Search PubMed⌕ Search

Biomedical subjects

J R Murphy

Publications and source records attributed to J R Murphy.

At least 37 records · Page 2Linked to original sources
PubMed1999

Solution structure and peptide binding studies of the C-terminal src homology 3-like domain of the diphtheria toxin repressor protein.

The diphtheria toxin repressor (DtxR) is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria. DtxR contains two domains that are separated by a short, unstructured linker. The N-terminal domain is structurally well-defined and is responsible for Fe2+ binding, dimerization, and DNA binding. The C-terminal domain adopts a fold similar to eukaryotic Src homology 3 domains, but the functional role of the C-terminal domain in repressor activity is unknown. The solution structure of the C-terminal domain, consisting of residues N130-L226 plus a 13-residue N-terminal extension, has been determined by using NMR spectroscopy. Residues before A147 are highly mobile and adopt a random coil conformation, but residues A147-L226 form a single structured domain consisting of five beta-strands and three helices arranged into a partially orthogonal, two-sheet beta-barrel, similar to the structure observed in the crystalline Co2+ complex of full-length DtxR. Chemical shift perturbation studies demonstrate that a proline-rich peptide corresponding to residues R125-G139 of intact DtxR binds to the C-terminal domain in a pocket formed by residues in beta-strands 2, 3, and 5, and helix 3. Binding of the proline-rich peptide by the C-terminal domain of DtxR presents an example of peptide binding by a prokaryotic Src homology 3-like protein. The results of this study, combined with previous x-ray studies of intact DtxR, provide insights into a possible biological function of the C-terminal domain in regulating repressor activity.

Amino Acid Sequence↗
PubMed1999

HIV-1 seroprevalence in Zambian patients with acute diarrhea: a community-based study.

The seroprevalence of HIV-1 in sub-Saharan African patients with diarrhea in the community remains largely unknown. We present the findings of a 2-month study that we undertook to ascertain the seroprevalence of HIV-1 in Zambian patients presenting with acute diarrhea in a community-based health center. A total of 256 patients with diarrhea and 140 apparently healthy controls was seen. Of the patients with diarrhea, 161 were < 16 years old and 95 were adults. Most children with diarrhea were < 6 years old (147 of 161; 91%). Overall, 81 of 256 (32%) patients with diarrhea were HIV-1-seropositive. When results from children < 18 months old and possibly having maternal anti-HIV-1 antibodies were excluded, 64 of 172 (37%) patients with diarrhea were HIV-seropositive. Rates of HIV-1 seropositivity for patients with diarrhea were significantly higher than were rates for diarrhea-free controls (p < .001 for both the total population; odds ratio [OR], 95% confidence interval [CI], 1.42 < 2.48 < 4.35) and population > 18 months old (OR, 95% CI, 1.54 < 2.90 < 5.49). Among children between 18 months and 5 years old, 14 of 63 (22%) were HIV-1-seropositive compared with 8 of 62 (13%) without diarrhea (p > .05, not significant). Moreover, 49 of 95 (52%) adults with acute diarrhea were HIV-1-seropositive compared with 10 of 44 (23%) healthy adult controls (p < .003; OR, 95% CI, 1.51 < 3.62 < 8.87). No significant differences were found in HIV-1 seroprevalence rates between males and females in all age groups. These data show a close association between acute diarrhea and HIV seropositivity in Zambian adults in the community.

Acute Disease↗
PubMed1999

Epidemiology of enterotoxigenic Escherichia coli diarrhea in a pediatric cohort in a periurban area of lower Egypt.

Enterotoxigenic Escherichia coli (ETEC) are diverse pathogens that express heat-labile (LT) and/or heat-stable (ST) enterotoxins, yet little is known about whether epidemiologic patterns of pediatric ETEC diarrhea vary by the expressed ETEC toxin phenotype. In total, 242 Egyptian children aged <3 years were prospectively followed in 1993-1995. ETEC episodes were detected during twice-weekly home visits, and asymptomatic ETEC excretion was identified from monthly cross-sectional surveys. ETEC episodes were 0.6 per child-year. ST-only ETEC was 2.6 times (P<.001) more common in warmer than cooler months, while LT-only ETEC showed no seasonal variation. Ownership of a household sanitary latrine, but not breast-feeding, was associated with a lower risk of both enterotoxin phenotypes. Coexpression of a colonization factor by LT- or ST-only ETEC strengthened the association with diarrhea. These findings indicate that the epidemiologic patterns of LT-only and ST-only ETEC are not identical and that disease interventions should include improved household sanitation.

