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J R Murphy

Publications and source records attributed to J R Murphy.

At least 181 records · Page 10Linked to original sources
PubMed1988

Interleukin 2 toxin: a step toward selective immunomodulation.

We have used protein engineering and recombinant DNA methodologies to genetically replace the eukaryotic cell receptor binding domain of diphtheria toxin with interleukin 2 (IL-2). The toxin-related T cell growth factor fusion gene has been cloned in Escherichia coli K12. Recombinant strains of E coli produce a 68,086 K hybrid toxin, IL-2 toxin that retains immunologic properties intrinsic to both its diphtheria toxin and IL-2 components. IL-2 toxin has been found to selectively inhibit protein synthesis in both human and murine T cell lines that bear high affinity IL-2 receptors, whereas the hybrid toxin is not active against cells that do not bear this receptor. The cytotoxic action of IL-2 toxin is specifically blocked by free IL-2 and monoclonal antibodies that bind to the p55 (Tac antigen) subunit of the high affinity IL-2 receptor. In addition, IL-2 toxin, like diphtheria toxin itself, must pass through an acidic compartment in order to deliver its adenosine diphosphate ribosyl transferase activity to the cytosol of target T cells. In a murine delayed type hypersensitivity (DTH) model system, we have shown that IL-2 toxin treatment induces a marked immunosuppression.

Adjuvants, Immunologic↗
PubMed1988

Interleukin 2-diphtheria toxin fusion protein can abolish cell-mediated immunity in vivo.

De novo expression of the interleukin 2 receptor (IL-2R) is a critical and pivotal event in initiation of an immune response. Targeting the low-affinity IL-2-binding p55 subunit of the high-affinity IL-2R with the rat anti-mouse IgM monoclonal antibody M7/20 suppresses a variety of T-cell-mediated reactions, including transplant rejection, autoimmunity, and delayed-type hypersensitivity (DTH). A hybrid IL-2-toxin gene was constructed from the diphtheria toxin gene by replacing the DNA encoding the diphtheria toxin receptor-binding domain with the DNA encoding the receptor-binding domain of IL-2, and the fusion protein encoded by the hybrid gene was expressed in Escherichia coli [Williams, D.P., Parker, K., Bacha, P., Bishai, W., Borowski, M., Genbauffe, F., Strom, T.B. & Murphy, J.R. (1987) Protein Eng. 1, 493-498]. We examined the action of the chimeric IL-2-toxin fusion protein on an in vivo T-cell mediated response, DTH. The IL-2-toxin fusion protein was found to be a potent immunosuppressive agent. Treatment of mice with the IL-2-toxin blocks DTH and prevents expansion of IL-2R+ T cells. Indeed, IL-2-toxin treatment targets IL-2R+ T cells in vivo and is shown to selectively eliminate their appearance in draining lymph nodes. DTH suppression was observed even in mice possessing high titers of antibodies to diphtheria toxoid.

Animals↗
PubMed1988

Expression of diphtheria toxin fragment A and hormone-toxin fusion proteins in toxin-resistant yeast mutants.

Mutants of the eukaryote Saccharomyces cerevisiae, previously selected for resistance to diphtheria toxin, were investigated for their suitability as hosts for the expression of tox-related proteins. The structural gene for the toxin, encoding the fragment A catalytic domain, was modified for efficient intracellular expression in eukaryotes and placed downstream of the yeast GAL1 promoter element in a plasmid. Transformed mutant yeast grown in galactose, which induces that promoter, were viable and contained active fragment A. In contrast, sensitive, wild-type cells harboring this plasmid grew normally under repressing conditions but were killed when the GAL1 promoter was induced. Additional constructions were also prepared that included sequences encoding either the lymphocyte growth factor interleukin 2 or alpha-melanocyte-stimulating hormone along with the lipid-associating domains of fragment B and the leader peptide of the Kluyveromyces lactis killer toxin. Resistant mutant strains transformed with these plasmids efficiently expressed and secreted the expected chimeric toxins.

Amino Acid Sequence↗
PubMed1988

Identification of novel prognostic indicators in burned patients.

