摘要:

Of 33 streptococcal cultures belonging to serological group L, all bound human immunoglobulin (Ig) G, fibrinogen, and fibronectin; 32 bound bovine IgG; 31 bound α2-macroglobulin; 5 bound albumin; and none bound either haptoglobin or IgA. The binding sites for IgG could be isolated from the L streptococci by trypsinization and purified by affinity chromatography on human IgG – Sepharose®. The purified Fc receptors reacted with IgG subclasses 1, 2, 3, 4 of humans, 1 and 2 of bovines, ovines, and caprines as well as a, b, c, and T of equines. They had a molecular mass of approximately 49 000 Da. Thus, the Fc receptors from L streptococci corresponded to type III Fc receptors ofStreptococcus dysgalactiae.

ISSN:0008-4166    

DOI:10.1139/m88-001

出版商:NRC Research Press    

年代:1988

数据来源: NRC

摘要:

Monoclonal antibodies against a mycoplasmalike organism that causes maize bushy stunt disease were produced by using partially purified preparations from infected corn plants as immunogen. Splenic cells from immunized mice were fused with P3/NS-1/1-Ag4-1 murine myeloma cells. Four stable hybridoma clones that secreted specific antibodies against the maize bushy stunt mycoplasmalike organism were selected from several thousand clones. The monoclonal antibodies were isotyped and determined to belong to immunoglobulin classes IgM and IgG2a. With these monoclonal antibodies, the maize bushy stunt mycoplasmalike organism was specifically identified from infected corn tissue by enzyme-linked immunosorbent assayin vitroand by immunofluorescent stainingin situ. Mycoplasmalike organisms associated with aster yellows (New Jersey strain), clover phyllody, eastern X disease, elm yellows, paulownia witches'-broom, and sweet potato witches'-broom were differentiated from maize bushy stunt agent by serology using these monoclonal antibodies. Monoclonal antibodies specific to maize bushy stunt mycoplasmalike organism also did not react with two plant pathogenic spiroplasmas:Spiroplasma kunkeliiandSpiroplasma citri.

ISSN:0008-4166    

DOI:10.1139/m88-002

出版商:NRC Research Press    

年代:1988

数据来源: NRC

摘要:

Amikacin, an aminoglycoside known to inhibit protein synthesis, was found to perturb the outer membrane of a sensitivePseudomonas aeruginosastrain (ATCC 9027). This perturbation was monitored using electron microscopy and biochemical analyses. Following exposure to 20 μg amikacin/mL for 15 min, the outer membrane of exponentially growing cells lost 15% of its protein, 18% of its lipopolysaccharide, and 18% of its phosphate. Sodium dodecyl sulphate – polyacrylamide gel electrophoresis showed that the whole spectrum of outer membrane protein and lipopolysaccharide was affected. Similarly, atomic absorption spectrophotometry revealed that magnesium and calcium were also lost. When cells were treated with amikacin, electron microscopy of negative stains showed a substantial increase in outer membrane blebbing. Freeze fractures revealed changes in membrane fracture pattern and particle distribution, and thin sections revealed a sequential disruption of the cell envelope beginning at the outer membrane and ending at the plasma membrane. This study supports the proposal that aminoglycoside antibiotics cross the outer membrane ofPseudomonas aeruginosaby displacing metal cations necessary to stabilize the organic constituents of the membrane. Their removal results in loss of the outer membrane and the formation of transient small holes which permit the antibiotic access to the cytoplasmic membrane where it is transported into the cytoplasm.

ISSN:0008-4166    

DOI:10.1139/m88-003

出版商:NRC Research Press    

年代:1988

数据来源: NRC

摘要:

