Cellular distribution of beta-lactamase RP4 is mediated by an outer membrane protease.

RP4 beta-lactamase extracted from the outer membrane of wild-type Escherichia coli can be resolved into several interconvertible forms that differ in their stabilities, substrate profiles and apparent molecular weights. beta-Lactamase isolated from outer membrane of strains which are lacking a protease that is involved in the cleavage of colicins differs from the beta-lactamase of parental cells in substrate profile, apparent molecular weight and the ability to interconvert. The cellular distribution of beta-lactamase also differs between wild-type and protease-deficient mutants. Both strains have equivalent amounts of beta-lactamase in their outer membranes, however the parental strain also has considerable beta-lactamase in the cytoplasmic membrane while the mutant does not. In addition the mutant contains only 30% of the parental level of enzyme in the periplasm. It is proposed that the reduced level of periplasmic enzyme is the result of a defect in processing of membrane-associated beta-lactamase. This conclusion is supported by the observation that the beta-lactamase isolated from the mutant can be converted to forms resembling those found in the parent by incubation with extracts or outer membrane isolated from the parent.

Precolicin E1, the major gene product of plasmid-ColE1 deoxyribonucleic acid in vitro.

Coupled transcription and translation of plasmid-ColE1 DNA in vitro under optimized conditions gave one major product. This has an apparent weight of 71 000, the same N-terminal sequence as colicin E1 and was not digested by deoxyribonuclease or ribonuclease. It differed from colicin E1 in its C-terminal residue and amino acid composition. It had lower specific activities in cell killing and in the fluorescence-enhancement in vitro assay of Phillips & Cramer [(1973) Biochemistry 12, 1170--1176] than did colicin E1, but both proteins bound in equimolar amounts to colicin-sensitive and colicin-resistant cells. The product of plasmid-ColE1-DNA-directed protein synthesis was converted into a protein indistinguishable in structure and activity from colicin E1 by incubation in the reaction mixture, after deoxyribonuclease and ribonuclease treatment, for a further 20 h at 37 degrees C. A protein with similar properties to the 71 000-dalton product in vitro was identified in extracts of a ColE1+ colicin-tolerant mutant of Escherichia coli K12. It is concluded that this protein probably represents a pre-form of colicin E1 which may be involved in colicin-E1 secretion or cellular colicin-E1 immunity in colicin-E-producing cells, or both of these processes.

Toxic fungal metabolites in food.

About 100 fungal metabolites may cause cancer, embryological defects, or other histopathological effects in mammals. They are produced by a wide variety of fungi. Few of these metabolites have significant acute toxicity. With the exception of aflatoxin B1 and sterigmatocystin, there is no conclusive evidence that any of them is carcinogenic. However, several of the compounds are mutagenic. Cytochalasin D and T-2 toxin are probably teratogenic. A wide variety of other histopathological effects have been shown. Liver damage has been most frequently reported. In almost all cases the molecular bases of these effects have not been extensively investigated. Although much is known about the routes by which some of the compounds are synthesized in vivo, nothing is known about control at the molecular level of these biosynthetic routes. Little is known about the biological degradation of these compounds or about the levels and incidences of them in food and animal feed. Future work in all these areas will depend on the further development of sensitive assay methods that are applicable to their measurement in food, in animal feed, and in animal tissues and body fluids and on the application of these methods to define exposure to these compounds in the diet.

An Assessment of Food Contamination by Toxic Products of Alternaria.

Invasion of food crops by the common mold Alternaria has been widely observed. Consequent contamination of food by five toxic Alternaria metabolities, i.e., tenuazonic acid, alternariol and its monomethyl ether, altenuene and altertoxin I, has been demonstrated in a few instances. These compounds have been found together in several food samples and they may have synergistic toxic effects. Suggestions for further work on these synergistic effects and on the effects of long-term feeding of the individual compounds to mammals are made. It is also suggested that more extensive surveys of food for these compounds are needed to establish the likely intakes of these compounds by man.

Protein purification using immobilised triazine dyes.

This review attempts to identify proteins which selectively interact with immobilised triazine dyes such as Cibacron blue F3GA and Procion red HE 3B. Different support matrices are compared by examining the capacities of these dyes for proteins. Various approaches to the immobilisation of triazine dyes are considered together with the use of spacers. Some theories of the mechanism of protein retardation by immobilised dyes are discussed. A number of methods are suggested for the measurement of dye concentrations and for the modification of the binding of proteins to dye columns. The variety of elution methods is compared with a view to optimizing purifications. The scope of applications is reviewed as well as the choice of dye. Some advantages of triazine dyes over other affinity ligands are given. It is concluded that although no satisfactory mechanism for the binding of triazine dyes to proteins has yet been proposed, these dyes possess considerable potential for protein purification, particularly when applied on the large scale.