The size and depth of burn and patient age are useful early prognostic indicators in burned patients, but have limited value in predicting which patients in a given cohort are likely to die. The objective of this study was to identify additional variables in the first 10 days of burn injury which could better predict patient outcome. Variables consisting of demographic information, routine laboratory data, and clinical observations on 89 burned patients (63 survivors and 26 nonsurvivors) were analyzed. Compared to survivors, nonsurvivors had significantly larger burns (58 +/- 23% vs. 37 +/- 17%; p less than 0.0002) and a higher incidence of Gram-negative septicemia (20 of 26 [77%] vs. 24 of 63 [38%]; p less than 0.001). Potential prognostic variables were subjected to multivariate logistic regression analysis for each of the first 10 days following burn injury in order to identify a combination of parameters which predicted patient outcome. The regression analyses revealed that, as previously demonstrated, patient age and burn size were significant predictors of mortality on admission and throughout the first 10 days postburn. In addition, absolute monocyte count (AMC), absolute lymphocyte count (ALC), maximum daily temperature (Tmax), and BUN were also significant predictors (p less than 0.05). These data indicate that logistic regression models can identify simple prognostic variables in burned patients which may improve clinicians' ability to identify high-risk patients early in the course of their burn injuries.

Adult↗
PubMed1988

Stage-selective inhibition of rodent malaria by cyclosporine.

The relative susceptibility of different developmental stages of Plasmodium berghei to cyclosporine was investigated in vivo. Within 12 h of receiving a single 25-mg/kg (body weight) dose of cyclosporine, mice with patent P. berghei infections uniformly exhibited a rapid fall in asexual parasite stages. Initially, ring forms and mature schizonts disappeared. Subsequently, trophozoites disappeared between 21 and 24 h, whereas gametocytes persisted for 36 h. In contrast, when cyclosporine was administered to mice 1 day before inoculation (100 mg/kg) with P. berghei sporozoites and for 2 consecutive days after inoculation (25 mg/kg), infections developed normally. When mice with patent infections were placed on prolonged cyclosporine therapy (25 mg/kg per day), parasitemia initially disappeared but often recrudesced. Recrudescent parasites were frequently resistant to cyclosporine (Csr). The Csr phenotype remained stable after serial passage of parasites in mice and after transmission through Anopheles stephensi mosquitoes, in which the capacity to produce oocysts was reduced. When infections of untreated mice were initiated with equal numbers of Csr and cyclosporine-susceptible (Css) parasites and then carried through two serial cycles of mosquito-to-mouse transmission without cyclosporine treatment, the Csr phenotype was lost. The results indicate that cyclosporine selectively inhibits asexual blood stages of P. berghei and favors the emergence of Csr parasites with diminished infectivity for mosquitoes.

Animals↗
PubMed1988

Nucleotide sequences and comparison of the hemolysin determinants of Vibrio cholerae El Tor RV79(Hly+) and RV79(Hly-) and classical 569B(Hly-).

We determined the nucleotide sequences of the hemolysin structural gene, hlyA, of Vibrio cholerae El Tor biotype strains RV79(Hly+) and RV79(Hly-) and the hly determinant of the nonhemolytic classical biotype strain 569B(Hly-). The sequences of the hlyA gene from El Tor strains RV79(Hly+) and RV79(Hly-) have an identical 2,223-base open reading frame which is predicted to encode an 81,977-dalton precursor form of hemolysin. This value is in excellent agreement with the 84,000-Mr hemolysin described in the earlier report of Goldberg and Murphy (S. L. Goldberg and J. R. Murphy, J. Bacteriol. 162:35-41, 1985). In contrast, the sequence of the hly determinant of the classical 569B(Hly-) strain has an 11-base-pair deletion within the hlyA structural gene. In this instance the hly determinant is predicted to encode a 26,765-dalton precursor form of a truncated hemolysin. In each case, the regulatory region encoding the putative hlyA promoter and the predicted 25-amino-acid signal sequence are identical.

Amino Acid Sequence↗
PubMed1988

Analysis of the diphtheria tox promoter by site-directed mutagenesis.

By oligonucleotide-directed mutagenesis, we introduced alterations in the two putative -10 regions of the diphtheria tox promoter which are positioned at -50 and -56 from the GUG tox initiation signal. The -10 region positioned at -50 is favored in the expression of ADP-ribosyltransferase activity from the wild-type tox promoter in recombinant Escherichia coli; however, the promoter down mutation at position -50 is compensated for by increased activity of the -10 region positioned at -56.

