELB protein accessible to biochemical analysis, the protein was purified from cells that overexpressed the selB gene from a phage T7 promoter plasmid. It was calculated that the overproduced SELB protein was purified 20-fold. The N-terminal amino acid sequence of the purified protein was determined, and it confirmed that the initiation codon of selB mRNA translation overlaps the stop codon of the preceding selA gene by 4 bases. Structural similarity between SELB and elongation factors was demonstrated by limited proteolysis of SELB by trypsin. The cleavage sites within SELB were identified by N-terminal sequencing of the two proteolytic products. The position in the SELB protein of the major cleavage site was homologous to a tryptic cleavage site which is characteristic for elongation factors. Immunological analysis showed that the levels of SELB are equivalent in aerobically and anaerobically grown cells; the amount of the protein was estimated to be approximately 1100 copies/E. coli cell. Upon fractionation of cell extracts, SELB was found to be partially associated with the ribosomes. The results therefore indicate that SELB is the first known elongation factor-like protein that has specificity for a particular charged tRNA.

A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues.

Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.

Purification and characterization of Go alpha and three types of Gi alpha after expression in Escherichia coli.

Complementary DNAs for the G protein alpha subunits Gi alpha 1, Gi alpha 2, Gi alpha 3, and Go alpha were expressed in Escherichia coli, and the four proteins were purified to homogeneity. The recombinant proteins exchange and hydrolyze guanine nucleotide, are ADP-ribosylated by pertussis toxin, and interact with beta gamma subunits. The rates of dissociation of GDP from Gi alpha 1 and Gi alpha 3 (0.03 min-1) are an order of magnitude slower than that from rGo alpha; release of GDP from Gi alpha 2 is also relatively slow (0.07 min-1). However, the values of kcat for the hydrolysis of GTP by rGo alpha and the three rGi alpha proteins are approximately the same, about 2 min-1 at 20 degrees C. The recombinant proteins restore inhibition of Ca2+ currents in pertussis toxin-treated dorsal root ganglion neurons in response to neuropeptide Y and bradykinin, indicating that the proteins can interact functionally with all necessary components of at least one signal transduction system. The two different receptors function with different arrays of G proteins to mediate their responses, since all four G proteins restored responses to bradykinin, while Gi alpha 2 was inactive with neuropeptide Y. Despite these results, high concentrations of activated Gi alpha proteins are without effect on adenylyl cyclase activity, either in the presence or absence of forskolin or Gs alpha, the G protein that activates adenylyl cyclase. These results are consistent with the hypothesis that G protein beta gamma subunits are primarily responsible for inhibition of adenylyl cyclase activity.

Biochemical and biological activities of recombinant S1 subunit of pertussis toxin.

degP-deficient strains of Escherichia coli grown in M-9 medium supplemented with ZnCl2 expressed the recombinant S1 subunit of pertussis toxin (rS1) in a form electrophoretically identical to the authentic S1 subunit. Subcellular fractionation showed that the full-length form of rS1 was membrane associated, while proteolytic fragments of rS1 were present in the periplasm. rS1 was extracted from outer membrane preparations with 8 M urea and purified by gel filtration chromatography. Purified rS1 ADP-ribosylated transducin at a similar molar efficiency relative to authentic pertussis toxin and, when associated with the native B oligomer of pertussis toxin, elicited Chinese hamster ovary cell clustering.

Induction of non-bilayer lipid structures by functional signal peptides.

Using 31P NMR and freeze-fracture electron microscopy we investigated the effect of several synthetic signal peptides on lipid structure in model membranes mimicking the lipid composition of the Escherichia coli inner membrane. It is demonstrated that the signal peptide of the E. coli outer membrane protein PhoE, as well as that of the M13 phage coat protein, strongly promote the formation of non-bilayer lipid structures. This effect appears to be correlated to in vivo translocation efficiency, since a less functional analogue of the PhoE signal peptide was found to be less active in destabilizing the bilayer. It is proposed that signal sequences can induce local changes in lipid structure that are involved in protein translocation across the membrane.

Recognition properties of peptides hydropathically complementary to residues 356-375 of the c-raf protein.