Cohort Studies↗
PubMed1998

Isolation and characterization of iron-independent positive dominant mutants of the diphtheria toxin repressor DtxR.

It is well known that the functional activity of the diphtheria toxin repressor DtxR is controlled by iron, which serves as an essential cofactor necessary for activation of target DNA binding by this regulatory element. In this communication, we describe the isolation and characterization of a unique series of DtxR mutants that are constitutively active and repress the expression of beta-galactosidase from a diphtheria tox promoter/operator-lacZ transcriptional fusion, even in the absence of iron. These self-activating mutants of DtxR (SAD) were isolated through the use of a positive selection system for the cloning of functional dtxR alleles and target DNA operator sites. Of the four independently isolated SAD mutants that were characterized, two (SAD2 and SAD11) were found to carry a single missense mutation (E175K) in their respective C-terminal SH3-like domains. In contrast, the mutant allele encoding SAD3 was found to carry a total of six missense mutations distributed throughout the N- and C-terminal domains of the repressor. Partial diploid analysis of strains carrying both native dtxR and alleles encoding either SAD2 or SAD3 demonstrate that these iron-independent mutants possess a positive dominant phenotype in the regulation of beta-galactosidase expression from a diphtheria tox promoter/operator-lacZ transcriptional fusion.

Bacterial Proteins↗
PubMed1998

Structure of the metal-ion-activated diphtheria toxin repressor/tox operator complex.

The virulent phenotype of the pathogenic bacterium Corynebacterium diphtheriae is conferred by diphtheria toxin, whose expression is an adaptive response to low concentrations of iron. The expression of the toxin gene (tox) is regulated by the repressor DtxR, which is activated by transition metal ions. X-ray crystal structures of DtxR with and without (apo-form) its coordinated transition metal ion have established the general architecture of the repressor, identified the location of the metal-binding sites, and revealed a metal-ion-triggered subunit-subunit 'caliper-like' conformational change. Here we report the three-dimensional crystal structure of the complex between a biologically active Ni(II)-bound DtxR(C102D) mutant, in which a cysteine is replaced by an aspartate at residue 102, and a 33-base-pair DNA segment containing the toxin operator toxO. This structure shows that DNA interacts with two dimeric repressor proteins bound to opposite sides of the tox operator. We propose that a metal-ion-induced helix-to-coil structural transition in the amino-terminal region of the protein is partly responsible for the unique mode of repressor activation by transition metal ions.

Bacterial Proteins↗
PubMed1998

Direct and indirect estimates of HIV-1 incidence in a high-prevalence population.

While the worldwide AIDS epidemic continues to expand, directly measured incidence data are difficult to obtain. Methods to reliably estimate human immunodeficiency virus type 1 (HIV-1) incidence from more easily available data are particularly relevant in those parts of the world where prevalence is rising in heterosexually exposed populations. The authors set out to estimate HIV-1 incidence in a population of heterosexual sexually transmitted disease clinic attendees in Trinidad who had a known high prevalence of HIV-1 subtype B. Over the period 1987-1995, HIV-1 incidence estimates from serial cross-sectional studies of HIV-1 prevalence, passive follow-up of clinic recidivists, modeling of early markers of HIV-1 infection (p24 antigen screening), and a cohort study of seronegative genital ulcer disease cases were compared. Measuring incidence density in the genital ulcer disease cases directly gave the highest estimate, 6.9% per annum. Screening for the detection of early HIV-1 markers yielded an incidence of 5.0% per annum, while estimating incidence from serial cross-sectional prevalence data and clinic recidivists gave estimates of 3.5% and 4.5% per annum, respectively. These results were found to be internally consistent. Indirect estimates of incidence based on prevalence data can give accurate surrogates of true incidence. Within limitations, even crude measures of incidence are robust enough for health planning and evaluation purposes. For planning vaccine efficacy trials, consistent conservative estimates may be used to evaluate populations before targeting them for cohort studies.

