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A A Weiss

Publications and source records attributed to A A Weiss.

At least 55 records · Page 3Linked to original sources
PubMed1989

Use of the promoter fusion transposon Tn5 lac to identify mutations in Bordetella pertussis vir-regulated genes.

Mutants of Bordetella pertussis deficient in virulence-associated factors were identified by using the transposon Tn5 lac. Tn5 lac is a derivative of Tn5 which generates promoter fusions for beta-galactosidase. Tn5 lac insertions in the vir-regulated genes of B. pertussis were identified by selecting for kanamycin-resistant mutants that expressed beta-galactosidase when the vir-regulated genes were expressed but not when the vir-regulated genes were turned off. Fourteen different mutations in vir-regulated genes were identified. Two mutants were deficient in the production of the filamentous hemagglutinin, two mutants were deficient in the production of adenylate cyclase toxin and hemolysin, and one mutant was deficient in the production of dermonecrotic toxin. One insertion mapped adjacent to the pertussis toxin gene, but the mutant produced pertussis toxin. The phenotypes of the remaining eight mutants were not determined, but the mutants did not appear to be deficient in the production of the 69,000-dalton outer membrane protein (agglutinogen 3) or the capsule. Screening for mutations in either of the fimbrial genes proved to be problematic since the parental strain was found to switch from a fimbriated to a nonfimbriated state at a high frequency, which was suggestive of the metastable expression of pili in other bacteria. We used Southern blot analysis with a 30-mer specific for the fimbrial sequences. No bands with the predicted increase in size due to the 12 kilobases from Tn5 lac were observed, which suggests that none of these genes were mutated. Southern blot analysis also revealed that seven of the eight unidentified mutations mapped to different restriction fragments, which suggests that they could be deficient in as many as seven different genes.

Bacterial Proteins↗
PubMed1989

Invasion of HeLa 229 cells by virulent Bordetella pertussis.

Phase-dependent invasive behavior of Bordetella pertussis was demonstrated by recovery of viable organisms from gentamicin-treated HeLa cell monolayers and by transmission electron microscopy. Several mutants of B. pertussis with Tn5 or Tn5 lac inserted into various vir-regulated genes were evaluated for differences in their invasive abilities. Mutants lacking filamentous hemagglutinin, pertussis toxin, and two as yet uncharacterized vir-regulated products had levels of invasion significantly lower than that of the parent strain BP338. In contrast, invasion by mutants lacking adenylate cyclase toxin was significantly increased compared with that of wild-type B. pertussis. This increase in invasion was eliminated when concentrations of intracellular cyclic 3'-5' AMP were stimulated by treating HeLa cells with cholera toxin or forskolin. Entry of B. pertussis occurred through a microfilament-dependent phagocytic process, as evidenced by the marked reduction in uptake following treatment of HeLa cells with cytochalasin D. Invasion was inhibited with polyclonal anti-B. pertussis and anti-filamentous hemagglutinin antisera. In addition, a monoclonal antibody against lipooligosaccharide A reduced uptake by 65.5%. The preservation of HeLa cell integrity and the limited replication of intracellular bacteria suggest that invasion may represent a means by which B. pertussis evades an active host immune response.

Actin Cytoskeleton↗
PubMed1989

Environmental regulation of expression of virulence determinants in Bordetella pertussis.

The trans-activator vir is required for expression of all virulence-associated genes in Bordetella pertussis. The nature of the global regulation of these factors by vir and environmental signals was examined by Northern blot analysis and with beta-galactosidase transcriptional fusions in five vir-regulated genes. Northern blots suggested that vir regulates at the level of transcription since Vir- organisms did not exhibit detectable mRNA from vir-regulated loci. Environmental signals such as high levels of salts, nicotinic acid, and 6-chloronicotinic acid or growth at low temperatures were examined. Of all of the cations and anions examined, only SO4 ions eliminated transcription of vir-regulated genes and reduced transcription of vir itself, suggesting that global regulation is obtained by modifying expression of the essential component, vir. Organisms grown on 6-chloronicotinic acid or quinaldic acid did not have detectable transcription from vir-regulated loci. Modulation by nicotinic acid, on the other hand, was strain dependent, acting at the level of transcription in strain 18-323 but not in Tohama I derivatives. Growth at lower temperatures reduced, but did not eliminate, transcription from vir-regulated loci. At 28 degrees C the ratio of pertussis toxin mRNA to recA mRNA (a non-vir-regulated factor) was equivalent to that at 37 degrees C, suggesting that transcription at low temperatures is reduced in a proportional manner and need not involve vir.

