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Biomedical subjects

J R Murphy

Publications and source records attributed to J R Murphy.

At least 91 records · Page 5Linked to original sources
PubMed1994

Crystallization and preliminary X-ray studies of the diphtheria Tox repressor from Corynebacterium diphtheriae.

Crystals of the diphtheria tox repressor (DtxR) from Corynebacterium diphtheriae suitable for structure determination have been obtained. DtxR activated with transition metal ions represses the expression of the structural gene for the diphtheria toxin, tox, which is encoded on the genome of a family of closely related corynebacteriophages. The space group of the obtained crystals is trigonal P3(1)21 or its enantiomorph P3(2)21 with a = b = 64.2 A, c = 220.5 A, alpha = beta = 90 degrees, gamma = 120 degrees. Two monomers comprise the asymmetric unit. The crystals diffract to a resolution of better than 3 A.

Bacterial Proteins↗
PubMed1994

Low doses of interferon-alpha are as effective as higher doses in inducing remissions and prolonging survival in chronic myeloid leukemia.

OBJECTIVE: To determine the toxicity and efficacy of low-dose interferon-alpha therapy in inducing remissions and prolonging survival in patients with chronic myeloid leukemia. DESIGN: Phase II evaluation and comparison with historical control patients and other series in which the investigators used higher interferon-alpha doses. SETTING: Tertiary care leukemia research clinic. PATIENTS: 41 patients with newly diagnosed or previously treated chronic-phase, Philadelphia chromosome-positive chronic myeloid leukemia received interferon-alpha at a dose of 2 x 10(6) U/m2 body surface area daily for 28 days and then three times weekly. MEASUREMENTS: Complete blood counts and physical examinations were done monthly to determine hematologic remission and toxicity. To determine karyotypic response, bone marrow cytogenetic analyses were done at 6 monthly intervals in patients who achieved a complete hematologic remission. In addition, Kaplan-Meier survival curves and median survival values were generated from diagnosis and the start of therapy with interferon-alpha. RESULTS: 70% of patients treated with low-dose interferon-alpha within 1 year of diagnosis achieved a complete hematologic remission, and 22% of these patients had a major or complete karyotypic response. Investigators who used higher interferon-alpha doses in similar patient populations have reported complete hematologic remission rates of 59% to 70% and major and complete cytogenetic response rates of 16% to 29%. The Kaplan-Meier estimated 5-year survival rate of minimally pretreated patients in our study is 73% (95% CI, 51% to 95%), which compares favorably with survivals reported by investigators who used higher doses. The estimated yearly cost of the interferon-alpha used in our study is $5953 compared with a median of $24,375 for the higher doses used by other investigators. Less toxicity was also observed. CONCLUSION: Low-dose interferon-alpha is as effective as higher-dose interferon-alpha in inducing remissions and prolonging survival in patients with chronic myeloid leukemia but is considerably less expensive and toxic.

Adolescent↗
PubMed1994

Determination of the minimal essential nucleotide sequence for diphtheria tox repressor binding by in vitro affinity selection.

The expression of diphtheria toxin in lysogenic toxigenic strains of Corynebacterium diphtheriae is controlled by the heavy metal ion-activated regulatory protein DtxR. In the presence of divalent heavy metal ions, DtxR specifically binds to the diphtheria tox operator and protects a 27-bp interrupted palindromic sequence from DNase I digestion. To determine the consensus DNA sequence for DtxR binding, we have used gel electrophoresis mobility-shift assay and polymerase chain reaction (PCR) amplification for in vitro affinity selection of DNA binding sequences from a universe of 6.9 x 10(10) variants. After 10 rounds of in vitro affinity selection, each round coupled with 30 cycles of PCR amplification, we isolated and characterized a family of DNA sequences that function as DtxR-responsive genetic elements both in vitro and in vivo. Moreover, these DNA sequences were found to bind activated DtxR with an affinity similar to that of the wild-type tox operator. The DNA sequence analysis of 21 unique in vitro affinity-selected binding sites has revealed the minimal essential nucleotide sequence for DtxR binding to be a 9-bp palindrome separated by a single base pair.