Two peptides with hydropathic complementarity to residues 356-375 of the c-raf protein were synthesized to determine if they recognize the raf-(356-375) peptide as well as the entire protein. One peptide was deduced from the complementary mRNA for the raf protein corresponding to residues 356-375, whereas the other was deduced solely from the amino acid sequence of the 20-mer segment using a computer program able to generate peptide sequences with hydropathic complementarity to a given sequence. Specific binding of both peptides to the raf 20-mer segment was demonstrated when either the raf 20-mer peptide or the complementary peptides were immobilized on a column. Binding affinities were in the millimolar-micromolar range. Identical binding properties were observed with peptides synthesized with either all D- or all L-amino acids, suggesting a lack of conformational dependence. Binding was also unaffected by the presence of 8 M urea or detergents, was dependent on solvent characteristics of pH and ionic strength, and was abolished by the presence of competing peptides in the eluting buffer. Recognition between raf complementary peptides was accompanied by spectral changes in the far and near UV region, as monitored by circular dichroism. Proteolytic degradation was retarded by the binding of these peptides. Once immobilized on a column, these peptides proved useful for the isolation by affinity chromatography of a recombinant c-raf protein from an Escherichia coli crude cell extract.

Complete structural determination of lipopolysaccharide obtained from deep rough mutant of Escherichia coli. Purification by high performance liquid chromatography and direct analysis by plasma desorption mass spectrometry.

Lipopolysaccharide (LPS) extracted from the deep rough mutant of Escherichia coli D31m4 was disaggregated with 0.1 M EDTA, pH 7.0, and fractionated on a diethylaminoethyl-cellulose column to yield the biphosphate form of LPS. After methylation, the derivative was purified by reverse-phase high performance liquid chromatography using a C18-bonded silica cartridge. A linear gradient of 50-100% isopropyl alcohol/water (93:7, v/v) in acetonitrile/water (93:7, v/v) was used over a period of 60 min. The derivatized LPS showed a single major peak by high performance liquid chromatography, and this hexamethyl hexaacyl LPS was recovered and subjected to chemical analysis, plasma desorption mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. Chemical analysis of the purified hexamethyl LPS quantitated certain key chemical compositions. Plasma desorption mass spectrometry showed a molecular ion (M + CH2 + Na)+ at m/z 2360, which established the molecular formula and Mr to be C116H214N2O39P2 and 2323, respectively. Thus, it contained two each of glucosamine, 2-keto-3-deoxyoctonate, and phosphate; four beta-hydroxymyristates; one laurate; and one myristate. NMR spectroscopy confirmed the locations of the four ester-linked fatty acyl groups. Based on these results and the known structure of free lipid A, the complete structure of the deep-rough chemotype LPS from E. coli can now be presented with confidence. This is the first report of a successful purification to homogeneity and the characterization of the simplest of the LPS at the intact level. This study shows that the natural distribution of the lipid A moiety of LPS from E. coli D31m4 is hexaacyl/pentaacyl in a molar ratio of greater than 90:less than 10. Acid hydrolysis of LPS causes the formation of the lower homologues of the free lipid A.

Effect of cysteine ethylester hydrochloride (Cystanin) on host defense mechanism (IV): Potentiating effects on the function of peritoneal or spleen macrophages.

L-Cysteine ethylester hydrochloride (ethylcysteine; 30 mg/kg, p.o.) increased the number of la-positive cells (antigen presenting cells) in spleen adherent cells (SAC) and that of Lyt 1.2-positive cells (helper T cells), but not that of Lyt 2.2-positive cells (suppressor T cells) of C57BL/6 mice immunized with sheep red blood cells. The production of hemolytic plaque forming cells (HPFC) in spleens of syngeneic recipient mice was enhanced by the transfer of SAC or spleen lymphocytes of the donor mice pretreated with ethylcysteine. This drug augmented phagocytosis of yeast particles by peritoneal macrophages of ICR mice at concentrations of 1-100 microM. In ex vivo experiments, this drug (30 mg/kg, p.o.) augmented the phagocytosis of yeast particles by mouse macrophages and showed a tendency to increase the macrophage number in the peritoneal cavity. Ethylcysteine (30 mg/kg, p.o.) significantly accelerated the decrease of viable E. coli number in the liver of normal mice 2 and 48 hr after challenge. Furthermore, this drug at the same dose restored the suppression of the decrease of E. coli number in the blood and liver of mice treated with cyclophosphamide (200 mg/kg, i.p.). These results suggest that ethylcysteine augments the functions of macrophages in vitro and ex vivo, and these enhancing effects may lead to the enhancement of host resistance to infections in compromised hosts.