Carcinogenic higher plant metabolites in the human diet in temperate countries: a review.

Interest has increased recently in the possible human carcinogenic effects of plant metabolites in the diet. Some psoralens, some pyrrolizidine alkaloids and 'bracken carcinogen' may be dietary carcinogens, the latter two via contamination of food products from animals ingesting toxic plants. With the exception of 'bracken carcinogen', the structures of these compounds have been established. Further research to quantify the levels of these compounds in foodstuffs and to correlate these with the amounts of the compounds having toxic effects in laboratory animals would be valuable. Epidemiological studies on critical-intake consumer groups would also be of value in defining the hazard, if any, the presence of these compounds in the diet poses to the consumer.

Cervical headache: an investigation of natural head posture and upper cervical flexor muscle performance.

In this study, 60 female subjects, aged between 25 and 40 years, were divided into two equal groups on the basis of absence or presence of headache. A passive accessory intervertebral mobility (PAIVM) examination was performed to confirm an upper cervical articular cause of the subjects' headache and a questionnaire was used to establish a profile of the headache population. Measurements of cranio-cervical posture and isometric strength and endurance of the upper cervical flexor muscles were compared between the two groups of subjects. The headache group was found to be significantly different from the non-headache group in respect to forward head posture (FHP) (t = -5.98, p < 0.00005), less isometric strength (t = 3.43, p < 0.001) and less endurance (t = 8.71, p < 0.0005) of the upper cervical flexors. A statistically significant relationship was also established between natural head posture and isometric endurance of the upper cervical flexor musculature which demonstrated that FHP corresponded with a low endurance capacity (chi 2 = 13.2; p < 0.01). The outcome of this study highlights the need to screen for cervical etiology in patients who are suspected of suffering from common migraine.

Herpes simplex virus resistance and sensitivity to phosphonoacetic acid.

Phosphonoacetic acid (PAA) inhibited the synthesis of herpes simplex virus DNA in infected cells and the activity of the virus-specific DNA polymerase in vitro. In the presence of concentrations of PAA sufficient to prevent virus growth and virus DNA synthesis, normal amounts of early virus proteins (alpha- and beta-groups) were made, but late virus proteins (gamma-group) were reduced to less than 15% of amounts made in untreated infected cells. This residual PAA-insensitive synthesis of gamma-polypeptides occurred early in the virus growth cycle when rates were identical in PAA-treated and untreated infected cells. Passage of virus in the presence of PAA resulted in selection of mutants resistant to the drug. Stable clones of mutant viruses with a range of drug sensitivities were isolated and the emergence of variants resistant to high concentrations of PAA involved the sequential selection of mutants progressively better adapted to growth in the presence of the drug. Increased drug resistance of virus yield or plaque formation was correlated with increased resistance of virus DNA synthesis, gamma-protein synthesis, and resistance of the virus DNA polymerase reaction in vitro to the inhibitory effects of the drug. PAA-resistant strains of herpes simplex virus type 1 (HSV-1) complemented the growth of sensitive strains of homologous and heterologous types in mixed infections in the presence of the drug. Complementation was markedly dependent upon the proportions of the resistant and sensitive partners participating in the mixed infection. Intratypic (HSV-1A X HSV-1B) recombination of the PAA resistance marker(s), Pr, occurred at high frequency relative to plaque morphology (syn) and bromodeoxyuridine resistance (Br, thymidine kinase-negative phenotype) markers, with the most likely order being syn-Br-Pr. Recombinant viruses were as resistant or sensitive to PAA as the parental viruses, and viruses recombinant for their PAA resistance phenotype were also recombinant for the PAA resistance character of the virus DNA polymerase. The results provide additional evidence that the herpesvirus DNA polymerase is the site of action of PAA and illustrate the potential usefulness of PAA-resistant mutants in genetic studies of herpesviruses.

Affinity chromatography of the Neurospora NADP-specific glutamate dehydrogenase, its mutational variants and hybrid hexamers.

The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-Sepharose 4B, is described. The assembly of the 2'-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2'-AMP and its derivatives, 8-bromoadenosine 2'-phosphate and 8-(l-aminohexyl)aminoadenosine 2'-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2'-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure from pairs of purified variants. Several chromatographically distinct peaks of differing enzymological properties were purified from each hybridization mixture in quantities of up to a few milligrams, and represented distinct species of hybrid hexamers differing in subunit ratio.

Subunit ratios of separated hybrid hexamers of Neurospora NADP-specific glutamate dehydrogenase containing complementing mutationally modified monomers.