Bordetella pertussis↗
PubMed1988

Sonographic guidance for infant hip reduction under anesthesia.

An initial attempt to reduce a hip dislocation in a 15-month-old by palpation under general anesthesia led to the subsequent discovery of a posterior subluxation by CT. During the second attempt, sonographic guidance in the operating room was used to show concentric reduction both before and after the application of a spica cast. Successful reduction was later confirmed by CT. Imaging by real-time sonography can be useful in monitoring infant hip reduction.

Female↗
PubMed1988

Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir.

The vir locus of Bordetella pertussis apparently encodes a trans-acting positive regulator that is required for the coordinate expression of genes associated with virulence: pertussis toxin, filamentous hemagglutinin (FHA), hemolysin, and adenylate cyclase toxin. DNA clones of vir and of genes required for the synthesis of some of the factors under vir control were obtained with DNA probes from the chromosomal DNA surrounding sites of Tn5 insertion mutations that inactivated those genes. Two vir clones were found which also contained genes required for the proper expression of FHA in B. pertussis. The plasmids which contained both the fha and vir genes expressed immunologically reactive FHA in Escherichia coli, as detected by colony blots, whereas plasmids which contained only fha or vir were negative in this assay. The regulation of FHA production in E. coli, as in B. pertussis, was temperature dependent and inhibited by high concentrations of either magnesium ions or nicotinic acid, indicating that the sequences cloned in E. coli contained the information required to preserve the physiological responses seen in B. pertussis. Further characterization of the vir-fha clones by Tn5 mutagenesis in E. coli and by the return of cloned sequences to B. pertussis in trans and to the B. pertussis chromosome led to the localization of the vir locus, the structural gene for FHA, and genes that are possibly required for the synthesis and export of FHA.

Bacterial Adhesion↗
PubMed1988

Aggressive lesion in osseous sarcoidosis.

We describe an unusual aggressive lytic bone lesion with "sunburst/hair-on-end" periosteal reaction in a patient with surgically proven osseous sarcoid and review the pertinent literature.

Adult↗
PubMed1987

Inhibition of monocyte oxidative responses by Bordetella pertussis adenylate cyclase toxin.

Bordetella pertussis and the other Bordetella species produce a novel adenylate cyclase toxin which enters target cells to catalyze the production of supraphysiologic levels of intracellular cyclic adenosine monophosphate (cAMP). In these studies, dialyzed extracts from B. pertussis containing the adenylate cyclase toxin, a partially purified preparation of adenylate cyclase toxin, and extracts from transposon Tn5 mutants of B. pertussis lacking the adenylate cyclase toxin, were used to assess the effects of adenylate cyclase toxin on human peripheral blood monocyte activities. Luminol-enhanced chemiluminescence of monocytes stimulated with opsonized zymosan was inhibited greater than 96% by exposure to adenylate cyclase toxin-containing extract, but not by extracts from adenylate cyclase toxin-deficient mutants. The chemiluminescence responses to particulate (opsonized zymosan, Leishmania donovani, and Staphylococcus aureus) and soluble (phorbol myristate acetate) stimuli were inhibited equivalently. The superoxide anion generation elicited by opsonized zymosan was inhibited 92% whereas that produced by phorbol myristate acetate was inhibited only 32% by B. pertussis extract. Inhibition of oxidative activity was associated with a greater than 500-fold increase in monocyte cAMP levels, but treated monocytes remained viable as assessed by their ability to exclude trypan blue and continued to ingest particulate stimuli. The major role of the adenylate cyclase toxin in the inhibition of monocyte oxidative responses was demonstrated by: 1) little or no inhibition by extracts from B. pertussis mutants lacking adenylate cyclase toxin; 2) high level inhibition with extract from B. parapertussis, a related species lacking pertussis toxin; and 3) a reciprocal relationship between monocyte cAMP levels and inhibition of opsonized zymosan-induced chemiluminescence using both crude extract and partially purified adenylate cyclase toxin. Pertussis toxin, which has been shown to inhibit phagocyte responses to some stimuli by a cAMP-independent mechanism, had only a small (less than 20%) inhibitory effect when added at concentrations up to 100-fold in excess of those present in B. pertussis extract. These data provide strong support for the hypothesis that B. pertussis adenylate cyclase toxin can increase cAMP levels in monocytes without compromising target cell viability or impairing ingestion of particles and that the resultant accumulated cAMP is responsible for the inhibition of oxidative responses to a variety of stimuli.

Adenylate Cyclase Toxin↗
PubMed1986

Real-time sonography of infant hip dislocation.