Blotting, Western↗
PubMed1998

The structure of the upper atmosphere of mars: In situ accelerometer measurements from mars global surveyor

The Mars Global Surveyor (MGS) z-axis accelerometer has obtained over 200 vertical structures of thermospheric density, temperature, and pressure, ranging from 110 to 170 kilometers, compared to only three previous such vertical structures. In November 1997, a regional dust storm in the Southern Hemisphere triggered an unexpectedly large thermospheric response at mid-northern latitudes, increasing the altitude of thermospheric pressure surfaces there by as much as 8 kilometers and indicating a strong global thermospheric response to a regional dust storm. Throughout the MGS mission, thermospheric density bulges have been detected on opposite sides of the planet near 90 degreesE and 90 degreesW, in the vicinity of maximum terrain heights. This wave 2 pattern may be caused by topographically-forced planetary waves propagating up from the lower atmosphere.

Journal Article↗
PubMed1998

Detection of reovirus RNA in hepatobiliary tissues from patients with extrahepatic biliary atresia and choledochal cysts.

Extrahepatic biliary atresia (EHBA) and choledochal cysts (CDC) are important causes of obstructive jaundice in pediatric patients. Viruses in general, and reoviruses in particular, have long been considered as possible etiologic agents responsible for inciting the inflammatory process that leads to these infantile obstructive cholangiopathies. In an effort to determine whether reovirus infection is associated with these disorders, we used a sensitive and specific reverse-transcriptase polymerase chain reaction (RT-PCR) technique designed to amplify a portion of the reovirus L1 gene segment from extracts of liver and/or biliary tissues. These tissues were obtained at the time of liver biopsy or surgical procedures from 23 patients with EHBA, 9 patients with CDC, and 33 patients with other hepatobiliary diseases. Hepatic and biliary tissues obtained at autopsy from 17 patients who died without known liver or biliary disease were also analyzed. Reovirus RNA was detected in hepatic and/or biliary tissues from 55% of patients with EHBA and 78% of patients with CDC. Reovirus RNA was found also in extracts of hepatic and/or biliary tissue from 21% of patients with other hepatobiliary diseases and in 12% of autopsy cases. The prevalence of reovirus RNA in tissues from patients with EHBA and CDC was significantly greater than that in patients with other hepatobiliary diseases (chi2 P = .012 EHBA vs. OTHER, P = .001 CDC vs. OTHER), or AUTOPSY cases (chi2 P = .006 EHBA vs. AUTOPSY, P < .001 CDC vs. AUTOPSY).

Biliary Atresia↗
PubMed1998

The diphtheria toxin transmembrane domain as a pH sensitive membrane anchor for human interleukin-2 and murine interleukin-3.

We have constructed two fusion proteins T-hIL-2 and T-mIL-3 in which human interleukin-2 (hIL-2) or murine interleukin-3 (mIL-3) are fused to the C-terminus of the diphtheria toxin transmembrane domain (T domain). Two additional fusion proteins, T-(Gly4-Ser)2-hIL-2 and T-(Gly4-Ser)2-mIL-3, were derived by introduction of the (Gly4-Ser)2 spacer between the T domain and cytokine components. Recognition of the hIL-2 receptor or the mIL-3 receptor by the corresponding recombinant proteins was demonstrated by their capacity to stimulate cytokine-dependent cell lines. All proteins retained the capacity of the T domain to insert into phospholipid membranes at acidic pH. Finally, anchoring of both cytokines to the membrane of lipid vesicles or living cells was assessed by specific antibody recognition. Our results show that the T domain fused to the N-terminus of a given protein can function as a pH sensitive membrane anchor for that protein.

Animals↗
PubMed1998

The effects of helix breaking mutations in the diphtheria toxin transmembrane domain helix layers of the fusion toxin DAB389IL-2.

The fusion protein toxin DAB389IL-2 is composed of the catalytic and transmembrane domains of diphtheria toxin genetically linked to human interleukin 2 (IL-2). This fusion toxin is selectively toxic for eukaryotic cells which express the high-affinity form of the IL-2 receptor and the mechanism of intoxication parallels that of native diphtheria toxin. We used site-directed mutagenesis to introduce Pro residues into each of the three helical layers of the transmembrane domain. Although each of the mutations results in the complete loss of cytotoxic activity, individual mutants were found to vary with respect to channel formation in planar lipid bilayers, binding affinity and melting temperature. We propose that each of the three helix layers plays a critical role in the productive delivery of the catalytic domain to the cell cytosol.