Characterization of flagella purified from enterohemorrhagic, vero-cytotoxin-producing Escherichia coli serotype O157:H7.

Escherichia coli of the serotype O157:H7 has recently been isolated in human fecal specimens in association with sporadic cases and outbreaks of hemorrhagic colitis and with the hemolytic uremic syndrome. The aim of this study was to characterize the flagellin protein subunit constituents of flagellar filaments from E. coli O157:H7 strain CL-56. Flagellin isolated from a reference Salmonella enteritidis strain was used for comparison. Flagella were dissociated by incubation of bacteria under acidic conditions, centrifugation, and differential ammonium sulfate precipitation. Reconstituted flagellar filaments were demonstrated by three complementary methods: transmission electron microscopy, antigenic reactivity with H7 antiserum by a dot blot immunoassay, and immunogold localization of antiserum raised to the purified antigen to intact flagella on whole E. coli O157:H7. On sodium dodecyl sulfate-polyacrylamide gels flagellin proteins from E. coli O157:H7 demonstrated an apparent Mr of 66,000. The isoelectric point of E. coli O157:H7 flagellin was 5.42. By immunoblotting, H7 flagellin proteins were shown to be immunogenic. They induced a systemic immune response both in rabbits challenged with whole bacteria and in a human previously infected with E. coli O157:H7.

19F nuclear magnetic resonance as a probe of anticodon structure in 5-fluorouracil-substituted Escherichia coli transfer RNA.

The use of 19F nuclear magnetic resonance (n.m.r.) spectroscopy as a probe of anticodon structure has been extended by investigating the effects of tetranucleotide binding to 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 (anticodon FAC). 19F n.m.r. spectra were obtained in the absence and presence of different concentrations of oligonucleotides having the sequence GpUpApX (X = A,G,C,U), which contain the valine codon GpUpA. Structural changes in the tRNA were monitored via the 5-fluorouracil residues located at positions 33 and 34 in the anticodon loop, as well as in all other loops and stems of the molecule. Binding of GpUpApA, which is complementary to the anticodon and the 5'-adjacent FUra 33, shifts two resonances in the 19F spectrum. One, peak H (3.90 p.p.m.), is also shifted by GpUpA and was previously assigned to FUra 34 at the wobble position of the anticodon. The effects of GpUpApA differ from those of GpUpA in that the tetranucleotide induces the downfield shift of a second resonance, peak F (4.5 p.p.m.), in the 19F spectrum of 19F-labeled tRNA(Val)1. Evidence that the codon-containing oligonucleotides bind to the anticodon was obtained from shifts in the methyl proton spectrum of the 6-methyladenosine residue adjacent to the anticodon and from cleavage of the tRNA at the anticodon by RNase H after binding dGpTpApA, a deoxy analog of the ribonucleotide codon. The association constant for the binding of GpUpApA to fluorinated tRNA(Val)1, obtained by Scatchard analysis of the n.m.r. results, is in good agreement with values obtained by other methods. On the basis of these results, we assign peak F in the 19F n.m.r. spectrum of 19F-labeled tRNA(Val)1 to FUra 33. This assignment and the previous assignment of peak H to FUra 34 are supported by the observation that the intensities of peaks F and H in the 19F spectrum of fluorinated tRNA(Val)1 are specifically decreased after partial hydrolysis with nucleass S1 under conditions leading to cleavage in the anticodon loop. The downfield shift of peak F occurs only with adenosine in the 3'-position of the tetranucleotide; binding of GpUpApG, GpUpApC, or GpUpApU results only in the upfield shift of peak H. The possibility is discussed that this base-specific interaction between the 3'-terminal adenosine and the 5-fluorouracil residue at position 33 involves a 5'-stacked conformation of the anticodon loop. Evidence also is presented for a temperature-dependent conformational change in the anticodon loop below the melting temperature of the tRNA.

A solution hybridization assay for ribosomal RNA from bacteria using biotinylated DNA probes and enzyme-labeled antibody to DNA:RNA.

