Search PubMed⌕ Search

Biomedical subjects

J R Murphy

Publications and source records attributed to J R Murphy.

At least 127 records · Page 7Linked to original sources
PubMed1992

Plasmodium falciparum: in vitro characterization and human infectivity of a cloned line.

The culture-adapted NF54 isolate of Plasmodium falciparum was subjected in vitro to three sequential limiting dilution titrations and the resulting clone was given the designation CVD1. DNA sequence analysis of the gene encoding the circumsporozoite (CS) protein revealed differences between CVD1 and the published NF54 CS gene. CVD1 had 1191 bp, 397 amino acids, and 42 repeat units while NF54 had 1218 bp, 405 amino acids, and 44 repeat units. The CVD1 clone was more sensitive to chloroquine than was the parental line, in vitro. Anopheles stephensi mosquitoes were infected equally by the cloned and uncloned parasites. Volunteers were readily infected by NF54 and CVD1 following infectious mosquito bites. The availability of a well-characterized, chloroquine-sensitive clone which safety infects humans should facilitate performance of experimental challenge studies to assess vaccine efficacy.

Amino Acid Sequence↗
PubMed1992

Anti-CD4 anti-idiotype antibodies in volunteers immunized with rgp160 of HIV-1 or infected with HIV-1.

We examined the sera of volunteers vaccinated with recombinant gp160 of human immunodeficiency virus type 1 (HIV-1) and control volunteers for the presence of anti-(anti-gp160 idiotype) antibodies which antigenically mimic gp160 and, therefore, bind to CD4 on human cells. Anti-CD4 antibodies were detected in the sera of 3 of 5 rgp160 recipients and 1 of 5 controls by indirect immunofluorescence using CD4-transfected HeLa cells or enzyme-linked immunosorbent assay (ELISA) using recombinant soluble CD4 as the solid phase. The control volunteer who was positive subsequently developed antibodies to HIV-1 by Western blot analysis. The anti-CD4 antibodies detected in the sera of the rgp160 vaccinees and the control volunteer appeared to be anti-idiotypic in nature, reacting with a paratope expressed on goat anti-gp160 antibodies but not on antibodies from normal goat serum. Binding to either transfected CD4+ HeLa cells or blotted anti-gp160 serum could be inhibited by preincubating the anti-CD4 serum with soluble CD4, or preincubating the cells or blotted anti-gp160 serum with recombinant gp160. Anti-CD4 antibodies were initially detectable only after the antibody response to gp160 began to decrease in the vaccinees, and the HIV-1-infected volunteer mounted a detectable anti-HIV-1 antibody response only after a decline in the anti-CD4 antibodies in his serum. These data strongly suggest that anti-CD4 antibodies which are anti-idiotypic to a paratope expressed on anti-gp160 antibodies are generated in response to both vaccination with rgp160 and infection with HIV-1.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS Vaccines↗
PubMed1992

Intoxication of high affinity IL-2 receptor positive thymocytes blocks early stages of T cell maturation.

Early murine fetal thymocytes express functional, high affinity IL-2 receptors as determined by: (i) the presence of IL-2R beta chain (p75) mRNA; (ii) IL-2 (10 U/ml) induced cell proliferation/cellular maturation in lobe submersion cultures (LSC). Under the influence of IL-2, early thymocytes differentiate in vitro into more mature, early single positive CD4-CD8+ followed in vivo by double positive CD4+CD8+ and single positive CD4+CD8-T and CD4-CD8+ thymocytes. Specific intoxication of high affinity IL-2R positive thymocytes by recombinant interleukin-2-diphtheria toxin-related fusion protein (DAB486-IL-2) results in transient, dose dependent blockade of in vivo and in vitro thymocyte maturation. DAB486-IL-2 induced effects upon in vivo maturation are reversible within 2 weeks after cessation of drug administration. Taken together, these results demonstrate the expression of functional, high affinity IL-2 receptors on early thymocytes. Elimination of high affinity IL-2 receptor positive thymocytes with DAB486-IL-2 results in transient blockade of T cell maturation. Since DAB486-IL-2 is now in clinical trial, it is reassuring to note that it does not permanently disrupt thymic maturation.