Two strains of street rabies virus from striped skunks (Mephitis mephitis) were used to infect either a murine neuroblastoma (NA 1300) or a baby hamster kidney (BHK-21/C13) cell culture and the cell infection rates were noted during 4 days postinfection. These cultures were then passaged for four consecutive passages, and the viruses obtained in the supernatant fluids of passage 4 were then treated as original isolates and used to infect both neuroblastoma and baby hamster kidney cells. The mortality period in Swiss white mice caused by the various virus suspensions was noted. The virus strain from the brain of skunks from Saskatchewan infected neuroblastoma and baby hamster kidney cells equally well, produced similar virus titres in supernatant fluids after four subcultures in both cell types, and appeared to produce similar mortality periods in mice from either the original brain tissue or from cell culture supernatant fluids. On the other hand, the virus from the brains of skunks from Ontario readily infected neuroblastoma but poorly infected baby hamster kidney cell cultures. Passage of this strain through four subcultures in both cell types produced virus titres in the supernatant fluids of equal magnitude. However, reisolation of the virus from the supernatant fluid of passage 4 in neuroblastoma cell cultures showed a similar pattern to that from the original brain, while the virus from baby hamster kidney cell passage supernatant fluid was considerably altered. Although the mortality period in mice was similar with virus from the brain and neuroblastoma cell cultures, this period was shortened when mice were inoculated with baby hamster kidney culture supernatant virus. Virus from the salivary glands of Ontario skunks readily infected both cell types, producing similar titres at 4 days postinfection. The mortality period of mice inoculated with salivary gland suspensions was shorter than of those inoculated with brain suspensions. These findings demonstrate differences in rabies street virus strains that may have affected diagnostic procedures.

ISSN:0008-4166    

DOI:10.1139/m88-004

出版商:NRC Research Press    

年代:1988

数据来源: NRC

摘要:

A procedure is described for the isolation of mutants ofAzospirillum brasilensestrains Sp7 (ATCC 29145) and Sp245 andAzospirillum lipoferumBr1702, which are unable to differentiate from vegetative vibrioids into encapsulated C forms. It is based on the characteristic of mutants, designated Enc−, to develop into white colonies in a background of wild-type red colonies (Enc+) on the surface of Congo Red plates. This behavior was ascribed to an inability of Enc−mutants to synthesize one or more as yet unidentified polysaccharides. The frequency of mutation from Enc+to Enc−was a function of the particular strain and of the culture conditions employed. Enc−mutants fromA.brasilenseandA.lipoferumwere at least as efficient as wild-type ancestors in adhering to and causing deformations of root hairs of germinated wheat seedlings.

ISSN:0008-4166    

DOI:10.1139/m88-005

出版商:NRC Research Press    

年代:1988

数据来源: NRC

摘要:

In continuing studies of patulin biosynthesis, the first enzyme of the pathway, 6-methylsalicylic acid synthetase, was found to be far more labile than were the later enzymes of the pathway. Attempts were made to stabilize 6-methylsalicylic acid synthetasein vitro. The combined addition of the cofactor NADPH, the substrates acetyl-CoA and malonyl-CoA, the reducing agent dithiothreitol, and the proteinase inhibitor phenylmethylsulfonyl fluoride to cell-free extracts was found to prolong the half-life of the enzyme as much as 12-fold. This suggested that proteolysis and the conformational integrity of the enzyme may play an important role in controlling the duration of antibiotic biosynthesisin vivo. This was in agreement with the finding that the intracellular proteinase content of antibiotic-producing cells ofPenicillium urticaerapidly increased just before the loss of 6-methylsalicylic acid synthetase content. Thesein vitrostabilization studies have provided some insight into the metabolic conditions that may stabilize these enzymesin vivo, and into possible ways of extending the life of these catalysts.

ISSN:0008-4166    

DOI:10.1139/m88-006

出版商:NRC Research Press    

年代:1988

数据来源: NRC

摘要:

Mutually antagonistic K1 and K2 killer strains compete when mixed and serially subcultured. At pH 4.6, where the K1 killer toxin is more stablein vitro, the K1 strain outcompeted the K2 strains at both 18 and 30 °C. At pH 4.0, closer to thein vitropH optimum of the K2 killer toxin, the K1 strain again predominated at 18 °C, but at 30 °C the K2 strains became the sole cell type on subculture. To show more clearly that these results were dependent upon the respective killer toxins, control experiments were conducted with isogenic, nonkiller strains cured of the dsRNA-based killer virions. Such nonkiller strains were unable to compete with antagonistic killers under conditions where their isogenic killer parents could, strongly suggesting that the killer phenotype was important in these competitions. Double K1–K2 killer strains cannot stably exist, as their dsRNA genomes compete at a replicative level. Using recombinant DNA methodology, a stable K1–K2 killer strain was constructed. This strain outcompeted both K1 and K2 killers when serially subcultured under conditions where either the K1 or the K2 strains would normally predominate in mixed cultures. Such a double killer may be useful in commercial fermentations, where there is a risk of contamination by killer yeasts.

