Assurance polyclonal enzyme immunoassay for detection of Listeria monocytogenes and related Listeria species in selected foods: collaborative study.

Six foods representing a variety of food products were analyzed by the Assurance Listeria polyclonal enzyme immunoassay (EIA) and by either the Bacteriological Analytical Manual or the U.S. Department of Agriculture culture method for detecting Listeria monocytogenes and related Listeria species. Samples of each food type, at each inoculation level, were analyzed simultaneously by both methods. A total of 19 laboratories representing federal government agencies and private industry in the United States and Canada participated. Food types were inoculated with Listeria species including L. monocytogenes, with the exception of 3 lots of green beans, which were naturally contaminated. During this study, 1764 samples and controls were analyzed and confirmed, of which 492 were positive and 947 were negative by both methods. There were 159 samples that were positive by culture method but negative by the EIA and 188 that were negative by culture method but positive by EIA. Twenty-two samples were negative by EIA and by culture method but confirmed positive when Assurance selective enrichment broths were subcultured to selective agar. The Assurance polyclonal EIA for detecting L. monocytogenes and related Listeria species in foods has been adopted first action by AOAC INTERNATIONAL.

Visual immunoprecipitate assay (VIP) for Listeria monocytogenes and related Listeria species detection in selected foods: collaborative study.

Six foods representing a variety of food products were analyzed by the Visual Immunoprecipitate Assay (VIP) and either the Bacteriological Analytical Manual (BAM) or the U.S. Department of Agriculture culture methods for detection of Listeria monocytogenes and related Listeria spp. Samples of each food type at each inoculation level were simultaneously analyzed by both methods. A total of 23 laboratories representing federal agencies and private industry in the United States and Canada participated in this collaborative study. Foods were inoculated with Listeria species including L. monocytogenes, with the exception of 3 lots of green beans that were naturally contaminated. During this study, 1509 samples and controls were analyzed and confirmed, of which 370 were positive and 921 were negative by both methods. One hundred and fifteen samples were positive by culture methods but negative by VIP. One hundred and thirty-two were negative by culture methods but positive by the VIP. Twenty-nine samples were negative by VIP and by culture methods but confirmed positive when VIP selective enrichment broths were subcultured to selective agars. The VIP method for detection of L. monocytogenes and related Listeria spp. in foods has been adopted first action by AOAC INTERNATIONAL.

Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants.

To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level. The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase. Labeling of purified synthase with either [32P]c-di-GMP or [alpha-32P]UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-kDa peptide. However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides. Further, the N-terminal amino acid sequences determined for the 90- and 67-kDa peptides share a high degree of homology with the amino acid sequence deduced from the gene. We suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB. The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton. The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.

Genetic organization of the cellulose synthase operon in Acetobacter xylinum.

An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the coding region of the first gene (bcsA) in the operon. Results from genetic complementation tests and gene disruption analyses demonstrate that all four genes in the operon are required for maximal bacterial cellulose synthesis in A. xylinum. The calculated molecular masses of the proteins encoded by bcsA, bcsB, bcsC, and bcsD are 84.4, 85.3, 141.0, and 17.3 kDa, respectively. The second gene in the operon (bcsB) encodes the catalytic subunit of cellulose synthase. The functions of the bcsA, bcsC, and bcsD gene products are unknown. Bacterial strains mutated in the bcsA locus were found to be deficient in cellulose synthesis due to the lack of cellulose synthase and diguanylate cyclase activities. Mutants in the bcsC and bcsD genes were impaired in cellulose production in vivo, even though they had the capacity to make all the necessary metabolic precursors and cyclic diguanylic acid, the activator of cellulose synthase, and exhibit cellulose synthase activity in vitro. When the entire operon was present on a multicopy plasmid in the bacterial cell, both cellulose synthase activity and cellulose biosynthesis increased. When the promoter of the cellulose synthase operon was replaced on the chromosome by E. coli tac or lac promoters, cellulose production was reduced in parallel with decreased cellulose synthase activity. These observations suggest that the expression of the bcs operon is rate-limiting for cellulose synthesis in A. xylinum.

Two functionally distinct forms of guanosine cyclic 3',5'-phosphate stimulated cation channels in a bovine rod photoreceptor disk preparation.