Animals↗
PubMed1992

Phe496 and Leu497 are essential for receptor binding and cytotoxic action of the murine interleukin-4 receptor targeted fusion toxin DAB389-mIL-4.

DAB389-mIL-4 is a murine interleukin-4 (mIL-4) diphtheria toxin-related fusion protein which has been shown to be selectively toxic to cells expressing the mIL-4 receptor. In this report, we have used site-directed and in-frame deletion mutagenesis to study the role of the putative C-terminal alpha-helix (helix E) of the mIL-4 component of DAB389-mIL-4 in the intoxication process. We demonstrate that deletion of the C-terminal 15 amino acids of the fusion toxin leads to loss of cytotoxicity. The substitution of Phe496 with either Pro, Ala or Tyr, results in a greater than 20-fold decrease in cytotoxic activity of the respective mutant fusion toxins. In addition, substitution of Leu497 with either Ala or Glu results in a similar loss of cytotoxic activity. All of these mutant forms of the mIL-4 fusion toxin demonstrate a significant decrease in binding affinity (Ki) to the mIL-4 receptor in a competitive radioligand binding assay. In marked contrast, however, the substitution of Asp495 with Asn results in a 4-fold increase in cytotoxic potency and binding affinity to mIL-4 receptor bearing cells in vitro.

Amino Acid Sequence↗
PubMed1992

DNA sequences and characterization of dtxR alleles from Corynebacterium diphtheriae PW8(-), 1030(-), and C7hm723(-).

The structural gene encoding DtxR, an iron-dependent diphtheria tox regulatory element, has recently been cloned and sequenced from the C7(-) strain of Corynebacterium diphtheriae (J. M. Boyd, M. Oza, and J. R. Murphy, Proc. Natl. Acad. Sci. USA 87:5972, 1990). We report here the molecular cloning, DNA sequence analysis, and characterization of DtxR from the PW8(-), 1030(-), and C7hm723 strains of C. diphtheriae. While the sequence of dtxR from PW8(-) is identical to that of the C7(-) allele, the sequence of dtxR from the 1030(-) strain is only 91.4% identical; however, the deduced amino acid sequence of DtxR from 1030(-) differs by only 6 of 678 amino acids. Moreover, DtxR from all three strains is shown to regulate expression of beta-galactosidase from a tox promoter-operator (toxPO)-lacZ transcriptional fusion. In contrast, the dtxR allele from the iron-insensitive tox constitutive mutant C7hm723 was found to have a single G----A transition, resulting in a substitution of Arg-47 to His and the loss of tox regulatory activity in recombinant Escherichia coli.

Alleles↗
PubMed1991

Immunotoxins and cytokine toxin fusion proteins.

Paul Ehrlich first suggested the simple and elegant concept of creating specific cell toxins or "magic bullets" through the fusion of cell-specific antibodies and toxins. In practice it has proven difficult to create safe and effective "magic bullets." In the past several years, several immunotoxins have been applied to clinical testing. These immunotoxins have been created by the biochemical coupling of cell- or lineage-specific monoclonal antibodies to plant toxins or fragments thereof. These immunotoxins have been used to treat bone marrow transplant recipients and patients with autoimmune disorders. In recent years, another strategy has also been pursued to create hybrid toxins. Rather than use antibodies as the targeting moiety, cytokines have been used to target a select population of cells bearing a high copy number of receptors for the specific cytokine. Rather than biochemically couple a cytokine to the toxin, the cytokine and toxin are fused by a peptide bond established via genetic engineering. A prototype IL-2 diphtheria toxin-related fusion protein is now being tested in the clinic for treatment of hematopoietic malignancies and autoimmune disorders.

Animals↗
PubMed1991

Diphtheria toxin-related alpha-melanocyte-stimulating hormone fusion toxin. Internal in-frame deletion from Thr387 to His485 results in the formation of a highly potent fusion toxin which is resistant to proteolytic degradation.

