Biomedical subjects

J R Murphy

Publications and source records attributed to J R Murphy.

At least 217 records · Page 12Linked to original sources

PubMed1983

Studies on the molecular epidemiology of diphtheria.

DNA was extracted from toxigenic and nontoxigenic (tox+ and tox-) diphtheria bacilli isolated during a carrier survey that followed recovery of a tox+ Corynebacterium diphtheriae mitis from a baby with membranous tonsillitis. The electrophoretic gel patterns of restriction enzyme digests were indistinguishable from one another. They were, however, readily distinguishable from similar gels of DNAs extracted from diphtheria bacilli associated with outbreaks elsewhere. Hybridisation of a labelled nick-translated corynephage-beta c-DNA probe to nitrocellulose blots of these gels occurred only to blots from tox+ strains. Other hybridisation studies showed that all of seven strains, each isolated from a diphtheria case or carrier in a different part of the world, carried a prophage with DNA closely related to phage beta tox+. When an individual carrying a tox+ diphtheria bacillus arrives in an immunised community, spread of the tox gene to other individuals may be via phage conversion of tox- C diphtheriae already prevalent among the nasopharyngeal bacterial flora of the local populace, rather than by colonisation with the tox+ strain itself.

Autoradiography↗

PubMed1983

Nucleotide sequence and expression of the diphtheria tox228 gene in Escherichia coli.

The complete nucleotide sequence of the diphtheria tox228 gene encoding the nontoxic serologically related protein CRM228 has been determined. A comparison of the predicted amino acid sequence with the available amino acid sequences from the wild-type toxin made it possible to deduce essentially the entire nucleotide sequence of the wild-type tox gene. The signal peptide of pro-diphtheria toxin and the putative tox promoter have been identified, a highly symmetrical nucleotide sequence downstream of the toxin gene has been detected; this region may be the corynebacteriophage beta attachment site (attP). The cloned toxin gene was expressed at a low level in Escherichia coli.

Base Sequence↗

PubMed1983

Cloned fragment A of diphtheria toxin is expressed and secreted into the periplasmic space of Escherichia coli K12.

An 831-base pair segment of the corynebacteriophage beta tox-45 genome encoding fragment A of diphtheria toxin was cloned into plasmid pUC8 in Escherichia coli K12. Strains containing recombinant plasmids expressed the adenosine diphosphate ribosyl transferase activity characteristic of fragment A; this activity could be inhibited by polyvalent antiserum to fragment A as well as by the appropriate monoclonal antibodies to diphtheria toxin. The transferase activity was secreted into the periplasmic space of E. coli. These findings have implications for the future construction of genetically engineered chimeric toxins.

Antibodies, Monoclonal↗

PubMed1983

Thyrotropin-releasing hormone-diphtheria toxin-related polypeptide conjugates. Potential role of the hydrophobic domain in toxin entry.

We have separately coupled a modified thyrotropin-releasing hormone (TRH) molecule to two diphtheria toxin-related polypeptides, CRM26 and CRM45. Both polypeptides are enzymatically active but only CRM45 contains the hydrophobic domain of the toxin molecule. The TRH-CRM45 conjugate caused a 50% inhibition of protein synthesis in GH3 rat pituitary cells at 3 X 10(-9) M. CRM45 alone was 200 to 500 times less toxic than the conjugate. Intoxication by TRH-CRM45 was prevented by excess TRH, preincubation with diphtheria antitoxin, or reduction of the disulfide cross-link. In addition, TRH-CRM45 was no more toxic than CRM45 itself to 3T3 cells which lack TRH receptors. In contrast, TRH-CRM26, although it retained enzymatic activity, was nontoxic for GH3 cells even at 10(-7) M. Binding experiments showed that both conjugates compete for TRH receptors with comparable affinities. The potential role of the hydrophobic domain in toxin entry is discussed.

Animals↗

PubMed1983

The relationship of relative risk and positive predictive value in 2 X 2 tables.

Both relative risk and positive predictive value are used to assess the relationship between an attribute and a disease state. While they can be equated only in one uninteresting situation, they do have an explicit algebraic relationship which is demonstrated in this paper. This relationship may provide an interesting link between epidemiology and medical decision analysis.

