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J D MacMillan

Publications and source records attributed to J D MacMillan.

6 recordsLinked to original sources
PubMed1993

Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus.

Previous evidence suggests that hemolysin BL, which consists of a binding component, B, and two lytic components, L1 and L2, is the enterotoxin responsible for the diarrheal form of gastroenteritis caused by food-borne strains of Bacillus cereus. To prove that hemolysin BL and the enterotoxin are the same requires large amounts of these components free of other B. cereus proteins. For this purpose, we sought to clone the gene encoding the B component and to express it in Escherichia coli. A partial genomic library was constructed and a 29-base, 1,152-fold-degenerate oligonucleotide probe, designed from the N-terminal amino acid sequence of the B component, was used to identify recombinant clones containing the gene. Detection of gene products reactive with a monoclonal antibody specific for the B component and analysis of the nucleotide sequence confirmed that isolated clones, reactive with the oligonucleotide probe, did contain the gene encoding the B component. The protein, expressed in E. coli, apparently from the B. cereus promoter, produces a ring-shaped zone of hemolysis when combined with purified L components from B. cereus, a reaction typical of hemolysin BL. Northern (RNA) blot analysis of B. cereus RNA showed a large (5.1-kb) transcript which hybridized with a 500-bp probe internal to the B-component-coding sequence, suggesting that the hblA gene encoding the B component may be transcribed as part of a polycistronic message, possibly including the structural genes for the two lytic components. Higher levels of expression and disruption of the hblA gene are being pursued to resolve whether hemolysin BL is indeed the enterotoxin.

Amino Acid Sequence↗
PubMed1990

A novel bicomponent hemolysin from Bacillus cereus.

A procedure combining isoelectric focusing (Sephadex IEF) and fast protein liquid chromatography (Superose 12; Mono-Q) removed hemolytic activity (presumably a contaminant) from partially purified preparations of the multicomponent diarrheal enterotoxin produced by Bacillus cereus. However, when the separated fractions were recombined, hemolytic activity was restored, suggesting that hemolysis is a property of the enterotoxin components. Combined fractions exhibited a unique ring pattern in gel diffusion assays in blood agar. During diffusion of the hemolysin from an agar well, the erythrocytes closest to the well were not lysed initially. After diffusion, hemolysis was observed as a sharp ring beginning several millimeters away from the edge of the well. With time the cells closer to the well were also lysed. This novel hemolysin consists of a protein (component B) which binds to or alters cells, allowing subsequent lysis by a second protein (component L). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and Western blot analysis showed that hemolysin BL has properties similar to those described previously for the enterotoxin and that both components are distinct from cereolysin and cereolysin AB.

Antibodies, Monoclonal↗
PubMed1985

Production of monoclonal antibodies in culture.

Factors that affected the production of monoclonal antibodies by a mouse-mouse hybridoma cell line, propagated in vitro in stirred vessels, were investigated. The purpose of the research was to estimate the efficiency of this system for large scale production of monoclonal antibodies. The antibody produced by these hybridoma cells was an IgG2a, specific for a surface antigen on Rhizobium japonicum NR-7 cells. Antibody content in the culture supernatant was determined by a radial-immunodiffusion assay using rabbit anti-mouse IgG antibodies in the immobile phase and mouse IgG (the monoclonal antibody) as the antigen in the mobile phase. This method was found to be more reproducible and reliable compared with an ELISA method. Cells were adapted to grow in an inexpensive, low protein content medium based on Dulbecco's Modified Eagle Medium (DMEM) supplemented with 0.25% Primatone RL, 0.01% Pluronic polyol F-68 and fetal bovine serum as low as 1%. Doubling time for the cells averaged 24 hrs, and final yields reached 2 X 10(6) cells per ml. The hybridoma cells were grown in the newly developed medium in 3 liter fermentors. Monoclonal antibody was produced during the early growth phase (3 days), however, most of the antibody was produced during a later growth phase (3-10 days) when 30 to 90% of the cells were dead. Final antibody yields were estimated to be 100-200 micrograms/ml. A low level of dissolved oxygen (25% air saturation) in the culture was found to increase the amount of antibody produced as compared with cells propagated at 60% air saturation (the optimal level for cell propagation) since the cells were kept alive for longer periods at the lower dissolved oxygen concentration. Using a fed-batch propagation method we were able to keep cells alive for long periods (up to 1 month) at a concentration of about 1 X 10(6) cells per ml, and thus to increase further monoclonal antibody production. Yields of 300-400 micrograms/ml were obtained.

Animals↗
PubMed1984

Development of a monoclonal antibody capable of interacting with five serotypes of Staphylococcus aureus enterotoxin.

A monoclonal antibody capable of binding to determinants shared in common by staphylococcal enterotoxin serotypes A, B, C1, D, and E was developed. To accomplish this, BALB/c mice were immunized by alternating injections of serotypes A and D to enrich for spleen lymphocytes programmed to produce antibody to possible common determinants. These cells, fused with mutant myeloma cells (P3-X-63Ag8.653) produced hybrids that formed monoclonal antibodies to either serotype A only or to both serotypes A and D. A cloned hybridoma from the latter group produced an antibody (subclass immunoglobulin G1) which interacted with five serotypes. Highest affinity was to B and C1. An immunomatrix consisting of this antibody cross-linked to protein A-Sepharose CL-4B with dimethyl pimelimidate was capable of binding enterotoxin. Bound toxin was eluted with diethylamine. Because of its ability to interact with all five serotypes, the monoclonal antibody should prove useful in the development of a rapid method for screening foods for the presence of staphylococcal enterotoxin.

Animals↗
PubMed1982

Evaluation of glutaraldehyde and hydrogen peroxide for sanitizing packaging materials of medical devices in sterility testing.

External surfaces of packaging materials used for sterile medical devices may introduce contaminants into working areas used for sterility testing. Light wiping with tissues moistened with alkaline 2% glutaraldehyde (Cidex) or 3% hydrogen peroxide effectively reduced counts on 5 X 8 cm strips of packaging material (Tyvek) inoculated with 10(7) spores of Bacillus subtilis. The ability of antimicrobial agents to penetrate packaging material and kill contaminants on the medical device was tested by inoculating filter membranes with ca 100 cells of Pseudomonas aeruginosa or Staphylococcus aureus or ca 100 spores of Bacillus subtilis. A sterile square of test packaging material placed over the inoculated membrane (direct method) or 0.5 cm above the membrane (indirect method) was wiped with the antimicrobial agent. Except for polyethylene film (3 mil), all materials tested, including glassine and several types of coated and uncoated Tyvek, were penetrated by the agents, killing cells on the inoculated membranes. Death rates varied, depending on the organism, packaging material, and testing method. It is suggested that penetration tests be performed before using antimicrobial agents for sanitizing packaging materials during sterility tests.

Aldehydes↗