Charm Safe-Level beta-Lactam Test for amoxicillin, ampicillin, ceftiofur, cephapirin, and penicillin G in raw commingled milk.

The Charm Safe-Level beta-Lactam Test was evaluated by a U.S. Food and Drug Administration (FDA) test protocol administered by the AOAC-Research Institute. The sensitivity and selectivity of the test were evaluated with >800 negative raw commingled and drug-fortified milk samples by the manufacturer and an independent laboratory. Probit analysis by the independent laboratory determined the following 90% positive levels with 95% confidence: amoxicillin, 5.6 ppb; ampicillin, 8.5 ppb; cephapirin, 13.7 ppb; ceftiofur, 46.2 ppb; and penicillin G, 3.6 ppb. These values were within a range of +/- 20% of the manufacturer's data. Selection of negative samples met confidence specifications. Ruggedness parameters were studied and defined, and the stability of frozen milk was verified. There were no interferences from somatic cells (1,000,000 somatic cell count/mL) or bacteria (300,000 colony-forming units/mL), or from 27 other non-beta-lactam animal drugs. Test performance with raw milk samples containing incurred penicillin, ampicillin, and amoxicillin was consistent with the dose responses determined with fortified milk samples. Incurred cephalosporin in raw milk samples was detected at lower levels than was cephalosporin in fortified milk samples, presumably because of the presence of metabolite, as verified by other test methods. Quality control data support consistency in manufacture between batches and the stability of refrigerated test reagents for up to 1 year. Successful fulfillment of these criteria led to FDA certification of the test when used with a reader in U.S. milk testing programs.

Genetic Transformation of the Biocontrol Fungus Gliocladium virens to Benomyl Resistance.

Methodology was developed to isolate and regenerate protoplasts from the biocontrol fungus Gliocladium virens and to transform them to benomyl resistance with a Neurospora crassa beta-tubulin gene. Southern blots demonstrated that multiple copies of the vector integrated into the chromosomal DNA of stable biotypes but not of abortive transformants. Analysis of nuclear condition in vegetative and asexual structures demonstrated that no structure of G. virens is dependably uninucleate and thus preferentially suitable for transformation.

Genetic separation of Escherichia coli recA functions for SOS mutagenesis and repressor cleavage.

Evidence is presented that recA functions which promote the SOS functions of mutagenesis, LexA protein proteolysis, and lambda cI repressor proteolysis are each genetically separable from the others. This separation was observed in recombination-proficient recA mutants and rec+ (F' recA56) heterodiploids. recA430, recA433, and recA435 mutants and recA+ (F' recA56) heterodiploids were inducible for only one or two of the three functions and defective for mutagenesis. recA80 and recA432 mutants were constitutively activated for two of the three functions in that these mutants did not have to be induced to express the functions. We propose that binding of RecA protein to damaged DNA and subsequent interaction with small inducer molecules gives rise to conformational changes in RecA protein. These changes promote surface-surface interactions with other target proteins, such as cI and LexA proteins. By this model, the recA mutants are likely to have incorrect amino acids substituted as sites in the RecA protein structure which affect surface regions required for protein-protein interactions. The constitutively activated mutants could likewise insert altered amino acids at sites in RecA which are involved in the activation of RecA protein by binding small molecules or polynucleotides which metabolically regulate RecA protein.

Mutations in uvrD induce the SOS response in Escherichia coli.

We have isolated three new mutations in uvrD that increase expression of the Escherichia coli SOS response in the absence of DNA damage. Like other uvrD (DNA helicase II) mutants, these strains are sensitive to UV irradiation and have high spontaneous mutation frequencies. Complementation studies with uvrD+ showed that UV sensitivity and spontaneous mutator activity were recessive in these new mutants. The SOS-induction phenotype, however, was not completely complemented, which indicated that the mutant proteins were functioning in some capacity. The viability of one of the mutants in combination with rep-5 suggests that the protein is functional in DNA replication. We suggest that these mutant proteins are deficient in DNA repair activities (since UV sensitivity is complemented) but are able to participate in DNA replication. We believe that defective DNA replication in these mutants increases SOS expression.

The Escherichia coli K-12 lexA2 gene encodes a hypocleavable repressor.

LexA2 repressor was partially inactivated after mitomycin C or UV light treatment in a recA+ or recA85(Prtc) (protease constitutive) host background. LexA2 protein was cleaved, but the reaction was slower than that observed for LexA+ repressor. lexA2 had a C-to-T transition at nucleotide 461 (Thr-154 to Ile).

Benzo[a]pyrene diol epoxide induces viral reactivation at concentrations that block DNA elongation in mammalian cells.

The survival of UV-irradiated Simian virus 40 (SV40) in CV-1P African green monkey kidney cells treated with (+/-)7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP-diol epoxide I) was studied. Enhanced survival of UV damaged SV40 was detected when CV-1P cells were treated with dose levels of BP-diol epoxide I corresponding to the exponential portion (0.33-1.11 microM) of a CV-1P cell survival curve. Dose levels of BP-diol epoxide I corresponding to the shoulder region (less than or equal to 0.16 microM) of a CV-1P survival curve did not induce viral reactivation. The shoulder region concentrations of BP-diol epoxide I selectively inhibited DNA initiation while the concentrations on the exponential portion of the curve preferentially inhibited DNA elongation. It was shown in a time course of enhanced viral survival at 0.66 microM BP-diol epoxide I that the reactivation response was fully induced by 24 h. In conclusion, the viral reactivation response was associated with concentrations of BP-diol epoxide I which induced lethal damage and preferentially inhibited DNA elongation.

Concentration dependent alterations of DNA replication initiation and elongation by benzo[a]pyrene diol epoxide.

Vero cells treated with various concentrations of (+/-)7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy, 7,8,9,10-dihydrobenzo[a]pyrene (BP-diol epoxide I) exhibited dose-dependent inhibition in both the rate of DNA synthesis and in the size of nascent DNA. The maximum inhibition was seen 2--3 h after addition of BP-diol epoxide I. A recovery in both the rate of synthesis and size of nascent DNA was observed 5--10 h after treatment. The pH step alkaline elution assay which separates different nascent DNA replication intermediates was used to investigate whether the inhibition and recovery noted above could be accounted for by alterations in DNA replication initiation (DNA synthesis within a replicon) or elongation (rejoining of replicons). At lowest dose studied (0.166 muM BP-diol epoxide I) a reversible inhibition in DNA initiation was observed. At the higher dose levels (0.66 muM and 1.66 muM BP-diol epoxide I) inhibition of both DNA initiation and elongation were observed and inhibition of elongation predominated. The inhibition in elongation was detected by an increase in the relative amount of low molecular weight nascent DNA associated with DNA synthesis within a replicon and a relative decrease in the higher molecular weight elongated DNA. A reversal in the inhibition of both initiation and elongation was noted.