Coffee↗

PubMed1983

Host defenses in murine malaria: characteristics of protracted states of immunity to Plasmodium berghei.

Vaccination of 8-week-old B6D2 (C57BL/6 x DBA/2) mice with Formalin-killed asexual erythrocytic stages of Plasmodium berghei provided a capacity to survive challenge with lethal P. berghei for more than 542 days. Although long-lived, this immunity did not provide a capacity to immediately neutralize parasites in a challenge; significant levels of erythrocytic infection occurred transiently after each challenge. The quality of long-lived immunity was not enhanced by an injection of live parasites 2 weeks after the last injection of killed antigen. The results suggest that it will be difficult to design a vaccination regimen which will provide long-lived protection from overt erythrocytic-stage infection.

Animals↗

PubMed1983

Molecular epidemiological studies of United States Gulf Coast Vibrio cholerae strains: integration site of mutator vibriophage VcA-3.

Environmental and clinical Vibrio cholerae O-1 strains isolated from the U.S. Gulf Coast region were found to be lysogenic for a vibriophage which we have designated VcA-3. Comparison of VcA-3 with the previously described vibriophages VcA-1 and VcA-2 has shown that VcA-1 and VcA-3 are homoimmune, have extensive sequence homology, but have markedly different restriction endonuclease digestion patterns. VcA-3 was found to randomly integrate into the V. cholerae RV79 chromosome and to introduce stable auxotrophic mutations. We show that all U.S. Gulf Coast environmental and clinical isolates that are lysogenic for VcA-3, including both tox+ and tox- isolates, contain the prophage integrated at an identical chromosomal site. Given the known stability of temperate mutator bacteriophages, these results suggest that there is a clonal relationship among the V. cholerae O-1 strains examined in this study, including the tox+ and tox- isolates.

Bacteriophages↗

PubMed1983

Integration of corynebacteriophages beta tox+, omega tox+, and gamma tox- into two attachment sites on the Corynebacterium diphtheriae chromosome.

The bacterial attachment sites of independently isolated Corynebacterium diphtheriae strains C7s and (belfanti)1030 lysogenic for corynebacteriophages beta tox+, omega tox+, and gamma tox- were determined by Southern blot analysis. Both corynebacterial strains contained two distinct bacterial attachment sites (attB1 and attB2). We found that infection by any of the three closely related corynebacteriophages may give rise to single, double, and triple lysogens. In the case of toxigenic C. diphtheriae strains C7s(beta tox+) and C7s(omega tox+), the final yields of diphtheria toxin produced under optimal conditions were equivalent and varied by one-, two-, or threefold depending upon the number of integrated prophage.

Bacteriophages↗

PubMed1983

Restriction endonuclease map of corynebacteriophage omega ctox+ isolated from the Park-Williams no. 8 strain of Corynebacterium diphtheriae.

The toxigenic corynebacteriophage omega tox+ was isolated from the hypertoxigenic Park-Williams no. 8 (PW8) strain of Corynebacterium diphtheriae and compared with the toxigenic corynebacteriophage beta tox+. The physical size and host range of both phages were found to be identical. An endonuclease restriction map of omega tox+ was constructed, and the locations of the cohesive ends (cos), phage attachment site (attP), and the diphtheria tox operon were identified. The genome of omega tox+ was found to differ from that of beta tox+ in three regions. In addition, omega tox+ was shown to be integrated into two nontandem corynebacterial phage attachment sites (attB1, attB2) in the PW8 chromosome. The differences in the restriction endonuclease digestion maps of omega tox+ and beta tox+ and the contribution of double lysogeny are discussed in relation to the hypertoxigenicity of the PW8 strain.

Bacteriophages↗

PubMed1983

Organ-specific binding of a thyrotropin-releasing hormone-diphtheria toxin complex after intravenous administration to rats.