Rapid, convenient and non-isotopic nucleic-acid hybridization methods are needed for this technology to have practical use in clinical diagnostic tests. A method for hybridization of RNA with a DNA probe in solution followed by capture and measurement of the hybrid is described. DNA probes complementary to 23S rRNAs from Escherichia coli and Bacillus subtilis were labeled with a photoactivable biotin reagent. Hybridization of the biotinylated probes with rRNA was complete in less than 5 min. The resultant hybrids were allowed to bind simultaneously to succinylated avidin immobilized on latex and to beta-galactosidase-labeled Fab' fragments of a monoclonal antibody-specific for DNA:RNA. Finally, beta-galactosidase associated with the captured hybrids was measured colorimetrically. The hybridization method can detect less than 1000 bacteria per assay and has broad specificity to permit detection of the various genera of bacteria that infect the urinary tract.

Electron probe analysis, X-ray mapping, and electron energy-loss spectroscopy of calcium, magnesium, and monovalent ions in log-phase and in dividing Escherichia coli B cells.

The elemental composition of individual cells of rapidly frozen and cryosectioned Escherichia coli B was measured with electron optical microanalytic methods. The Ca content was high (26.2 mmol/kg) in a 10-nm-wide region of the cell envelope. Amounts of cytoplasmic Ca in actively dividing cells were significantly higher (32.6 mmol/kg [dry weight]) than in the log-phase (1.5 mmol/kg) cells. Cellular Mg was 205 mmol/kg (dry weight) and it was uniformly distributed throughout the cell. Cells washed in distilled water before freezing lost monovalent ions (Na, Cl, and K), but the membrane-bound Ca and cellular Mg were not reduced, indicating that cellular Mg and membrane Ca are more tightly bound.

Heterogeneity of lipid A: structural determination by 13C and 31P NMR of lipid A fractions from lipopolysaccharide of Escherichia coli 0111.

Purified lipid A from Escherichia coli 0111 was fractionated by thin-layer chromatography, and seven major bands were studied by 13C and 31P NMR. All lipid A fractions except one had fatty acids, 3-hydroxytetradecanoic acid, 3-(acyloxy)tetradecanoic acid, and phosphate groups bonded to the diglucosamine backbone. The remaining fraction was shown to be phosphatidylethanolamine. The number of substituents found showed that in all fractions all sites available for C-acylation (C-3, C-4, and C-3') and N-acylation (C-2 and C-2') carried acylic substituents. The number, ranging from four to six, and type of ester-bound carboxylic acid residues as well as the number of phosphate groups differed among the fractions. The three fastest moving bands all had three unsubstituted hydroxy fatty acids and one phosphate group (C-4'), while the slower moving bands had four hydroxy fatty acids and two phosphate groups. Unsubstituted 3-hydroxytetradecanoic acid residues were amide-bound to the disaccharide in all but one of the fractions. In summary, the heterogeneity of E. coli 0111 lipid A is found to be a consequence of a variation of the number and composition of carboxylic acid residues and of varying phosphate content.

Identification and synthesis of a naturally occurring selenonucleoside in bacterial tRNAs: 5-[(methylamino)methyl]-2-selenouridine.

Escherichia coli, Clostridium sticklandii, and Methanococcus vannielii synthesize 75Se-labeled amino acid transfer ribonucleic acids [( 75Se]tRNAs) when grown with low levels (approximately equal to 1 microM) of 75SeO32-. When E. coli [75Se]tRNA was digested to nucleosides and analyzed by reversed-phase high-performance liquid chromatography, a single selenonucleoside accounted for 70-90% of the 75Se label in the bulk tRNA. This nucleoside was shown to be indistinguishable in a number of its properties from authentic 5-[(methylamino)methyl]-2-selenouridine. Preparation of the authentic selenonucleoside was accomplished and the synthetic compound characterized by its UV and 1H NMR spectral properties. The new selenonucleoside also accounted for 40-60% of the 75Se found in [75Se]tRNA from C. sticklandii or M. vannielii. Each of these anaerobic bacteria contains one additional selenonucleoside in their tRNA populations distinct from 5-[(methylamino)methyl]-2-selenouridine. Pure seleno-tRNAGlu isolated from C. sticklandii contains one 5-[(methylamino)methyl]-2-selenouridine and one 4-thiouridine per tRNA molecule.