We have previously reported the genetic construction and properties of a fusion protein which was composed of the enzymatically active and membrane translocation domains of the diphtheria toxin and the receptor-specific ligand alpha-melanocyte-stimulating hormone (alpha-MSH) (Murphy, J.R., Bishai, W., Borowski, M., Miyanohara, A., Boyd, J., and Nagle, S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8258-8262). While this fusion toxin was found to be selectively toxic for MSH receptor-bearing cells in vitro, it was subject to profound proteolytic degradation in recombinant Escherichia coli making purification difficult. We now report that the deletion of diphtheria toxin fragment B sequences between Thr387 and His485 results in a protease-resistant form of the fusion toxin, DAB389-alpha-MSH. We show that DAB389-alpha-MSH is expressed in high yield in recombinant Escherichia coli, that it is readily purified from crude bacterial lysates by immunoaffinity and high performance liquid chromatography, and its cytotoxic activity toward both human and murine malignant melanoma cell lines is mediated through the MSH receptor.

Animals↗
PubMed1991

Selective elimination of HIV-1-infected cells with an interleukin-2 receptor-specific cytotoxin.

Infection by human immunodeficiency virus type 1 (HIV-1) is associated with cellular activation and expression of the interleukin-2 (IL-2) receptor. A genetically engineered fusion toxin, DAB486 IL-2, that contains the enzymatic site and translocation domain of diphtheria toxin and the receptor binding domain of IL-2 specifically kills cells that express high-affinity IL-2 receptors. This toxin selectively eliminated the HIV-1-infected cells from mixed cultures of infected and uninfected cells and inhibited production of viral proteins and infectious virus. Thus, cellular activation antigens present a target for early antiviral intervention.

Cell Survival↗
PubMed1991

Interleukin 4 receptor targeted cytotoxicity: genetic construction and in vivo immunosuppressive activity of a diphtheria toxin-related murine interleukin 4 fusion protein.

The interleukin 4 (IL 4) receptor is expressed on various cells of the immune system, including T and B lymphocytes, macrophages and mast cells. We have constructed a recombinant protein, DAB389-mIL 4, that is composed of the enzymatically active and membrane translocation domains of diphtheria toxin fused to murine IL 4. We demonstrate that this fusion toxin selectively inhibits protein synsthesis in eukaryotic cells which express the murine IL 4 receptor. The cytotoxic potency of this fusion toxin is shown to be directly proportional to the reported number of IL 4 receptors on the surface of target cells. Since the action of DAB389-mIL 4 can be blocked with either excess mIL 4 or antibody to mIL 4, we conclude that its entry into target cells is mediated through the mIL 4 receptor. A mutant form of DAB389-mIL 4, DA(197)B389-mIL 4, in which the fragment A-associated ADP-ribosyltransferase is inactive, is not cytotoxic to murine IL 4 receptor-bearing cells. Finally, we demonstrate that DAB389-mIL 4 administered subcutaneously to DBA/2 mice results in suppression of delayed-type hypersensitivity (DTH); whereas, the non-toxic DA(197)B389-mIL 4 fails to dampen the DTH response.

Animals↗
PubMed1991

Conformational perturbation of interleukin-2: a strategy for the design of cytokine analogs.

Interleukin-2 (IL-2) is a representative of a growing family of small proteins termed lymphokines which are responsible for mediating cell differentiation, growth and function in the immune system. Many of these proteins are being evaluated for their clinical potential. From the perspective of drug development, structure-function analysis of these molecules and their receptors require the use methodologies different than those traditionally employed for small peptides and other natural products. However, similar pharmacologic principles apply and an understanding of ligand-receptor interactions and the associated responses is required in order to efficiently pursue agonist and antagonist design. Although IL-2 is a protein of only 133 amino acid residues for which a low resolution X-ray structure does exist, the complexity of its receptor system has provided an added challenge to structure-function studies. Consequently, little is known concerning the receptor contact residues for this protein. We have attempted to utilize established principles of protein and peptide structure to manipulate the conformation of IL-2 in a manner which has provided analogs helpful for receptor interaction studies. These proteins have not only providing useful information on the nature of the IL-2 receptor but have also revealed potential strategies for the design of IL-2 agonists and antagonists.