ADP Ribose Transferases↗
PubMed1988

Cell receptor specific targeted toxins: genetic construction and characterization of an interleukin 2 diphtheria toxin-related fusion protein.

We have genetically replaced the diphtheria toxin receptor binding domain with a DNA insert encoding the T-cell growth factor interleukin-2 (IL-2). The toxin-related IL-2 fusion gene encodes a 70,586 dalton protein, pro-IL-2-toxin. The mature form of IL-2-toxin is exported to the periplasmic space of recombinant Escherichia coli and has a molecular weight of 68,086. IL-2-toxin has been partially purified from periplasmic extracts of recombinant E. coli, and it is shown to contain immunologic determinants intrinsic to both its diphtheria toxin and IL-2 components. The chimeric toxin is targeted toward IL-2 receptor bearing T-cells in vitro.

Cell Line↗
PubMed1988

A model for Plasmodium falciparum sporozoite challenge and very early therapy of parasitaemia for efficacy studies of sporozoite vaccines.

Plasmodium falciparum parasitaemias were induced in four non-immune volunteers by the bites of mosquitoes infected from cultured gametocytes. Radical cure was accomplished before three of the four volunteers developed clinical malaria. Despite very low peak levels of parasitaemia, the plasmodium was recultured from the blood of all volunteers. This volunteer model of early detection of parasitaemia and prompt treatment will contribute to the safe and practical efficacy testing of sporozoite vaccines, thus facilitating the selection of candidate vaccines for large scale definitive field trials.

Adult↗
PubMed1987

Diphtheria toxin receptor binding domain substitution with interleukin-2: genetic construction and properties of a diphtheria toxin-related interleukin-2 fusion protein.

We have genetically replaced the diphtheria toxin receptor binding domain with a synthetic gene encoding interleukin-2 (IL-2) and a translational stop signal. The diphtheria toxin-related T-cell growth factor fusion gene encodes a 70 586-d polypeptide, pro-IL-2-toxin. The mature form of IL-2-toxin has a deduced mol. wt of 68,086 and is shown to be exported to the periplasmic compartment of Escherichia coli (pABI508), and contain immunologic determinants intrinsic to both its diphtheria toxin and IL-2 components. IL-2-toxin has been purified from periplasmic extracts of recombinant strains of E. coli (pABI508) by immunoaffinity chromatography using immobilized anti-IL-2. The purified chimeric toxin is shown to selectively inhibit protein synthesis in IL-2 receptor bearing targeted cells, whereas cell lines which do not express the IL-2 receptor are resistant to IL-2-toxin action.

Bacterial Proteins↗
PubMed1987

High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli.

ABM508 is a recombinant fusion protein consisting of the N-terminal 485 amino acids of diphtheria toxin joined to alpha-melanocyte-stimulating hormone. When expressed in Escherichia coli under the control of the tox promoter and signal sequence, ABM508 is severely degraded. When overexpressed from a thermoinducible lambda pR promoter fusion, ABM508 is largely insoluble. We compared the expression of ABM508 (501 amino acids) to a full-length mutant form of the toxin (CRM197; 535 amino acids) and found that CRM197 showed minimal proteolysis. Thus, the removal of the C-terminal 50 amino acids of the toxin destabilizes the protein, making it a target for proteases. Proteolysis of ABM508 could be reduced by removal of the tox signal sequence (thereby directing the protein to the cytoplasm) and growth in lon and htpR mutant strains of E. coli. We also showed that the solubility of tox gene products expressed in E. coli was directly related to the growth temperature of the culture. Thus, a fragment A fusion protein (223 amino acids), ABM508, and CRM197 were found in soluble extracts when expressed at 30 degrees C but could not be released by the same procedures after growth at 42 degrees C. On the basis of these observations, we fused the coding sequences for mature ABM508 to the trc promoter (inducible at 30 degrees C by isopropyl-beta-D-thiogalactoside) and expressed this construct in a lon htpR strain of E. coli. This plasmid made 10 mg of soluble tox protein per liter of culture (7.7% of the total cell protein) or 14 times more than our previous maximal level. Extracts from lon htpR cells harboring this plasmid had high levels of ADP-ribosyltransferase activity, and although proteolysis still occurred, the major tox product corresponded to full-length ABM508.