Bacterial Proteins↗
PubMed1994

An intact transmembrane helix 9 is essential for the efficient delivery of the diphtheria toxin catalytic domain to the cytosol of target cells.

To investigate structure/function relationships involved in the delivery of the diphtheria toxin (DT) catalytic (C) domain to the cytosol of target cells, we have constructed and characterized internal in-frame deletion mutants in the transmembrane (T) domain of the fusion toxin DAB389IL-2. This fusion protein is composed of the C and T domains of DT to which human interleukin-2 (IL-2) has been genetically fused. The mutant fusion toxins were compared to DAB389IL-2 with respect to cytotoxic potency, receptor binding affinity, channel formation in planar lipid membranes, and sensitivity to proteolytic digestion. We demonstrate that genetic fusion of human IL-2 sequences to a diphtheria toxin-related fragment that contains less than full-length transmembrane helix 9 results in a fusion protein that binds to the high affinity IL-2 receptor, but has lost > or = 3 logs of cytotoxic potency and has decreased ability to insert into planar membranes and form stable channels. These observations are consistent with the hypothesis that an intact transmembrane helix 9 is essential for the formation of stable channels that are required for the efficient delivery of the C domain to the cytosol of target cells.

Amino Acid Sequence↗
PubMed1994

Sample size determination for an exponential survival model with an unrestricted covariate.

We derive formulae for estimating sample size and power for detecting the effect of an unrestricted covariate on survival time. These are useful in designing survival studies with different patterns of recruitment and follow-up when survival time is exponentially distributed. We use the asymptotic covariance matrix, conditional expectation and Taylor's expansion techniques to develop these formulae. Computer simulations indicate that the asymptotic approximations used in developing the formulae are good over a range of parameter values and different patterns of recruitment and follow-up that are relevant to survival studies.

Computer Simulation↗
PubMed1994

Diphtheria toxin-related cytokine fusion proteins: elongation factor 2 as a target for the treatment of neoplastic disease.

We have used protein engineering and recombinant DNA methodologies in order to construct a fusion protein in which human interleukin-2 (IL-2) is genetically linked to the catalytic and transmembrane domains of diphtheria toxin. The fusion toxin, DAB486IL-2, is highly cytotoxic for only those cells which display the high affinity interleukin-2 receptor (IL-2R) on their surface. In phase I/II clinical studies the intravenous administration of DAB486IL-2 has been found to be safe, well tolerated and may lead to the induction of durable remissions in patients presenting with a variety of IL-2R positive lymphomas.

Antineoplastic Agents↗
PubMed1994

Treatment of ALS with high dose pulse cyclophosphamide.

Autoimmune abnormalities have been described in sporadically acquired amyotrophic lateral sclerosis (ALS), but ALS patients do not benefit from conventional immunosuppressive therapy. Because Multifocal Motor Neuropathy causes a syndrome mimicking the lower motor neuron involvement in ALS and responds to high dose cyclophosphamide, we tested whether ALS patients would respond to the same treatment. Eighteen patients with classical ALS initially received a high loading dose of intravenous cyclophosphamide (3 g/m2) followed by 6-monthly injections of 750-1000 mg/m2. We monitored isometric strength, fine motor coordination and pulmonary function monthly for 3 months prior to the study, during the treatment phase, and for 6 months after treatment. Treatment subjects were matched to control subjects from the WALS natural history data base and compared on the basis of decline rate (megaslopes). Treatment did not alter the course of ALS.

Adult↗
PubMed1994

Inhibition of AIDS-associated Kaposi's sarcoma cell growth by DAB389-interleukin 6.

