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Biomedical subjects

J R Murphy

Publications and source records attributed to J R Murphy.

At least 55 records · Page 3Linked to original sources
PubMed1997

Characterization and receptor specific toxicity of two diphtheria toxin-related interleukin-3 fusion proteins DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3.

We have constructed two fusion proteins, DAB389-mIL-3 and DAB389-(Gly4Ser)2-mIL-3, in which the receptor-binding domain of diphtheria toxin is replaced by mouse interleukin-3 (IL-3). Cytotoxic activity of the fusion toxins was observed on three out of six cell lines assayed. This toxicity was mediated through binding to the IL-3 receptor as it was inhibited in a dose-dependent manner with murine IL-3 or anti-IL-3 neutralizing antibodies. DAB389-(Gly4Ser)2-mIL-3 was up to 5 times more toxic than DAB389-mIL-3, depending on the cell line (0.8 x 10(-10) M < IC50 < 3 x 10(-10) M). These proteins can be used for the detection of IL-3 receptors on mouse cells and should allow for the selective elimination of IL-3 receptor-positive pluripotent hematopoietic stem cells prior to bone marrow transplantation.

Animals↗
PubMed1997

Inhibition of protein synthesis in small cell lung cancer cells induced by the diphtheria toxin-related fusion protein DAB389 GRP.

DAB389 GRP is composed of the catalytic and transmembrane domains of diphtheria toxin fused to gastrin-releasing peptide (GRP). DAB389 GRP is selectively targeted to, and inhibits protein synthesis in, cell lines expressing GRP receptors. Protein synthesis in 5'ET4 cells (BALB/3T3 fibroblasts transfected with the gene encoding the GRP receptor) was inhibited by 50% in the presence of 20 pM DAB389 GRP (IC50, 20 pM). DAB389 GRP did not inhibit protein synthesis in untransfected BALB/3T3 cells. A second neuropeptide-conjugated toxin, DAB389 SP, directed to cells expressing substance P receptors, was not cytotoxic to 5'ET4 cells, nor was DAB389 GRP cytotoxic to substance P receptor-bearing cells. DAB389 GRP cytotoxic effects were receptor specific and were inhibited either by excess GRP or anti-GRP antibody. Cytotoxicity was mediated by passage through an acidic vesicle, because addition of 10 microM chloroquine to the reaction inhibited cytotoxicity. DAB389 GRP and DAB389 SP were tested on a number of tumor cell lines. DAB389 GRP inhibited protein synthesis in AR42J rat pancreatic acinar cells and HuTu 80 human duodenal adenocarcinoma cells with IC50s of 65 and 200 pM, respectively. DAB389 SP had an IC50 of 9.5 pM for the AR42J cells and 12 nM for the HuTu 80 cell line. A number of small cell lung cancer cell (SCLC) lines were tested, and the IC50 for DAB389 GRP ranged from 1.1 to 85 nM. Sensitivity to DAB389 GRP appeared to be based on receptor number and receptor type (i.e., GRP or neuromedin B preferring). SCLC cells were also sensitive to DAB389 SP, with IC50s ranging from 2.4 to 11.5 nM. These results suggest that a potential use exists for diphtheria-based fusion toxins as therapeutic agents for treatment of SCLC and other neuropeptide receptor-bearing cancers.

3T3 Cells↗
PubMed1997

A logistic dose-ranging method for phase I clinical investigations trials.

This paper describes an alternative to the continual reassessment method (CRM) for phase I trials. The logistic dose ranging strategy (LDRS) uses logistic regression and a dose allocation scheme similar to the CRM. It can easily be implemented from any logistic regression program. The LDRS can be a stand alone dose allocation scheme or it can be incorporated into standard three on a dose strategies to indicate when escalation can proceed more rapidly. Finally, the effect of covariates such as age or comorbid conditions on the toxicity expected for the dose selected for a phase II trial can be examined.

Bayes Theorem↗
PubMed1997

Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery.

After reaching early endosomes by receptor-mediated endocytosis, diphtheria toxin (DT) molecules have two possible fates. A large pool enters the degradative pathway whereas a few molecules become cytotoxic by translocating their catalytic fragment A (DTA) into the cytosol. Impairment of DT degradation by microtubule depolymerization does not block DT cytotoxicity. Therefore, DTA membrane translocation into the cytosol occurs from an endocytic compartment located upstream of late endosomes. Comparisons between early endosomes and endocytic carrier vesicles in a cell-free translocation assay have demonstrated that early endosomes are the earliest endocytic compartment from which DTA translocates. DTA translocation is ATP-dependent, requires early endosomal acidification, and is increased by the addition of cytosol. Cytosol-dependent DTA translocation is GTP gamma S-insensitive but is blocked by anti-beta COP antibodies.