Amino Acid Sequence↗
PubMed1987

Cloning and expression in Escherichia coli of three fragments of diphtheria toxin truncated within fragment B.

We have constructed three different truncated versions of diphtheria toxin (a 535-amino-acid polypeptide) which correspond to the N-terminal 290, 377, and 485 amino acids of the toxin. These lengths include one, three, and all four of the putative membrane-spanning sequences of the toxin which are thought to play a role in the translocation of fragment A into cells. Each of these three genes has been modified at its 3' end to code for a C-terminal cysteine (to allow for disulfide linkage of a targeting ligand) or a gene fusion with alpha-melanocyte-stimulating hormone. We have also substituted the native diphtheria tox promoter (ptox) with the lambda pR promoter in an effort to overexpress these proteins. The truncated genes are expressed in Escherichia coli from both the tox promoter in a constitutive fashion and from the pR promoter by using the heat-inducible cI857 repressor. The clones produce proteins which react with anti-diphtheria toxin serum, which migrate at the anticipated Mr on Western blots, and which have ADP-ribosyltransferase activity. Constitutive synthesis from ptox leads to severe proteolytic degradation even in a protease-deficient strain. High-level expression from the pR promoter in the same lon htpR strain allows the full-length polypeptides to accumulate but also stops the growth of the cells. It appears that removal of as few as 50 amino acids from the C-terminus of diphtheria toxin alters its conformation, making it a target for proteases and causing overexpression lethality in the host cells.

Cloning, Molecular↗
PubMed1987

Evaluating human choriogonadotropin heterogeneity in nongestational malignancy with monoclonal immunoassays.

The ectopic production of human choriogonadotropin (hCG) and its free alpha- and beta-subunits was evaluated in 195 patients with nongestational malignancy. Dimer hCG was the predominant species secreted. Significant levels of free beta-hCG were detected in 11% of patients, and represented 1.6-12% of immunoreactive hCG. In these patients the ratio of free beta-subunit to hCG remained constant during their follow-up period. Significant levels of free alpha-hCG occurred in 1.5% of patients, but were secreted independently of free beta-hCG. These findings indicate that immunoassays specific for dimer hCG would not be significantly disadvantaged in monitoring malignancy, and that free beta-hCG subunit levels have limited application as a tumor marker.

Antibodies, Monoclonal↗
PubMed1987

Safety, infectivity, immunogenicity, and in vivo stability of two attenuated auxotrophic mutant strains of Salmonella typhi, 541Ty and 543Ty, as live oral vaccines in humans.

Two Salmonella typhi mutants, 541Ty (Vi+) and 543Ty (Vi-), auxotrophic for p-aminobenzoate and adenine, were evaluated as live oral vaccines. 33 volunteers ingested single doses of 10(8), 10(9), or 10(10) vaccine organisms, while four others received two 2 X 10(9) organism doses 4 d apart. No adverse reactions were observed. Vaccine was recovered from coprocultures of 29 of 37 vaccinees (78%) and from duodenal string cultures of two; repeated blood cultures were negative. The humoral antibody response to S. typhi O, H, Vi, and lysate antigens in serum and intestinal fluid was meager. In contrast, all vaccinees manifested cell-mediated immune responses. After vaccination, 69% of vaccinees overall and 89% of recipients of doses greater than or equal to 10(9) responded to S. typhi particulate or purified O polysaccharide antigens in lymphocyte replication studies but not to antigens of other Salmonella or Escherichia coli. All individuals, postvaccination, demonstrated a significant plasma-dependent mononuclear cell inhibition of wild S. typhi.

Antibodies, Bacterial↗
PubMed1987

Genetic assembly and selective toxicity of diphtheria-toxin-related polypeptide hormone fusion proteins.