ISSN:0008-4166    

DOI:10.1139/m88-007

出版商:NRC Research Press    

年代:1988

数据来源: NRC

摘要:

A defined medium, which allowed growth in both stationary and shaken cultures in the absence of fetal calf serum, was developed for protoplasts of the fungusEntomophaga aulicae. The protein "requirement" was obviated and growth was enhanced by the addition of hematin (0.5 μg/mL) and oleic acid (1.0 μg/mL). The utilization and production of ninhydrin-positive compounds was monitored at various stages of medium simplification. The protoplasts were autotrophic for all vitamins and organic acids. Fructose was not required for protoplast growth; however, it could function as a carbon source. The optimum nitrogen level was 635.08 mg N/L, and the optimum carbon level was 280 mg C/L under the conditions utilized. The protoplasts did not utilize either inorganic nitrogen (ammonium ion) or sulfur (sulfate ion) at the levels tested. No amino acid could function as a sole nitrogen source. Analysis of the amino acid composition of a general (> 14 000 molecular weight) protein extract gave no evidence of a skewed amino acid content. The final simplified medium included 8 amino acids (aspartic acid, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, and threonine) of the 21 in the original medium. This medium is useful in isolating vitamin auxotrophs for use as markers in virulence and in protoplast fusion studies, and in studies on the chemical nature of the limiting membrane and its outer surface.

ISSN:0008-4166    

DOI:10.1139/m88-008

出版商:NRC Research Press    

年代:1988

数据来源: NRC

摘要:

Antisera were prepared in rabbits against formalized and heat-killed bacteria ofYersinia enterocoliticaserotype O:5,27 and against formalized bacteria of serotype O:8. Both strains used for immunization demonstrated adhesion to and invasion of HeLa cells. Coating of the bacteria with antibody did not greatly alter adhesion (i.e., extracellular attachment) to HeLa cells; however, antibody against formalized bacteria of both serotypes inhibited HeLa cell invasion by the homologous and heterologous strains. The Fab fragments from purified immunoglobulins also demonstrated cross-reacting inhibition of HeLa cell invasion. Antibody against heat-killed bacteria of serotype O:5,27 had no inhibitory activity. Adsorption of the antiserum against formalized bacteria of serotype O:5,27 with lipopolysaccharide from the homologous strain removed anti-lipopolysaccharide antibody but did not remove the inhibitory activity. The antiserum against formalized bacteria of serotype O:8 showed no antibody against lipopolysaccharide from serotype O:5,27 and no agglutinins against heat-killed bacteria of this strain. From these results, it is tentatively suggested that protein structures are important in mediating epithelial cell invasion byY.enterocolitica.

ISSN:0008-4166    

DOI:10.1139/m88-009

出版商:NRC Research Press    

年代:1988

数据来源: NRC

摘要:

It is well established that plasmids are involved in the expression of lipopolysaccharide in certain species ofShigella. InShigella sonnei, both the biosynthesis of oligosaccharide side chains (O antigen), and cell invasiveness are controlled exclusively by a 120 megadalton (MDa) plasmid. InShigella dysenteriae1, a 10 kilobase (kb) plasmid is required for O-antigen production.Shigella dysenteriae1 strains devoid of this plasmid lose the ability to synthesize O antigen. Interestingly, this 10-kb plasmid is not stably maintained inEscherichia coliK-12 strains, where it is lost spontaneously at a high frequency. Our genetic analyses ofShigella dysenteriae1 strain IDBM11 and its derivatives indicate that the stability of this plasmid is associated with the histidine region of the chromosome which is unique toShigella dysenteriae. Furthermore, the 10-kb plasmid is stably maintained in wild-type IDBM11 with an intact histidine locus. However, this plasmid is not stable in IDBM11 derivatives (e.g., IDBM11-1 and IDBM11-2), in which thehislocus has been substituted with the histidine region of anE.coliK-12 chromosome. TheS.dysenteriaeIDBM11 strain, and its derivatives (lacking a 10-kb plasmid), displayed an invasive property as demonstrated by their internalization by HeLa cells in anin vitroassay. Thus the 10-kb plasmid ofShigella dysenteriae1 is required for O-antigen synthesis but not for cell invasion.

ISSN:0008-4166    

DOI:10.1139/m88-010

出版商:NRC Research Press    

年代:1988

数据来源: NRC