We have previously demonstrated that a hybrid protein consisting of TRH linked to CRM45, a fragment of diphtheria toxin which lacks its native cell-binding moiety, specifically binds to TRH receptors in vitro. In this study we have examined its in vivo binding and shown that after iv injection, this complex labeled with 125I is selectively concentrated in the normal anterior pituitary and that concomitant administration of cold TRH reduces its uptake. Displaceable uptake was also demonstrated in the hypothalamus and testis, whereas nondisplaceable binding exceeding that of CRM45 alone was shown in the ovary and the breast parenchyma of lactating rats. Similar experiments with tritiated TRH were performed. We found that although there was uptake of the material by many tissues, almost all of the radioactivity was in the form of TRH degradation products. Therefore, we conclude that TRH linked to a large carrier like CRM45 may be a more revealing indicator of in vivo binding affinities than native TRH.

Animals↗

PubMed1982

Isolation of enterotoxin structural gene deletion mutations in Vibrio cholerae induced by two mutagenic vibriophages.

Phenotypically nontoxinogenic mutants of Vibrio cholerae were isolated after infection with either of two mutagenic vibriophages, VcAI and VcA2ctsl. DNA isolated from these mutants was analyzed for toxin gene sequences by the Southern blotting method with 32P-labeled probes derived from the cloned A and B subunit genes for the heat-labile enterotoxin of Escherichia coli, designated LT. Several of the mutant isolates were shown by this method to have lost all sequences hybridizing to the LT probes, indicating that these clones contain deletion mutations that removed the structural gene(s) for cholera toxin. The mutants were prototrophic and grew normally, in vitro, demonstrating that the toxin is not essential for the growth and viability of V. cholerae. Moreover, the toxin gene deletion mutants multiplied well in vivo in ligated rabbit intestine. Because of these growth properties and the stability of deletion mutations, these strains are promising candidates for testing as live oral vaccine strains for protection against cholera.

Bacteriophages↗

PubMed1982

Isolation of hybridoma cell lines and characterization of monoclonal antibodies against cholera enterotoxin and its subunits.

Hybridoma cell lines which produced monoclonal antibodies against cholera toxin were isolated. These cell lines were detected with an enzyme-linked immunosorbent assay screening procedure with purified cholera toxin or subunit A of cholera toxin. Seven cell lines were characterized with respect to their reactivity with cholera toxin subunits by Western blot analysis. Five clones produced antibodies which were directed against subunit A, and two clones produced antibodies which reacted with subunit B. These antibodies were also characterized by Western blot analysis for reactivity with the heat-labile enterotoxin produced by porcine and human enterotoxinogenic strains of Escherichia coli. Monoclonal antibodies which reacted with subunit A of cholera toxin also reacted with subunit A of both porcine and human heat-labile enterotoxins. In contrast, monoclonal antibodies to subunit B of cholera toxin did not react with subunit B of the heat-labile enterotoxin. Antibodies directed against subunit B neutralized the biological activity of cholera toxin in vitro in the S49 mouse lymphosarcoma assay. In contrast to polyclonal anti-subunit A antisera, monoclonal anti-subunit A from four of five clones had small but measurable neutralizing capacities in vitro.

Animals↗

PubMed1982

Restriction endonuclease map of the nontoxigenic corynephage gamma c and its relationship to the toxigenic corynephage beta c.

Clear-plaque-forming mutant gamma tox- corynephages were isolated independently from nontoxigenic lysogenic Corynebacterium diphtheriae strains C7s(gamma tox-) and C4(gamma tox-). A physical map was constructed by using restriction endonucleases BamHI, EcoRI, HindIII, and KpnI. A comparison of nontoxigenic gamma c with toxigenic corynephage beta c revealed large areas of homology, including common regions for cohesive ends (cos) and attachment sites (att). Localization of the att sites on the beta prophage and correlation of the physical and genetic maps defined the orientation of the diphtheria tox operon. Diphtheria tox sequence homologies were mapped on gamma c by hybridizing 32P-labeled diphtheria tox mRNA to restriction fragments of gamma c DNA. Two regions of heterogeneity between phages beta c and gamma c were localized and these regions accounted for the 3-kilobase larger molecular size of gamma c compared with beta c. One change occurs near the tox promoter and may explain the nontoxigenic phenotype of corynephage gamma tox-.

Bacteriophages↗

PubMed1982

Neonatal hyperviscosity. II. Effect of partial plasma exchange transfusion.