The reports of Miyanohara et al. (1986) and Murphy et al. (1986) were the first to describe the genetic construction, expression, and receptor-specific selective toxicity of a chimaeric toxin. In the present report, we have extended these earlier observations and have shown that the fusion of a modified gene encoding IL-2 to a truncated diphtheria toxin gene also results in the expression of a biologically active chimaeric IL-2 toxin. In both instances we have used receptor-binding-domain substitution and have genetically coupled those portions of the diphtheria toxin structural gene that encode the ADP-ribosyl transferase activity of fragment A and lipid-associating domains of fragment B to modified genes which encode either the polypeptide hormone alpha-MSH or the T-cell growth factor IL-2. The chimaeric toxins expressed from these gene fusions have been shown to be selectively targeted to those eukaryotic cells that carry specific surface receptors for the ligand compounds of the hybrid. For example, in the case of the IL-2 toxin, it is clear that the selective action of this hybrid protein is based upon both its diphtheria-toxin and IL-2-related components. Following binding to the IL-2R on activated and/or malignant T-cell, IL-2 toxin is internalized by receptor-mediated endocytosis. Upon acidification of the endosome, diphtheria toxin fragment B portions of the chimaeric toxin facilitate the delivery of fragment A to the cytosol where it catalyses the ADP ribosylation of EF-2. The assembly of chimaeric toxins at the level of the gene offers several advantages over chemical linkage. Since chemical linkage of the toxophore and ligand components of the conjugate toxins requires activation of the epsilon-amino moiety of lysine residues with reagents that will allow for subsequent disulphide linkage, the precise site of coupling is generally not known. In addition, there has been considerable concern over the lability of the disulphide bond between the toxophore and ligand components in vivo due to the action of disulphide reductases. The assembly of chimaeric toxins at the level of the gene allows for precise linkage of the toxophore and ligand components. Since the linkage between the toxophore and ligand is a peptide bond, the chimaeric toxin should be stable in vivo. In addition, the genetic construction of chimaeric toxins also allows for further protein engineering through site-directed mutagenesis.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1986

Central, stereoselective receptors mediate the acute effects of opiate antagonists on luteinizing hormone secretion.

Although a central site of acute opiate action in regulating luteinizing hormone (LH) secretion has been suggested by the ability of centrally implanted opiate antagonists to increase LH levels, opiate antagonists are lipophilic and could influence the pituitary in situ. Also, the physiological significance of opiate receptor blockade with antagonists rests on the assumed, but untested, stereoselectivity of these receptors. Therefore, a lipophobic quaternized derivative of naltrexone (MRZ 2663-Naltrexone methobromide) and dextro- (+) and levo- (-) stereoisomers of naloxone were used to study the site- and stereoselectivity of gonadotropin responses to opiate antagonists in vivo. Male rats were injected intracerebroventricularly (icv) or intravenously (iv) with the quaternary or tertiary congeners of naltrexone and subcutaneously (sc) with (-) or (+)-naloxone. Rats injected icv with 20 ug of quaternary naltrexone displayed significant increases in serum luteinizing hormone (LH). The onset of the response was rapid with serum LH levels being significantly elevated 15 minutes after the injection and returning to basal levels 30 minutes later. Rats injected iv with 10 mg/kg of quaternary naltrexone failed to show significant LH responses. Rats injected either centrally or periphally with equivalent doses of tertiary naltrexone showed LH responses that were similar to those found in animals injected icv with quaternary naltrexone. As little as 0.5 mg/kg of (-)-naloxone resulted in significant elevations in serum LH that were higher than those elicited by up to 10 mg/kg of (+)-naloxone, indicating that this effect of naloxone is stereoselective. These data support the argument that opioids can acutely modulate LH secretion through actions at stereoselective opioid receptors in the central nervous system.

Animals↗
PubMed1986

Clinical importance of polymicrobial bacteremia.

Ninety-one episodes of polymicrobial bacteremia and fungemia were compared with 407 unimicrobial episodes to assess differences in the microbiological, epidemiological, and clinical features of the two syndromes. Enterobacteriaceae, nongroup A streptococci, anaerobic bacteria, and pseudomonads were disproportionately common in polymicrobial bacteremia. Polymicrobial episodes were significantly more likely to be hospital-acquired, to emanate from bowel or multiple foci, and to occur in patients with nonhematologic malignancies or multiple underlying diseases. Deaths directly related to sepsis were twofold higher in polymicrobial versus unimicrobial bacteremia. Factors associated with increased mortality in polymicrobial sepsis included age greater than 40 yr; absent or diminished febrile response to sepsis; absolute granulocytopenia; inadequate antimicrobial therapy for all microorganisms isolated; and a primary focus of infection in the bowel, the respiratory tract, an abscess, or an occult site. The occurrence and type of polymicrobial bacteremia can suggest a source of sepsis as well as additional diagnostic and therapeutic maneuvers.

Abscess↗