Real-time sonography offers many advantages in the evaluation of suspected hip dislocation: The procedure is short, cost-effective, requires no sedation and does not expose the patient to ionizing radiation.

Hip Dislocation, Congenital↗
PubMed1986

Prokaryotic adenylate cyclase toxin stimulates anterior pituitary cells in culture.

Bordetella pertussis synthesizes a variety of virulence factors including a calmodulin-dependent adenylate cyclase (AC) toxin. Treatment of anterior pituitary cells with this AC toxin resulted in an increase in cellular cAMP levels that was associated with accelerated exocytosis of growth hormone (GH), prolactin, adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH). The kinetics of release of these hormones, however, were markedly different; GH and prolactin were rapidly released, while LH and ACTH secretion was more gradually elevated. Neither dopamine agonists nor somatostatin changed the ability of AC toxin to generate cAMP (up to 2 h). Low concentrations of AC toxin amplified the secretory response to hypophysiotrophic hormones. We conclude that bacterial AC toxin can rapidly elevate cAMP levels in anterior pituitary cells and that it is this response that explains the subsequent acceleration of hormone release.

Adenylate Cyclase Toxin↗
PubMed1985

Genetic studies of the molecular basis of whooping cough.

The etiologic agent of whooping cough, Bordetella pertussis, synthesizes several biochemically and biologically active factors believed to contribute to the disease process. They include filamentous hemagglutinin (FHA), pertussis toxin (PTX), adenylate cyclase toxin (AC), and hemolysin (HLY). We have used a genetic approach to evaluate the contribution of these factors to the virulence of B. pertussis. A series of B. pertussis mutants prepared by transposon Tn5 insertion mutagenesis were characterized and determined to be specifically deficient in the elaboration of these factors. The wild type strain and the mutants were tested for their ability to produce a lethal infection in the classical infant mouse intranasal infection model. The dose of organisms required to cause lethal infection in 50% of the animals (LD50) for the wild type strain was 2 X 10(3) bacteria. A mutant deficient in production of the proposed adherence factor, FHA, was equivalent in virulence to the wild type strain (BP353, LD50 = 8 X 10(3)). In contrast, mutants deficient in production of PTX alone (BP357, LD50 = 5 X 10(6)), AC and HLY (BP348, LD50 greater than 3 X 10(7)), or all known toxins and virulence factors (BP347, LD50 greater than 3 X 10(7)) were severely impaired in their ability to cause a lethal infection. Our data indicate the hemolysin and adenylate cyclase as well as pertussis toxin are important virulence factors and provide the basis for a working model of the molecular basis of the disease caused by Bordetella pertussis.

Adenylate Cyclase Toxin↗
PubMed1985

Bordetella extracytoplasmic adenylate cyclase: actions as a bacterial toxin.

Virulent Bordetella organisms produce an adenylate cyclase which is extracytoplasmic in location, activated by the eukaryotic regulatory protein, calmodulin, and able to act as a toxin, promoting cyclic AMP accumulation in target mammalian cells. Initial steps in purification of this novel adenylate cyclase toxin reveal two forms: one which possesses only enzymatic adenylate cyclase activity, but has no effect on intact target cells; and the other which has both enzymatic and intoxicating activity. These data suggest that this toxin may conform to the A/B model for bacterial toxins. A variety of mammalian cell types can be affected by the adenylate cyclase toxin, including neutrophils, macrophages, monocytes, lymphocytes, lymphoma cells, and pituitary cells. Although the consequence of intoxication in many cells is inhibition of normal function, the enhancement of pituitary hormone secretion by this toxin suggests that its biological effects are the result of cAMP accumulation. These data confirm the hypothesis that Bordetella adenylate cyclase is, indeed, a toxin and illustrate its role as a novel research probe.

Adenylyl Cyclases↗
PubMed1985

Muscle biopsy: proper surgical technique.

Muscle biopsy is often incorrectly performed despite the fact that it is a relatively simple procedure. A consistently reproducible surgical technique in 93 open vastus lateralis muscle biopsies implemented in conjunction with neuromuscular and malignant hyperthermia research demonstrated 14 cases of malignant hyperthermia and a variety of forms of neuromuscular pathology. No wound complications or disabilities have resulted from this procedure. The procedure includes careful selection of biopsy site, regional anesthesia, atraumatic dissection, and immediate processing of the biopsy sample. A new muscle biopsy clamp is described. The authors recommend the vastus lateralis for the biopsy site unless another area of involvement is specifically indicated. Careful attention to technical details is required for optimal results.