Cyclic nucleotide stimulated efflux of 22Na+ and 45Ca2+ from a purified bovine rod outer segment disk preparation was measured on the 25-100-ms time scale by a novel rapid superfusion method. Activation of cation efflux by 8-bromoguanosine cyclic 3',5'-phosphate (8-Br-cGMP) was maximal within 25 ms. Over a wide range of concentrations of 8-Br-cGMP, the kinetics of termination of efflux precisely conformed to the sum of two exponential decay processes: a rapid phase (decay constant of 200 ms) and a slower phase (decay constant of 1.6 s). The kinetics of the biphasic decay of efflux cannot be explained by depletion of a pool of releasable 22Na but appear to reflect an intrinsic process for inactivation of the channels. 8-Br-cGMP-stimulated release of actively accumulated 45Ca exhibited identical biphasic decay kinetics. The maximum rate of Ca release [5 nmol.(mg of disk protein)-1.min-1] may be sufficient to produce a 1 microM change in local cytoplasmic [Ca] within 20 ms. The Ca:Na selectivity ratio is approximately 0.5:1 for both decay phases. 8-Br-cGMP demonstrated a lower potency (EC50 of 8.4 microM vs 2.8 microM) but a higher degree of cooperativity in its activation of the rapid vs the slower decay phase of 22Na efflux. The slower phase of decay was selectively inhibited by 25 microM l-cis-diltiazem, a relatively weak inhibitor of the rapid decay phase. Sodium ion (5-10 mM) selectively inhibited the rapid decay phase of 8-Br-cGMP-stimulated 45Ca release. These two kinetically and pharmacologically distinct phases of decay are hypothesized to represent two functionally distinct forms of cGMP-stimulated cation channels.

Chemical modification of rhodopsin and its effect on regeneration and G protein activation.

The studies reported are concerned with the functional consequences of the chemical modifications of the lysines and carboxyl-containing amino acids of bovine rhodopsin. The 10 non-active-site lysine residues of rhodopsin can be completely dimethylated and partially acetimidated (8-9 residues) with no loss in the ability of the proteins to activate the G protein when photolyzed or to regenerate with 11-cis-retinal. These modifications do not alter the net charge on the protein. Surprisingly, heavy acetylation of these lysines (eight to nine residues) with acetic anhydride, which neutralizes the positive charges of the lysine residues, yields a modified rhodopsin fully capable of activating the G protein and being regenerated. It is concluded that the non-active-site lysine residues of rhodopsin are not importantly and directly involved in interactions with the G protein during photolysis. However, this is not to say that they are unimportant in maintaining the tertiary structure of the protein because heavy modification of these residues by succinylation and trinitrophenylation produces proteins incapable of G protein activation, although the succinylated protein still regenerated. The active-site lysine of rhodopsin was readily modified and prevented from regenerating with 11-cis-retinal and with o-salicylaldehyde and o-phthalaldehyde/mercaptoethanol, two sterically similar aromatic aldehyde containing reagents which react by entirely different mechanisms. It is suggested that rhodopsin contains an aromatic binding site within its active-site region. Monoethylation, but not monomethylation, of the active-site lysine also prevented regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

Deprotonation of the Schiff base of rhodopsin is obligate in the activation of the G protein.

Photolysis of rhodopsin leads to the formation of an activated intermediate that activates a G protein, thus beginning the visual cascade. This activated form of rhodopsin appears coincident in time with the spectroscopically defined intermediate, metarhodopsin II. Metarhodopsin I, the precursor of metarhodopsin II, contains a protonated Schiff base, whereas metarhodopsin II does not. The question of whether the deprotonation of the protonated Schiff base is obligate in the formation of activated rhodopsin was addressed by monomethylating the active-site lysine of permethylated rhodopsin and determining whether this pigment can activate the G protein upon photolysis. The photolysis of the new pigment, which absorbs at 520 nm, led to the formation of a relatively stable metarhodopsin I-like intermediate with a lambda max of approximately equal to 485 nm, with no apparent formation of either metarhodopsin II- or metarhodopsin III-like intermediates. The only probe available to detect formation of the active form of rhodopsin is G protein activation. Photolysis of the pigment in the presence of the G protein did not lead to measurable activation of the GTPase activity of the latter. These studies establish a functional link between Schiff base deprotonation and activation of the G protein. It is concluded that proton transfer from the protonated Schiff base of rhodopsin is obligate for the initiation of visual transduction.

all-trans-retinoids and dihydroretinoids as probes of the role of chromophore structure in rhodopsin activation.