Acquired immune deficiency syndrome-associated Kaposi's sarcoma (AIDS-KS)-derived spindle cells produce and use interleukin 6 (IL-6) among several other cytokines as a growth factor. In this study we show that AIDS-KS cells express approximately 1100 high-affinity IL-6 receptors (IL-6R) per cell with a dissociation constant (Kd) of 110 pM. Furthermore, AIDS-KS cells express the IL-6R alpha subunit, detected as a single 5.0-kb messenger ribonucleic acid species, and the high-affinity converting, signal-transducing IL-6R beta subunit designated as gp130. Similarly, tumor tissue obtained from patients with KS and AIDS expresses IL-6R messenger ribonucleic acid. We have exploited the chimeric fusion toxin DAB389-IL-6, which exerts cellular toxicity only to the cells expressing IL-6R. This chimeric protein was engineered by fusion of a truncated diphtheria toxin structural gene, in which the region encoding the native receptor-binding domain was removed and replaced with the gene encoding IL-6. DAB389-IL-6 inhibited protein synthesis in AIDS-KS-derived spindle cells at very low concentrations (IC50 of 3.4 x 10(-11) M). Similarly, inhibition of cell viability by DAB389-IL-6 was observed at equivalent dose levels (IC50 of 5 x 10(-11)). These effects on protein synthesis and cell viability can be abrogated by recombinant human IL-6, indicating receptor specificity. Thus, DAB389-IL-6 is a potential agent for the treatment of AIDS-associated KS.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Maintenance of the hydrophobic face of the diphtheria toxin amphipathic transmembrane helix 1 is essential for the efficient delivery of the catalytic domain to the cytosol of target cells.

The transmembrane (T) domain of diphtheria toxin (DT) comprises nine alpha-helices and has been shown to play an essential role in the efficient delivery of the catalytic (C) domain of DT across the eukaryotic cell membrane and into the cytosol. We have demonstrated recently that the first three amphipathic helixes of the T domain, although not necessary for either channel formation or receptor binding, are required for the efficient transmembrane delivery of the C domain. In the present study, we have performed a detailed structure-function analysis of T domain helix 1 (TH1) of the DT-related fusion protein DAB389IL-2. We performed exchange and site-directed mutagenesis of TH1 and the resulting mutant fusion toxins were analyzed by gel electrophoresis and tested for their efficiencies in the delivery of the C domain to the cell cytosol. We demonstrate that the overall charge distribution and hydrophobicity of amino acids in the amphipathic helix TH1, rather than a specific amino acid sequence, are critical for the function of this helix. The insertion of a charged residue in the hydrophobic face of TH1 abolishes cytotoxic activity, whereas replacement of a hydrophobic residue by a charged amino acid in the hydrophilic face of the helix has little, if any, effect on cytotoxic activity. In addition, we have identified Ser220 by site-directed mutagenesis as a residue that appears to be critical for correct folding of the fusion toxin. Mutations in this position result in fusion proteins that are extremely sensitive to proteolytic attack.

Amino Acid Sequence↗
PubMed1994

Iron, DtxR, and the regulation of diphtheria toxin expression.

In recent years considerable advances have been made in the understanding of the molecular basis of iron-mediated regulation of diphtheria toxin expression. The tox gene has been shown to be regulated by the heavy metal ion-activated regulatory element DtxR. In the presence of divalent heavy metal ions, DtxR becomes activated and binds to a 9 bp interrupted palindromic sequence. The consensus-binding site has been determined by both the sequence analysis of DtxR-responsive operators cloned from genomic libraries of Corynebacterium diphtheriae as well as by in vitro genetic methods using cyclic amplification of selected targets (CASTing). It is now clear that DtxR functions as a global iron-sensitive regulatory element in the control of gene expression in C. diphtheriae. In addition, the metal ion-activation domain of DtxR is being characterized by both mutational analysis and determination of the X-ray structure at 3.0 A resolution.

Bacterial Proteins↗
PubMed1994

A structured literature review for risk assessment: EMF and human health risk.