Animals↗
PubMed1997

Standardization of measurement of immunoglobulin-secreting cells in human peripheral circulation.

A sensitive, and at times the most sensitive, measurement of human vaccine immunogenicity is enumeration of antibody-secreting cells (ASC) in peripheral blood. However, this assay, which is inherently capable of measurement of the absolute number of antigen-specific ASC, is not standardized. Thus, quantitative comparison of results between laboratories is not currently possible. To address this issue, isotype-specific ASC were enumerated from paired fresh and cryopreserved mononuclear cell (MNC) preparations from healthy adult volunteers resident in either the United States (US group) or Egypt (EG group). Analysis of fresh cells from US volunteers revealed mean numbers of ASC per 10(6) MNC of 617, 7,738, and 868 for immunoglobulin M (IgM), IgG, and IgA, respectively, whereas EG volunteers had 2,086, 7,580, and 1,677 ASC/10(6) MNC for the respective isotypes. Cryopreservation resulted in a slight reduction in group mean IgM, IgG, and IgA ASC (maximum reduction in group mean, 14%), but in no instance were results obtained with cryopreserved cells significantly lower than those obtained with fresh cells. To determine if cryopreservation affected the number of bacterial antigen-specific ASC detected, cells from a group of US adult volunteers who received a single oral dose of a mutated Escherichia coli heat-labile enterotoxin (LT(R192G)) were tested. There was no significant difference (P > 0.05) in the number of antigen-specific IgA or IgG ASC detected between fresh and cryopreserved MNC. The results support the views that ASC assays can be standardized to yield quantitative results and that the methodology can be changed to make the test more practical.

Adult↗
PubMed1997

Sleep in detoxified alcoholics: impairment of most standard sleep parameters and increased risk for sleep apnea, but not for myoclonias--a controlled study.

OBJECTIVE: To assess recently alcohol-abstinent chronic alcoholic patients for selected parameters indicative of sleep quality. METHOD: Patients (n = 24, 14 male), abstinent 3-6 weeks, and healthy controls (n = 20) were admitted to a clinical sleep unit. Measurements included sleep respiratory events and periodic limb movements, using strict methodology. RESULTS: Clear signs of sleep deterioration and a high prevalence of apneic/hypopneic episodes were observed. Apneas were found at the same frequency for men and women; this has not been described before. No periodic limb movement was found. CONCLUSIONS: Considering the high degree of morbidity and mortality observed in sleep apneic syndrome, systematic screening for sleep apneas is recommended for alcoholics seeking help.

Adult↗
PubMed1996

Genetic construction and properties of a diphtheria toxin-related substance P fusion protein: in vitro destruction of cells bearing substance P receptors.

We have genetically replaced the native receptor binding domain of diphtheria toxin with an extended form of substance P (SP): SP-glycine (SP-Gly). The resulting fusion protein, DAB389SP-Gly, is composed of the catalytic and transmembrane domains of diphtheria toxin genetically coupled to SP-Gly. Because native SP requires a C-terminal amide moiety to bind with high affinity to the SP receptor, the precursor form of the fusion toxin, DAB389SP-Gly, was converted to DAB389SP by treatment with peptidylglycine-alpha-amidating monooxygenase. We demonstrate that following conversion, DAB389SP is selectively cytotoxic for cell lines that express either the rat or the human SP receptor. We also demonstrate that the cytotoxic action of DAB389SP is mediated via the SP receptor and dependent upon passage through an acidic compartment. To our knowledge, this is the first reported use of a neuropeptide as the targeting ligand for a fusion toxin; and the first instance in which an inactive precursor form of a fusion toxin is converted to the active form by a posttranslational modification.

Amino Acid Sequence↗
PubMed1996

DAB389 interleukin-2 receptor binding domain mutations. Cytotoxic probes for studies of ligand-receptor interactions.