The am1 and am3 mutational variants of the Neurospora crassa NADP-specific glutamate dehydrogenase show complementation activity in hybrid hexamers. A freeze-thaw hybridization method was used to construct hybrids from purified enzymes and the products were separated into species of different monomer ratio by affinity chromatography. Hexamers with am1:am3 ratios of 1:5, 2:4, 3:3, 4:2 and 5:1 were all recovered as resolved or partially resolved peaks in quantities approximating to a binomial distribution. Reassociation of monomers during the hybridization process was random, except for some differential loss of am3 protein by precipitation and an apparent absence of reassociated am1 homohexamers. Complementation activity was shown by hybrids of all five monomer ratios, owing to activation of am3 monomers by conformational constraints arising from the intrinsically inactive am1 monomers. The activating effect of such constraints was greatest in hexamers containing only a single am1 monomer and least in the 5 am1:1am3 species. When fully activated by L-glutamate all am3 monomers were equivalent in intrinsic catalytic activity, irrespective of the number of am1 monomers per hexamer.

Some structural antigens of herpes simplex virus type 1.

Several of the major structural polypeptides of herpes simplex virus were obtained in purified form by polyacrylamide gel electrophoresis of purified virus particle polypeptides. Antisera made by footpad inoculation of these polypeptides into rabbits were used to study the antigenic properties of two envelope glycoproteins and of the major capsid protein.

Glycoproteins with type common and type specific antigenic sites excreted from cells infected with herpes simplex virus types 1 and 2.

Agar immunodiffusion tests demonstrated that BHK 21 cells infected with either HSV I or HSV 2 release only a few HSV-specified antigens into the extracellular fluid (infected cell released polypeptides-ICRP). Neutralization blocking experiments showed that the majority of antigens/(including the Band II common antigen) involved as target sites in antibody-mediated virus neutralization are present in the ICRP of both HSV I and HSV 2.SDS-PAGE identified six regions of virus-specified proteins in the ICRP from both HSV I- and HSV 2-infected BHK cells. All these specifically released proteins are glycosylated, although to varying degrees. The SDS-PAGE profiles of HSV I and HSV 2 ICRP are different but do show some similarities, the most notable being a highly glycosylated protein with an estimated mol. wt. of 50,000 to 54,000 in HSV I ICRP and 52,000 to 56,000 in HSV 2 ICRP. Immune precipitation demonstrated that these two proteins contain the Band II antigenic site. Similar studies showed that the major type I specific antigenic site, which is involved as a target site in the neutralization of virus infectivity, is located in the highest mol. wt. glycoprotein region of HSV I ICRP and has a similar mobility to the VP7/8 region of purified enveloped virus.

The selective retardation of NADP+-dependent dehydrogenases by immobilized procion red HE-3B.

The capacities of Procion Red HE-3B and Cibacron Blue F3G-A immobilized to Sepharose CL-4B and Matrex 201R for NAD+-, NADP+- and NAD(P)+-dependent dehydrogenases were measured. Procion Red HE-3B columns retarded NADP+-dependent dehydrogenases more effectively than NAD+-dependent dehydrogenases, whilst immobilized Cibacron Blue F3G-A retarded NAD+-dependent dehydrogenases more effectively than NADP+-dependent dehydrogenases. The capacity of procion Red HE-3B-Sepharose CL-4B for five dehydrogenases was highest in the region of 70nmol of immobilized ligand/ml of settled gel. The effects of using poly(ethyleneimine) as a spacer for both porous and pellicular supports were also examined. Four NADP+-dependent dehydrogenases were purified from yeast extract by using Procion Red HE-3B-Sepharose CL-4B. Two NAD+-dependent dehydrogenases were purified from the same source using Cibacron Blue F3G-A-Sepharose CL-4B. These results are discussed in relation to the use of immobilized Procion Red HE-3B to purify dehydrogenases. This immobilized dye's chromatograhic behaviour is compared with that of immobilized nucleotides. The most important feature of immobilized tirazine dyes seems to be their high operational capacities when compared with group-specific nucleotide adsorbents.

A note on inhibition test and electrophoretic detection limits of antibiotics used in British animal husbandry.

A four plate microbiological inhibition test (the FPT) and a bioelectrophoretic method were evaluated for their ability to detect a range of antibiotic agents, which may be present as residues in animal tissues following their therapeutic use in animal husbandry. Both methods exhibited a wide range of sensitivities and several of the tested antibiotics could not be detected by either method. The pattern of responses across the bacterial plates in the FPT could not be used to identify agents and the bioelectrophoretic inhibition zone diameters were generally too large to allow the use of Rs values for identification. The Bacillus subtilis pH 7.2 plate with trimethoprim added was as effective as the four bacterial plates used in the FPT in antibiotic detection.

Recombination and linkage between structural and regulatory genes of herpes simplex virus type 1: study of the functional organization of the genome.