Amino Acid Sequence↗
PubMed1998

Differentiation of Campylobacter isolates on the basis of sensitivity to boiling in water as measured by PCR-detectable DNA.

Differential sensitivity for the release of PCR-detectable genomic DNA upon boiling in water is reported for 45 Campylobacter jejuni and Campylobacter coli strains isolated in Egypt. All of the strains released PCR-detectable DNA when treated with proteinase K and sodium dodecyl sulfate. When DNA was extracted from these strains by boiling in water, nine (20%) of the strains were PCR negative or resistant to boiling, suggesting the presence of boiling-sensitive and boiling-resistant phenotypes.

Campylobacter coli↗
PubMed1998

Using a nonlinear mixed model to evaluate three models of human stature.

The modern mixed model approach is used to evaluate three current nonlinear models of development of human stature. By combining both fixed and random effects in the same model, the mixed approach incorporates variability between subjects in the estimation of the mean parameter values. This allows us to provide a single statistical test for the differences between each pair of statistical models. Asymptotic growth models from Preece and Baines (1978), Jolicoeur et al. (1988, 1991,1992), and Kanefuji and Shohoji (1990) were applied to height data collected from 28 males and 25 females. The NLINMIX Macro from SAS was used to evaluate the fit of each model allowing for two random components in addition to the fixed mean parameter values. In every case, the addition of random parameters improved the fit of each growth model. Models were evaluated by the calculation of the Akaike Information Criterion, differences in -2 log likelihood, and determination of the residual variance. For males, the Jolicoeur et al. model was superior, while for females, the Kanefuji and Shohoji model provided the best fit. This new approach is more parsimonious than previous techniques by allowing for individual variation in the estimation of model parameters in a population average model of growth.

Humans↗
PubMed1997

Disulfide bond assignment in human interleukin-7 by matrix-assisted laser desorption/ionization mass spectroscopy and site-directed cysteine to serine mutational analysis.

Interleukin-7 (IL-7) is a proteinaceous biological response modifier that has a bioactive tertiary structure dependent on disulfide bond formation. Disulfide bond assignments in human (h)IL-7 are based upon the results of matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and Cys to Ser mutational analyses. A gene encoding the hIL-7 was synthesized incorporating Escherichia coli codon usage bias and was used to express biologically active protein as determined by stimulation of precursor B-cell proliferation. MALDI mass spectroscopic analysis of trypsin-digested hIL-7 was performed and compared with the anticipated results of a simulated tryptic digestion. Many of the anticipated hIL-7 tryptic fragments were detected including one with a molecular mass equivalent to the sum of two polypeptides linked through a disulfide bond formed from Cys residues (Cys3 and Cys142). Subsequently, Cys to Ser substitution mutational analyses were performed. A hIL-7 variant with all six Cys substituted with Ser was found to be biologically inactive (EC50 > 1 x 10(-7) M). In contrast, a family of single disulfide bond-forming variants of hIL-7 were constructed by reintroducing Cys pairs (Cys3-Cys142, Cys35-Cys130, and Cys48-Cys93), and each could stimulate cell proliferation with an EC50 of 4 x 10(-9), 2 x 10(-8), and 2 x 10(-9) M, respectively. In single disulfide bond-forming mutants of hIL-7, the ability to stimulate cell proliferation was abolished in the presence of 2 mM dithiothreitol. The results presented strongly suggest that only a single disulfide bond is required for hIL-7 to form a tertiary structure capable of stimulating precursor B-cell proliferation.

Amino Acid Sequence↗
PubMed1997

The Mars Pathfinder atmospheric structure investigation/meteorology (ASI/MET) experiment.

The Mars Pathfinder atmospheric structure investigation/meteorology (ASI/MET) experiment measured the vertical density, pressure, and temperature structure of the martian atmosphere from the surface to 160 km, and monitored surface meteorology and climate for 83 sols (1 sol = 1 martian day = 24.7 hours). The atmospheric structure and the weather record are similar to those observed by the Viking 1 lander (VL-1) at the same latitude, altitude, and season 21 years ago, but there are differences related to diurnal effects and the surface properties of the landing site. These include a cold nighttime upper atmosphere; atmospheric temperatures that are 10 to 12 degrees kelvin warmer near the surface; light slope-controlled winds; and dust devils, identified by their pressure, wind, and temperature signatures. The results are consistent with the warm, moderately dusty atmosphere seen by VL-1.

Atmosphere↗