Site-directed mutagenesis was used to generate point mutations in the diphtheria toxin-related fusion protein, DAB389 interleukin-2 (IL-2). Thr-439, in the IL-2 receptor binding domain of the fusion toxin, was changed to a Pro residue. The resultant fusion toxin, DAB389 IL-2(T439P), was 300-fold less cytotoxic than wild type DAB389 IL-2, partially as the result of a 100-fold decrease in binding affinity for the high affinity form of the IL-2 receptor. However, DAB389 IL-2(T439P) stimulated DNA synthesis to a greater extent than expected. Studies of intoxication kinetics indicated that the increased stimulation might result from an increased contact time between the mutated IL-2 receptor binding domain and the receptor, perhaps due to a decreased internalization rate. Another mutant, DAB389 IL-2(Q514D), in which a Gln residue at position 514 was changed to an Asp, was 2000-fold less cytotoxic than wild type DAB389 IL-2. This mutant had a 50-fold decrease in binding affinity, did not stimulate DNA synthesis and also had a reduced rate of intoxication. Gln-514 appears to play a role in receptor binding and activation, whereas Thr-439 appears to be involved with receptor binding and signaling internalization of the fusion toxin-receptor complex.

Cell Line↗
PubMed1996

A clinical trial of verapamil in amyotrophic lateral sclerosis.

Seventy-two patients with amyotrophic lateral sclerosis (ALS) were enrolled in a clinical trial of the efficacy of verapamil in the treatment of ALS. In period 1 (pretreatment, months 1-3) and period 3 (posttreatment, months 10-12), patients received no drug. In period 2 (months 4-9), patients received verapamil. The slopes of declining pulmonary function and limb megascores were not significantly different during drug treatment compared to natural history and washout periods. Thus, verapamil was ineffective in slowing the clinical progression in ALS patients. Controlled trials using a natural history period may represent a faster and less expensive method of screening drugs for ALS compared to placebo-controlled trials.

Amyotrophic Lateral Sclerosis↗
PubMed1996

Use of kappa statistic in determining validity of quality filtering for meta-analysis: a case study of the health effects of electromagnetic radiation.

The objective of this study was to determine the validity and reliability achieved in employing trained graduate students to read and evaluate the methodologic content of papers analyzing the health effects of electromagnetic field radiation. The search identified 11,730 references from multiple sources, of which 8759 were unique and from which 97 were selected for inclusion in a meta-analysis. Without reference to the graduate students' assessments, an epidemiologist read and scored all 97 articles as well. A 10% random sample of the 97 articles was selected for a repeat assessment by the epidemiologist, who did not review her first assessment. Interrater validity was good (kappa > or = 0.45-0.75) for 15 of the 23 criteria. Intrarater reliability was excellent (kappa > 0.75) or good in 21 of the 23 rating criteria. We concluded that valid and reliable results can be obtained by training graduate students to screen papers for methodologic content.

Adult↗
PubMed1996

Protein engineering and design for drug delivery.

Over the past decade considerable effort has been focused on the genetic construction, expression, and characterization of fusion proteins designed to selectively target specific disease-causing cells and/or extracellular targets. In each instance, the coding sequences for the functional domains of different proteins have been genetically fused, resulting in the formation of fusion proteins which retain the function of their component segments. As anticipated from their design, many of these fusion proteins offer a marked therapeutic advantage over conventional agents in preclinical studies. While several recombinant fusion proteins have entered or will shortly begin Phase I/II human clinical trials, the first of these fusion proteins is currently being evaluated in Phase III human clinical trials for the treatment of cutaneous T cell lymphoma.

Amino Acid Sequence↗
PubMed1996

Identification of the primary metal ion-activation sites of the diphtheria tox repressor by X-ray crystallography and site-directed mutational analysis.

The diphtheria tox repressor, DtxR, is a 226 amino acid transition metal ion-activated regulatory protein that controls the expression of diphtheria toxin in toxigenic Corynebacterium diphtheriae. The previously solved three-dimensional DtxR structures have identified two potential metal ion binding sites which may play a role in the activation of DNA binding by the repressor. We have used both X-ray crystallographic and site-directed mutational analysis of DtxR(C102D)-Ni2+ complexes and DtxR to identify the metal ion-binding site which results in the activation of the repressor. We demonstrate that DtxR contains both a primary and an ancillary metal ion binding site. The primary site functions directly in the activation of DNA binding. In contrast, the ancillary site contributes weakly, if at all, to activation.