To determine the effect of partial plasma exchange transfusion, 20 newborn infants with neonatal hyperviscosity were randomly assigned to observation or treatment with partial plasma exchange transfusion within the first eight hours after birth. They were studied for organ involvement by roentgenogram, blood count, coagulation studies, and neurologic behavior and were followed up using the Brazelton Neonatal Behavior Assessment scale at 8, 24, and 72 hours and 2 weeks of age; in addition, ten control infants without hyperviscosity of similar birth weights and gestational ages were also studied. Exchange transfusion improved blood viscosity but both hyperviscous groups showed a higher proportion of abnormal results than did the control subjects. Infants receiving exchange transfusions subsequently improved during the period from 8 hours to 2 to 3 weeks of age, until they were indistinguishable from the control subjects. Neurologic improvement in hyperviscous infants who had not received exchange transfusions were significantly slower during this period. At 8 months of age, abnormal neurologic and developmental findings were impressive in both groups; no significant differences in neurologic abnormalities were noted at that time. Developmental delays, tremors, spastic diplegia, and monoparesis were found in four of six untreated infants and five of ten infants who had received exchange transfusions. A fine tremor was present in one control child.

Blood Viscosity↗

PubMed1982

A feasibility study of computer aided diagnosis in appendicitis.

In this study, the hypothesis that computer aided diagnosis could enable a more accurate differentiation between patients with acute appendicitis and those with abdominal pain but normal appendixes was examined. A data base was established by analyzing the records of 476 patients having an emergency measure appendectomy during a five year period. There were 360 or 76 per cent with acute appendicitis, 98 or 20 per cent with normal appendixes and 18 or 4 per cent with other diseases requiring operation. The records were analyzed with regard to history, physical examination and laboratory findings. The data base was then divided randomly into two parts. Part 1 was subjected to univariate discriminant analysis, using the chi-square test. The only quantities which were significantly different between appendicitis and a normal appendix were sex, duration of symptoms, anorexia and vomiting. Multivariate discriminant analysis was used to derive an abdominal pain index which discriminated between appendicitis and a normal appendix with a sensitivity of 0.82 and a specificity of 0.39. Using the abdominal pain index to evaluate the patients in part 2 of the data base, 23 or 40 per cent of the 58 patients with a normal appendix would have avoided operation. However, 31 or 18 per cent of the 169 patients with appendicitis would have not been operated upon; three of those 31 had perforated appendixes. Computer aided diagnosis was no more effective than unaided clinical diagnosis in appendicitis.

Abdomen↗

PubMed1982

The unnecessary laparotomy for appendicitis-can it be decreased?

There has been no decrease in the incidence of negative appendectomies in the adult population over the past two decades. Review of 484 appendectomies over a five-year period revealed that females between the ages of 13 to 40 have the lowest appendiceal perforation rate and the highest diagnostic error rate. More thorough preoperative assessment is indicated in this group. A program utilizing intensive observation, diagnostic barium enema, and laparoscopy may produce a reduction in negative laparotomies for appendicitis.

Adolescent↗

PubMed1981

Conducting clinical trials with practicing community oncologists.

The practicing community oncologist has limited time and opportunity to participate in clinical trials of new cancer therapy. However, the majority of patients treated are seen in the community setting that there is increasing interest in getting community oncologists involved in these trials. This paper outlines a currently active program that has succeeded in engaging the participation of 70% of the available oncologists in the Colorado region.

Attitude↗

PubMed1981

The relation of electronic fetal monitoring patterns to infant outcome measures in a random sample of term size infants born to high risk mothers.

In the rush to use a new medical technology the fact that relative risk is not a measure of the predictive validity of a variable is sometimes forgotten. In the present paper both the relative risk and predictive value of electronic fetal monitoring patterns are examined to see whether they are associated with problematic neonatal outcomes. It is shown that nonreassuring or ominous patterns are not good predictors of problematic neonatal outcomes. This suggests either that there is no risk associated with these patterns or if in fact there is such a risk, it is moderated by some as yet unknown variables between the time that the pattern is seen and the actual birth of the infant. This fact has implication for the management of labor and delivery using electronic monitoring equipment, since it appears that the use of such equipment will lead to a significant increase in the cesarean section rate. If this increase in the cesarean section rate comes about because of the assumed predictive value of a nonreassuring or ominous pattern, it may represent an unnecessary increase in cost and risk of delivery.

Colorado↗