Amino Acid Sequence↗
PubMed1991

Luteinizing hormone sensitivity to naloxone in maturing male chimpanzees.

We have systematically investigated the involvement of endogenous opioids in gonadotropin secretion during primate sexual maturation by examining LH/FSH responses to gonadotropin-releasing hormone (GnRH) and changes in LH secretion during infusions of saline or naloxone, an opiate antagonist, in ten male chimpanzees between one and nine years of age. Animals were anesthetized with ketamine (10 mg/kg) and injected or infused IV with GnRH, naloxone or saline. Circulating levels of serum LH were elevated to the same extent (approximately 400%) in response to GnRH (100 micrograms) in animals 1-5 years old (juvenile) and in animals 6-9 years old (pubertal). No differences were noted between the two groups in GnRH-stimulated levels of serum FSH. During treatment with naloxone (0.14 mg/kg bolus followed by 0.2 mg/kg/h maintenance infusion for 3 h), serum LH levels in pubertal animals were significantly (p less than 0.05) elevated by as much as 95% over LH levels found during treatment with saline. Juvenile animals, on the other hand, failed to demonstrate significant increases in serum LH following naloxone at the doses tested. A strong correlation (r = .84) was found between circulating testosterone and serum LH levels during naloxone treatment. These data indicate that opioid inhibition of LH secretion can be reversed by naloxone only when puberty is reached in chimpanzees and suggest an alteration in opioid regulation of GnRH near the time of puberty. The strong correlation between testosterone levels and LH responses to naloxone suggests that steroids may participate in the maturation of opioid control of LH during puberty of nonhuman primates.

Aging↗
PubMed1991

Protein engineering of diphtheria-toxin-related interleukin-2 fusion toxins to increase cytotoxic potency for high-affinity IL-2-receptor-bearing target cells.

We have used site-directed insertion and point mutagenesis in an attempt to increase the cytotoxic potency and receptor-binding affinity of the diphtheria-toxin-related interleukin-2 (IL-2) fusion toxins. Previous studies have demonstrated that both the DAB486-IL-2 and DAB389-IL-2 forms of the fusion toxin consist of three functional domains: the N-terminal fragment-A-associated ADP-ribosyltransferase, the hydrophobic-membrane-associating domains, and the C-terminal receptor-binding domain of human IL-2. By insertion mutagenesis we have increased the apparent flexibility of the polypeptide chain between the membrane-associating domains and the receptor-binding domain of this fusion toxin. In comparison to DAB486-IL-2, the cytotoxic potency of the insertion mutants was increased by approximately 17-fold for high-affinity IL-2-receptor-bearing cell lines in vitro. Moreover, competitive displacement experiments using [125I]rIL-2 demonstrate that the increase in cytotoxic potency correlates with an increase in receptor-binding affinity for both the high and intermediate forms of the IL-2 receptor.

Amino Acid Sequence↗
PubMed1991

Diphtheria toxin receptor-binding domain substitution with interleukin 6: genetic construction and interleukin 6 receptor-specific action of a diphtheria toxin-related interleukin 6 fusion protein.

We have genetically replaced that portion of the diphtheria toxin structural gene which encodes the native receptor-binding domain with a synthetic gene encoding the cytokine interleukin 6 (IL-6/IFN-beta 2/BSF-2). The resulting gene fusion encodes the chimeric toxin DAB389-IL-6. Following expression and purification, we demonstrate that DAB389-IL-6 is selectively cytotoxic for eukaryotic cells bearing the interleukin 6 receptor. In addition, the cytotoxic action of DAB389-IL-6 is shown to require binding to the IL-6 receptor, internalization by receptor-mediated endocytosis and passage through an acidic compartment. Following the delivery of the catalytically active fragment A to the cytosol of target cells, cellular protein synthesis is inhibited by the ADP-ribosylation of elongation factor 2. While eukaryotic cells which are devoid of the IL-6 receptor are uniformly resistant to the action of this fusion toxin, the data presented suggest that a minimal number of IL-6 receptors may be necessary to mediate the internalization of sufficient levels of DAB389-IL-6 to result in the intoxication of target cells.

Animals↗