Inhibition of the autoxidation of ascorbate and norepinephrine by extracts of Clostridium butyricum, Megasphaera elsdenii and Escherichia coli.

The autoxidation of ascorbate and of norepinephrine in Krebs Ringer phosphate medium, pH 7.4, was studied. The autoxidation of the two substances was determined spectrophotometrically at 265 and 480 nm respectively. The effect of dialyzed extracts (m.w. greater than 12,000) from Escherichia coli (aerobe), Megasphaera elsdenii, and Clostridium butyricum (obligate anaerobes) was examined and compared to similarly prepared extracts from rat serum and cerebral cortex. The assay medium contained cellular components diluted 10(3)-10(6)-fold. Up to 10(4)-fold dilution there was a substantial reduction in the rate of both autoxidation reactions, but the preparations from M. elsdenii and C. butyricum were conspicuously less effective. After 5 min heat treatment at 100 degrees C the anaerobic preparations produced less than 20% inhibition, while the activity of the other preparations remained unchanged at 75-95% inhibition. These and earlier experiments involving additional mammalian species (Mishra and Kovachich, Neurosci. Lett., 43: 103-108, 1983) and plants (Mishra and Kovachich, Life Sci., 34: 2207-2212, 1984) suggest that a high level of heat-stable antioxidant activity in one or both of these autoxidation tests (denatured plant extracts only inhibit ascorbate autoxidation) is a general characteristic of organisms that thrive in oxygen-rich atmosphere.

Electron spectroscopic imaging: parallel energy filtering and microanalysis in the fixed-beam electron microscope.

A new imaging modality in electron microscopy uses energy filtration to produce micrographs with elastically scattered electrons or with electrons that have lost a specific, often characteristic amount of energy in interacting with the specimen. No deleterious effects on microscope performance are encountered. Instead, microanalysis of specimens is made possible with a spatial resolution of 3 to 5 A and a sensitivity of detection of 2 X 10(-21) g corresponding to about 50 atoms of phosphorus. Elements detected range from hydrogen (Z = 1) to uranium (Z = 92). Examples of elemental mapping show membrane structure, DNA within nucleosomes, and RNA within ribosomal particles.

Codon-level analysis of histone primary sequence: evidence of a repeat tetrapeptide origin and later inclusion of transcribed sequence.

This work is directed to the question of protein sequence conservation. By reference to the genetic code the aminoacyl sequence of histones H2A, H4, H3, H2B and H1 (fragment) were rewritten as the codon sequences. The N-terminal regions were set aside on the grounds of different composition and sequence. The remainder of the molecule could be referred to simple repeat-tetrapeptide proteins by codon composition (high Gxy, low xGy content) and by sequence. Random segments of three to six residues occur characterized by composition and sequence as originating from the complimentary DNA strand, i.e. as codon "transcript". Ancestral features are probably best seen in H3, point mutations appear to be more extensive in H2B and H1. Segments in reverse order in H2A and in "transcript" in H4 distinguish these two from the other three histones. There is a tenuous possibility the N-terminals also originated as repeat-tetrapeptide now intensively modified. At codon-level the 50S ribosomal protein (L7/L12) of E. coli has features in common with histones (including a palindrome-containing N-terminal). It has the composition and sequence of a well-conserved tetrapeptide-repeat strand (statistical support). If interpretations made here are substantially correct, the 50S r-protein illustrates a significant stage in evolution of histone codon strands.

Collaborative study of the MPN, Anderson-Baird-Parker direct plating, and hydrophobic grid-membrane filter methods for the enumeration of Escherichia coli biotype I in foods.

Five Health Protection Branch laboratories compared two membrane filter methods (the Anderson-Baird-Parker direct plating, and a hydrophobic grid-membrane filter method) against the most probable number procedure (MPN) for enumerating Escherichia coli biotype I in foods. Results were available in 24 h by both membrane filter methods, compared with 10-14 days by the MPN procedure. For ground beef, Parmesan cheese, and cut green beans, the hydrophobic grid method generally gave the highest recovery, although the two membrane filter methods were not significantly different. Both these methods gave significantly higher recoveries than the MPN procedure, and for most foods, either method would be preferable. Further work is required before either membrane filter method can be recommended for bean and alfalfa sprouts, which may contain very high levels of Klebsiella spp.