The volume of publications on any given topic makes it difficult to select an appropriate subset of publications for review by an expert panel. This paper proposes a method for selecting a subset using a protocol that attempts to order publications based upon good research methods. The protocol was followed by graduate students with no expert knowledge of the area they were reviewing. They reduced 11730 articles on the topic of EMF and Health risk to 68 articles that were reviewed by an expert panel. The interrater reliability was 96% and 94% of the relevant articles were captured by this process.

Animals↗
PubMed1994

Epitope tagging of DAB389IL-2: new insights into C-domain delivery to the cytosol of target cells.

The fusion toxin DAB389IL-2 is composed of the catalytic (C) and transmembrane (T) domains of native diphtheria toxin to which human interleukin-2 (IL-2) has been genetically fused (1,2). Following binding to the IL-2 receptor, the fusion toxin is internalized by receptor mediated endocytosis, and upon acidification of the endocytic vesicle, the T domain spontaneously inserts into the membrane, and facilitates the delivery of the C domain to the cytosol (3,4). In order to further study the process by which the C domain is delivered to the target cell cytosol, we genetically fused an eleven amino acid epitope derived from the vesicular stomatitis virus (VSV) G protein to the N-terminal end of DAB389IL-2. The epitope labelled fusion toxin, VSV-G-DAB389IL-2, was found to retain IL-2 receptor specific binding and cytotoxic activity. Target cells were incubated for various times in the presence of VSV-G-DAB389, fixed and then treated with anti-VSV G and FITC conjugated secondary antibody. Laser scanning confocal microscopy was used to determine the location of the fluorescent signal. The VSV-G epitope tagged fusion toxin was found only to be associated with small vesicles that were situated adjacent to the plasma membrane. These results suggest that the C domain of the fusion toxin is associated with an early intracellular compartment and is rapidly delivered to the cytosol. Since channel formation by the T domain is necessary for the delivery of the C domain, it follows that T domain insertion into the membrane also occurs early in the intoxication pathway.

Amino Acid Sequence↗
PubMed1993

Cysteine-102 is positioned in the metal binding activation site of the Corynebacterium diphtheriae regulatory element DtxR.

DNA sequence analysis of dtxR has shown that the M(r) 25,316 regulatory protein contains a single cysteine residue at position 102. DtxR readily forms inactive disulfide-linked dimers. We have used saturation site-directed mutagenesis of the cysteine codon (TGC) at position 102 in order to determine the role of this residue in metal ion binding. We show that the insertion of amino acids other than cysteine or aspartic acid into this position abolishes DtxR function both in vitro and in recombinant Escherichia coli DH5 alpha:lambda RS45toxPO/lacZ. Only those mutant alleles in which the TGC codon for Cys-102 was replaced by either TGT (Cys) or GCA (Asp) were found to direct the expression of active forms of DtxR that regulate the expression of beta-galactosidase from the toxPO/lacZ transcriptional fusion.

2,2'-Dipyridyl↗
PubMed1993

beta-Carotene inhibits rectal mucosal ornithine decarboxylase activity in colon cancer patients.

Colonic adenocarcinoma affects approximately 6% of adults in many Western countries. beta-Carotene (BC), a safe, inexpensive, and widely available compound, has been proposed as a cancer chemopreventive agent. To evaluate whether BC shows promise as an inhibitor of colonic carcinogenesis, we studied 20 male subjects who had previously undergone resection of colonic adenocarcinoma. Each subject received beta-carotene, 30 mg orally, daily for 6 months. Rectal mucosa was sampled at multiple intervals prior to, during, and following BC administration. Mucosal ornithine decarboxylase (ODC) activity and serum and mucosal BC concentrations were determined at each interval. ODC activity was inhibited by 44% (P < 0.05) and 57% (P < 0.01) after 2 and 9 weeks, respectively, of BC administration and remained low compared with baseline even 6 months following discontinuation of BC. Serum and mucosal BC concentrations increased as expected during BC administration and remained elevated for 6 months following BC discontinuation. The demonstrated inhibition of rectal mucosal ODC activity in these patients with resected colon cancer suggests that BC may prove useful as a cancer chemopreventive agent.

Aged↗