Adolescent↗
PubMed1984

Pertussis toxin and extracytoplasmic adenylate cyclase as virulence factors of Bordetella pertussis.

A wild-type strain of Bordetella pertussis and a series of transposon Tn5-induced mutants deficient in the production of various factors believed to play a role in pertussis (whooping cough) were tested for virulence in infant mice. The 50% lethal dose of the wild-type strain in these animals was 2 X 10(3) bacteria. A mutant deficient in the production of the filamentous hemagglutinin was almost as virulent as the wild type. Avirulent phase mutants (i.e., deficient in the production of all toxins), a pertussis-toxin mutant, and a double mutant deficient in both hemolysin and adenylate cyclase were severely impaired in the ability to cause pertussis. These data underscore the role of pertussis toxin in the disease and provide the first direct evidence that adenylate cyclase and hemolysin are important in the pathogenesis of the infection.

Adenylate Cyclase Toxin↗
PubMed1984

Genetic analysis of phase change in Bordetella pertussis.

Avirulent-phase derivatives of Bordetella pertussis (those which have simultaneously lost the ability to synthesize several virulence-associated factors) and the genetic mechanism of the phase change were studied. Increased tolerance to erythromycin was shown to be an avirulent-phase marker. By the use of efficiency of plating on erythromycin, the proportion of avirulent-phase (Vir) variants in a virulent-phase (Vir+) population was determined to be between 10(-3) and 10(-6), depending on the strain. We showed that the phase shift is reversible and detected a complete Vir- to Vir+ to Vir- to cycle. In other experiments, hybridization studies with avirulent-phase mutants obtained by Tn5 mutagenesis suggested that a single region located at a unique site in the B. pertussis chromosome controls the phase change. One of the avirulent Tn5 mutants was used as a recipient in a conjugative cross with a virulent-phase donor. All recombinants which had reacquired the virulence-associated factors also lost Tn5, indicating the loss of Tn5 was required to restore the Vir+ phenotype. The Tn5 avirulent-phase mutants behave as if the insertion interrupted the function of a transacting gene product which is required for the expression of the other virulent-phase genes. A model of the molecular basis of the phase regulation is presented.

Anti-Bacterial Agents↗
PubMed1983

Tn5-induced mutations affecting virulence factors of Bordetella pertussis.

Transposon Tn5 was used to isolate mutants of Bordetella pertussis. Strains with Tn5 insertions were screened for loss of virulence-associated factors, including filamentous hemagglutinin, hemolysin, and pertussis toxin. Several mutants deficient for hemolysin production were obtained. All produced dermonecrotic toxin, pertussis toxin, and filamentous hemagglutinin, but were found to vary with respect to adenylate cyclase production. One hemolysin mutant had no detectable adenylate cyclase activity; others had 0.6% or 16% wild-type activity, whereas a fourth seemed to be unaffected in terms of adenylate cyclase activity. Mutants deficient in the ability to hemagglutinate sheep erythrocytes were also isolated. These mutants either failed to synthesize or produced reduced amounts of three protein species of 200,000, 130,000, and 100,000 daltons, all of which reacted with antiserum to filamentous hemagglutinin. Pertussis toxin mutants were identified by screening culture supernatants for failure to induce a clustered growth pattern in Chinese hamster ovary cells, and identification was confirmed by the standard histamine-sensitizing assay in mice. These mutants will be useful to determine the relative contribution of each virulence factor to pathogenicity as well as to determine the identity of the antigens important in protective immunity.

Adenylyl Cyclases↗
PubMed1983

Transposon insertion and subsequent donor formation promoted by Tn501 in Bordetella pertussis.

The mercuric chloride resistance transposon, Tn501, was introduced into Bordetella pertussis by using the chimeric plasmid pUW942, which is unable to replicate in this species. Tn501 insertions which conferred a thiamine requirement were the predominant insertion class. In many cases, the mercuric chloride-resistant transconjugants were also resistant to the other plasmid markers, but failure to detect plasmid DNA in these isolates indicated that integration of the entire plasmid into the chromosome had occurred. One such insertion was further characterized. Southern hybridization with a Tn501-specific probe indicated that chromosomal DNA from one strain containing the integrated plasmid had two copies of Tn501 and an intervening copy of the plasmid associated with the chromosome. The presence of the plasmid was unstable, and derivatives which had lost all of the plasmid markers except mercuric chloride resistance were obtained. These strains had a single copy of Tn501 and had lost all of the rest of the plasmid-specific sequences. Strains containing the plasmid in the integrated state could act as genetic donors and mobilize chromosomal genes.

Bordetella pertussis↗