Phenotypic and genetic properties of 12 markers in structural and regulatory functions of herpes simplex virus type 1 were characterized, and their recombination and segregation behavior was investigated and interpreted with reference to available information on their physical locations. The markers were: (i) ts markers in a structural glycoprotein (tsB5) and in alpha (immediate early; tsLB2, tsc75) or beta (early, delayed early; tsB1) functions with regulatory effects; together with (ii) plaque morphology (syn), phosphonoacetate resistance (Pr), and thymidine kinase (TK) phenotypes; and (iii) electrophoretically distinct variants of glycosylated (glycoprotein C, gpC; ICP10) and non-glycosylated [VP(13-14), VP23] structural and nonstructural [ICP(47-48)] polypeptides. Mean two-factor recombination frequencies ranged from 2% (for noncomplementing mutants tsLB2 and tsc75) to 35 to 40% (for unlinked markers) and were influenced by the relative contributions of parental viruses to the mixed infection. Even with control of this variable, standard deviations of mean measures of recombination frequency ranged from a minimum of 14% (with n greater than or equal to 10) to 65% (with n = 3) of mean values; no recombination frequencies higher than 55% were observed. Differences in mean two-factor recombination frequencies between a small number of loosely linked markers were, therefore, not reliable measures of real differences in linkage. Measurements of the segregation of unselected markers among recombinant progeny were, therefore, used as measures of linkage. These experiments (i) established a linkage group for markers in the long unique region of the genome additional to, but consistent with, existing physical data, i.e., TK-syn-tsB5-(tsB1.Pr)-[gpC.VP(13-14)]; (II) identified markers, e.g., ICP(47-48), linked to regulatory mutations (tsLB2, tsc75) in redundant DNA sequences; and (iii) used the segregation of these regulatory mutations and linked markers among unselected progeny to demonstrate the linkage groups: Pr-syn-TK-tsc75-ICP(47-48), [VP(13-14).gpC]-Pr-syn-TK, and TK-tsc75-[VP(13-14).gpC]. These results were most simply explained if bi- or intermolecular recombination occurred between circular molecules or molecules catenated "head-to-tail" and were incompatible with intermolecular recombination as the mechanism of isomerization of herpes simplex virus DNA.

Surveillance of potentially hazardous chemicals in food in the United Kingdom.

Surveillance of chemical contaminants in food plays an important role in helping to ensure a safe food supply in those countries that undertake it. This paper reviews the methods used in the UK as a means of highlighting the essential elements required by any food chemical surveillance programme. The following topics have been covered: quantifying food consumption, setting priorities in food surveillance, developing a common approach to the surveillance of different chemicals in the food supply (including the use of Total and Duplicate Diet Studies), estimating human intakes of chemicals from the diet, developing suitably sensitive and reliable methods of analysis, obtaining representative samples, and assessing and managing risk.

Some properties of recombinants between type 1 and type 2 herpes simplex viruses.

Four intertypic recombinants of herpes simplex virus have been shown to possess genetic information for functions characteristic of each of the two parental types. The functions were identified by (a) polyacrylamide gel electrophoresis of purified virus particles and of polypeptides synthesized in cells infected with the recombinants and (b) analysis of antigenic sites interacting with type specific neutralizing antibody. The analysis shows that each recombinant possess a different combination of these type specific markers. Finally we have been unable to detect recombination between herpes simplex type 1 and pseudorabies viruses.

Serological study of a mutant of herpesvirus unable to stimulate thymidine kinase.

A mutant of herpes simplex virus which was unable to produce thymidine kinase also failed to produce an antigen which blocked the enzyme-inhibiting activity of antiserum prepared against virus-infected cells.

Purification of herpes simplex virus glycoproteins B and C using monoclonal antibodies and their ability to protect mice against lethal challenge.

Monoclonal antibodies which react with herpes simplex virus type 1 (HSV-1) glycoproteins, but do not neutralize infectivity, were prepared. The protein to which each monoclonal antibody was directed was determined by various techniques including their reaction with polypeptides from glycoprotein-deficient mutants after protein blotting, and also tests using passive haemagglutination. Monoclonal antibodies A7 and alpha C3 were directed against a type-specific determinant on gC and a type-common determinant on gB respectively. In addition, A7 reacted only with the HFEM strain of HSV-1 and did not react with any of the 20 low-passage human isolates also tested. The monoclonal antibodies were used in immunoadsorption chromatography to purify individual glycoproteins from detergent extracts of HSV-1-infected cells. The ability of the monoclonal antibodies or purified glycoproteins to protect mice against a lethal encephalitis induced by intracerebral inoculation of HSV-1 was investigated. Passive immunization was not very effective; however, purified gC or a mixture of gB and its precursor pgB induced good levels of neutralizing antibody which persisted for at least 9 weeks and mice survived virus challenge.