The absorption of a photon of light by rhodopsin results in the cis to trans isomerization of the 11-cis-retinal Schiff base chromophore. In the studies reported here, an attempt is made to determine the mechanism of the energization of rhodopsin as it relates to the chemistry of the isomerization process and the geometrical state of the chromophore. Studies were performed with vitamin A analogues to probe this mechanism. Both 11-cis-7,8-dihydroretinal and 9-cis-7,8-dihydroretinal form bleachable pigments when combined with opsin. Photolysis of these pigments in the presence of G-protein results in the activation of the latter as revealed by its GTPase activity. Phosphodiesterase is also activated when it is included in the incubation. Therefore, the possibility that rhodopsin is energized by mechanisms involving photochemically induced charge transfer from the protonated Schiff base to the beta-ionone ring can be discarded. Further studies were conducted with all-trans-vitamin A derivatives to determine if these compounds can form the GTPase-activating state R*, a situation that is possible, in principle, by microscopic reversibility. Neither all-trans-retinal nor its oxime, when incubated with bovine opsin in the dark, caused activation of the GTPase, requiring at least a 5 kcal/mol energy gap between them. Furthermore, stoichiometric adducts of all-trans-retinoids and opsin were also unable to mediate activation of the GTPase. Since both all-trans-15,16-dihydroretinylopsin and all-trans-retinoylopsin possess an all-trans-retinoid permanently adducted to opsin, it can be concluded that the all-trans-retinoid chromophore-opsin linkage may be necessary but not sufficient to achieve activation of the visual pigment.

Biochemical properties of 9-cis- and all-trans-retinoylopsins.

The stoichiometry of the reaction between [14C]-9-cis-retinoyl fluoride, a close isostere of 9-cis-retinal, and bovine opsin and the biochemical and spectral properties of this new pigment were investigated. The stoichiometry of retinoid incorporation is approximately one in dodecyl maltoside, a detergent in which opsin is capable of regeneration with 11-cis-retinal. Interestingly, in Ammonyx LO, a detergent that does not permit rhodopsin regeneration, the stoichiometry of binding is still approximately one. By contrast, heat-denatured opsin does not irreversibly bind substantial [14C]retinoyl fluoride. This result strongly suggests that the nucleophilicity of the active site lysine is retained in Ammonyx LO but that further conformational changes in the protein, required to form rhodopsin, are not possible. These results are all consistent with an active site directed mechanism for the irreversible reaction of 9-cis-retinoyl fluoride with opsin probably at the active site lysine residue. The ultraviolet spectra of 9-cis-retinoylopsin and its all-trans congener show gamma max's at 373 and 380 nm, respectively, somewhat bathochromically shifted from their respective model N-butylretinamides which absorb at 347 and 351 nm. Photolysis of both 9-cis- and all-trans-retinoylopsins leads to the same photostationary state. This shows that, as expected, photoisomerization without bleaching occurs. The photolysis of either 9-cis- or all-trans-retinoylopsin in the presence of the G protein (transducin) does not lead to the activation of the latter. This is consistent with the notion that a protonated Schiff base is critical for the function of rhodopsin.

Ca2+ activated and pH sensitive cyclic AMP phosphodiesterase in the nervous system of the mollusc Pleurobranchaea.

Regulation of cyclic AMP through its synthesis is known to be important in modulating the activity of molluscan neurons; however, no data exists regarding the regulation of cyclic AMP degradation. We find that cyclic AMP phosphodiesterase (PDE) activity in homogenates of the nervous system of the mollusc Pleurobranchaea is significantly stimulated by calcium ion. Ca2+ stimulation is suppressed by the calmodulin antagonist trifluoperazine (TFP), indicating resemblance to the Ca2+-calmodulin PDEs of mammalian neurons. Ca2+ also accentuates the pH sensitivity of PDE. The qualities of Ca2+ and pH sensitivity of PDE are fitted into a model for cAMP regulation of neuronal activity in an identified feeding command neuron; the postulated role of PDE is consistent with effects of cAMP, TFP, and pH on the neuron's activity.