Relations between beta-lactamases and penicillin-binding proteins: beta-lactamase activity of penicillin-binding protein 5 from Escherichia coli.

The molecular mechanism of the hydrolysis of penicillin by penicillin-binding protein (PBP) 5 was investigated. Cysteine-115 had been previously implicated in the hydrolysis of the acyl-enzyme intermediate in PBP5. In studies with oligonucleotide-directed mutagenesis, the role of cysteine-115 in the mechanism of PBP5 was investigated by the mutation of this residue to either a serine or an alanine residue.

EMS response to a ski lift disaster in the Colorado mountains.

The EMS system in a Colorado mountain community was tested by the fall of a ski lift injuring 49 people. The response was complicated by the remote location of the accident and the number of injuries. Use of a preconceived disaster plan reduced morbidity and mortality.

Site-directed mutants of a soluble form of penicillin-binding protein 5 from Escherichia coli and their catalytic properties.

Soluble, truncated mutant and wild-type forms of penicillin-binding protein 5 (sPBP 5) from Escherichia coli were produced in large amounts by placing the dacA gene that encodes PBP 5 under the control of the trp-lac fusion promoter. The 3' end of the dacA gene used in this study contains a stop codon that results in the deletion of 15 amino acids from the carboxyl terminus and the production of a soluble protein. Using oligonucleotide-directed mutagenesis, the role of cysteine 115 in the mechanism of sPBP 5 was investigated.

An enzyme-linked immunosorbent assay for the detection of antibodies to avian reticuloendotheliosis virus using whole cell antigen.

An enzyme-linked immunosorbent assay (ELISA) using reticuloendotheliosis virus-infected chick embryo fibroblasts as coating antigen is described for the detection of antibodies to reticuloendotheliosis virus in chicken sera. The ELISA was specific and during the early stages of infection more sensitive than an indirect fluorescent antibody test.

Isolation of mutants of Candida glabrata resistant to miconazole.

Elucidation of the mode of action of azole antifungals would be aided by studying resistant mutants. It is difficult to obtain mutants of Candida albicans in the laboratory, and there have only been a few studies on clinical isolates which seem to be resistant because of impaired drug uptake. C.

Replication of avian encephalomyelitis virus in chick embryo neuroglial cell cultures. Brief report.

Avian encephalomyelitis virus replicated to relatively high titres in chick embryo neuroglial cell cultures, as measured by the indirect fluorescent antibody test, but induced few cytopathic effects. This cell system should provide a good source of virus for serological tests and vaccines.

Improved potency test for live avian encephalomyelitis vaccines.

Potency tests were carried out on live avian encephalomyelitis virus (AEV) vaccines. Vaccines were titrated in chick embryo brain cell cultures using an indirect fluorescent antibody test just before vaccination. Groups of chickens were inoculated orally with graded doses of each vaccine.

Drug resistance in the opportunistic pathogens Candida albicans and Candida glabrata.

There are three major classes of antifungal drug used to treat patients suffering from topical and systemic infections caused by Candida albicans. Both the polyene macrolide antibiotics and the synthetic imidazole derivatives interact with membranes of sensitive organisms causing an impairment of function and cessation of growth. It is possible to obtain mutants of C.

A comparison of titration methods for live avian encephalomyelitis virus vaccines.

Factors associated with the indirect fluorescent antibody test used for the titration of avian encephalomyelitis virus (AEV) in chick embryo brain cell cultures were examined for their influence on virus replication. It was found that virus should be inoculated onto semi-confluent cell cultures and adsorbed for two hours at room temperature. The cells should then be examined for fluorescence after five days' incubation.

Development of in vitro tests for detection of extraneous agents.

The use of animals in tests to detect extraneous agents is not only undesirable from the ethical viewpoint but also because of the expense and length of time involved in such tests. We have carried out tests on a variety of potential contaminating avian pathogens to determine whether tests in chicks offer any advantage over tests in embryos or cell cultures. In many cases, but not all, in vitro tests were shown to be more sensitive.

Detection of avian encephalomyelitis virus.

Methods for the detection of two strains of avian encephalomyelitis virus (AEV) in chick embryo brain cell cultures and chickens were compared. It was found that the agar gel precipitin test (AGPT) and the enzyme-linked immunosorbent assay (ELISA) carried out on the serum of inoculated chickens were more sensitive than either the indirect fluorescent antibody test in cell cultures or the detection of clinical signs in chicks. On the basis of results obtained in this experiment the effects were then determined of routes and time of inoculation of chickens on the detection of AEV.

Identification of the active site in penicillin-binding protein 3 of Escherichia coli.

We report the sequence of the active site tryptic peptide of penicillin-binding protein 3 from Escherichia coli. Purified penicillin-binding protein 3 was labeled with [14C]penicillin G and digested with trypsin, and the resulting radioactive peptides were isolated by a combination of gel filtration and high-pressure liquid chromatography. The major radioactive peak from high-pressure liquid chromatography was sequenced, and the peptide Thr-Ile-Thr-Asp-Val-Phe-Glu-Pro-Gly-Ser-Thr-Val-Lys, which comprises residues 298 to 310 in the amino acid sequence, was identified.

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 1b of Escherichia coli.

This paper reports the sequence of the active site peptide of penicillin-binding protein 1b from Escherichia coli. Purified penicillin-binding protein 1b was labeled with [14C]penicillin G, digested with trypsin, and partially purified by gel filtration. Upon further purification by high-pressure liquid chromatography, two radioactive peaks were observed, and the major peak, representing over 75% of the applied radioactivity, was submitted to amino acid analysis and sequencing.

Purification and sequencing of the active site tryptic peptide from penicillin-binding protein 5 from the dacA mutant strain of Escherichia coli (TMRL 1222).

The localization of the active site of penicillin-binding protein 5 from the dacA mutant of Escherichia coli strain TMRL 1222 has been determined. The protein was purified to homogeneity and labeled with [14C] penicillin G. The labeled protein was digested with trypsin, and the active site tryptic peptide was purified by a combination of gel filtration and high-pressure liquid chromatography.

Detection of antibodies against infectious bursal disease virus: a comparison of three serological methods.

Four different oil emulsion infectious bursal disease virus (IBDV) vaccines were inoculated into four-week-old specific pathogen-free chickens. At weekly intervals for five weeks, sera were obtained from the vaccinated birds and from uninoculated control birds and examined for antibodies against IBDV by enzyme-linked immunosorbent assay (ELISA), the quantitative agar gel precipitin (QAGP) test and the virus neutralisation (VN) test. There was a highly significant correlation between the mean responses to all tests; the highest correlation (0.

Detection of avian leukosis virus: comparison of five techniques.

Five techniques were compared for their ability to detect decreasing dilutions of RAV-I, an avian leukosis sarcoma virus, in serially passaged chick embryo fibroblast cell cultures. The indirect fluorescent antibody test, sandwich enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase assay were equally sensitive in detecting the virus. The indirect immunoperoxidase and complement fixation avian leukosis tests were less sensitive.