Bacterial Proteins↗
PubMed1996

Uniform matrix stability study designs.

Uniform matrix designs for drug stability studies are introduced and compared to standard matrix designs. Comparisons are made on the basis of design moment, D-efficiency, uncertainty, G-efficiency, and statistical power. It is shown that uniform matrix designs provide superior statistical properties with the same or fewer design points than standard matrix designs.

Chemistry, Pharmaceutical↗
PubMed1996

Murine intranasal challenge model for the study of Campylobacter pathogenesis and immunity.

Campylobacter jejuni infection of mice initiated by intranasal administration was investigated as a potential model for studies of pathogenesis and immunity. By using a standard challenge (5 x 10(9) CFU), C. jejuni 81-176 was more virulent for BALB/c (72% mortality) than for C3H/Hej (50%), CBA/CAJ (30%), or C58/J (0%). Intranasal challenge of BALB/c was used to compare the relative virulence of three reference strains; C.jejuni 81-176 was more virulent (killing 83% of challenged mice) than C. jejuni HC (0%) or C. coli VC-167 (0%). The course of intranasally initiated C. jejuni 81-176 infection in BALB/c was determined. C. jejuni was recovered from the lungs, intestinal tract, liver, and spleen at 4 h after challenge, the first interval evaluated. After this initial interval, three distinct patterns of infection were recognized: (i) a progressive decline in number of C. jejuni CFU (stomach, blood, lungs), (ii) decline followed by a second peak in the number of organisms recovered at 2 or 3 days postchallenge (intestine, liver, mesenteric lymph nodes), and (iii) persistence of approximately the same number of C.jejuni CFU during the course of the experiment (spleen). Intranasally induced infection initiated with a sublethal number of bacteria or intranasal immunization with killed Campylobacter preparations resulted in both the generation of Campylobacter antigen-specific immune responses and an acquired resistance to homologous rechallenge. The model was used to evaluate the relative virulence of nine low-in vitro-passage (no more than five passages) isolates of C. jejuni species from patients with diarrhea. The patient isolates were differentially virulent for mice; one killed all exposed mice, three were avirulent (no deaths) and the remainder showed an intermediate virulence, killing 17 to 33%. Mouse virulence of Campylobacter strains showed a trend toward isolates originating from individuals with watery diarrhea; however, no association was found between mouse virulence and other signs or symptoms. There were no observed relationships between mouse virulence and bacterial Lior serotype or Fla polymorphic group. Intranasal challenge of BALB/c with C. jejuni is a useful model for the study of infection and vaccination-acquired immunity to this agent.

Animals↗
PubMed1996

Restriction fragment length polymorphism of flagellin genes of Campylobacter jejuni and/or C. coli isolates from Egypt.

The conservation of flagellin genes from thermophilic Campylobacter spp. strains isolated in Egypt was evaluated by a restriction fragment length polymorphism (RFLP) assay. The flaA and flaB genes were amplified from 59 independent clinical isolates and digested with EcoRI and PstI, and the resulting patterns were compared with each other and with previously described RFLP groups. The results indicate that the isolates fell into 14 groups for flaA and 11 groups for flaB, 9 of which have been described, and that considerable genetic variability exists among isolates belonging to the same LIO serogroup. In most cases, the flaB gene displayed the same RFLP pattern as that of the flaA gene of the same strain, although some variability was observed. The data suggest that more variability of flagellin genes exists within the LIO serogroups common to Campylobacter field isolates from Egypt than has previously been reported for North American isolates.

Base Sequence↗
PubMed1996

Hantaan virus infection among human and rat populations in Alexandria.

Hantaan virus has recently been identified as the cause of the rodent borne viral zoonosis collectively termed Hantavirus disease (HVD). Although the disease is mainly endemic in two main areas, clinical forms as well as positive antibody testing from healthy persons throughout the world have been reported denoting a worldwide presence. Rattus species are almost universally implicated as hantavirus reservoirs. Screening for hantavirus antibodies was done on 637 samples of human sera and 861 rodent sera trapped from 26 locations throughout the city of Alexandria, Egypt. ELISA assays were performed and 12.2% of humans were found seropositive with no effect of age, duration of work at sea or residence on seropositivity. 12.8% of rodents were seropositive with Rattus norvegicus showing the highest prevalence and with no influence of sex, age or habitat. A significant association was found between the east district and seropositivity to Hantaan in rodents.

Adolescent↗