Search PubMed⌕ Search

Biomedical subjects

C Hughes

Publications and source records attributed to C Hughes.

At least 217 records · Page 12Linked to original sources
PubMed1991

Loss of secreted hemolysin activity in the mutant strain Hsb. 1 is due to a lesion in a plasmid copy number locus.

Further studies have been carried out on mutation hsb which was previously suggested to block hemolysin secretion (Muñoa et al., 1988, FEMS Microbiol. Lett. 56: 167-172). We show that the reported reduction in the extracellular hemolytic activity of mutant Hsb. 1 is due to lower hemolysin synthesis and that this is itself a consequence of a decrease in plasmid copy number. We suggest that the hsb is identical to the pcnB lesion located at minute 3.6 of the chromosome.

Chromosomes, Bacterial↗
PubMed1991

Activation of Escherichia coli prohaemolysin to the mature toxin by acyl carrier protein-dependent fatty acylation.

Haemolysin secreted by pathogenic Escherichia coli binds to mammalian cell membranes, disrupting cellular activities and lysing cells by pore-formation. It is synthesized as nontoxic prohaemolysin (proHlyA), which is activated intracellularly by a mechanism dependent on the cosynthesized HlyC. Haemolysin is one of a family of membrane-targeted toxins, including the leukotoxins of Pasteurella and Actinobacillus and the bifunctional adenylate cyclase haemolysin of Bordetella pertussis, which require this protoxin activation 1-5. HlyC alone cannot activate proHlyA, but requires a cytosolic activating factor6. Here we report the cytosolic activating factor is identical to the acyl carrier protein and that activation to mature toxin is achieved by the transfer of a fatty acyl group from acyl carrier protein to proHlyA. Only acyl carrier protein, not acyl-CoA, can promote HlyC-directed proHlyA acylation, but a range of acyl groups are effective.

Acyl Carrier Protein↗
PubMed1991

Amiloride differentially modulates ANP binding in human thyroid cells and bovine endothelial cells.

The diuretic and sodium channel inhibitor, amiloride, has been shown to increase atrial natriuretic peptide (ANP) binding several fold in certain cell types, but in other tissues it causes only marginal increases in specific ANP binding. In the present report we compare the effects of amiloride on ANP binding in bovine endothelial cells and human thyroid-derived cells, two cell types which differ in their predominant ANP receptor subtype. We found that amiloride (10(-3) M) increased specific [125I]ANP binding to 750% above control in endothelial cells, but among several thyroid cultures tested the maximal increase in ANP binding with amiloride was only 23% above control. Moreover, most of the thyroid cultures showed decreased ANP binding in the presence of amiloride. The increased ANP binding in endothelial cells exposed to amiloride is best explained by an increased affinity of the receptor for its ligand since the drug lowered the Kd of ANP binding from 0.73 nM to 0.16 nM without affecting the receptor binding capacity. The degree of amiloride enhancement of ANP binding in endothelial cells is increased with time in culture (200% above control at 5 days, 750% above at 30 days) suggesting the increase of an amiloride-sensitive receptor relative to an amiloride-insensitive receptor. The fact that the amiloride-induced decrease in ANP binding in thyroid cells was not exacerbated by pre-incubation with amiloride suggested that the observed amiloride effect was not due to increased receptor internalization with the drug. These results support a hypothesis that ANP receptor subtypes associated with separate signal transduction mechanisms might be modulated in an opposite manner by the binding of amiloride.

Amiloride↗
PubMed1991

Update on technology diffusion.

Magnetic resonance imaging (MRI) continues to expand in influence as a diagnostic tool, with the number of units installed in the United States growing rapidly, according to an AHA technology assessment report. Technology assessment is being used more and more in decision making regarding the use of MRI and other diagnostic technologies. Two perspectives on the phenomenon are explored.

American Hospital Association↗
PubMed1991

Energetically distinct early and late stages of HlyB/HlyD-dependent secretion across both Escherichia coli membranes.

The alternative secretion pathway which exports hemolysin across both Escherichia coli membranes into the surrounding medium is directed by an uncleaved C-terminal targeting signal and the membrane translocator proteins HlyD and HlyB. In order to identify stages and intermediates in this unconventional secretion process we have examined the effect of inhibition of the total proton motive force (delta P) and its components during the in vivo HlyB/HlyD-dependent export of a 22.4 kDa secretion competent HlyA C-terminal peptide (Actp). Secretion of Actp was severely inhibited by the proton ionophore carbonylcyanide m-chlorophenylhydrazone (CCCP), which collapses simultaneously membrane potential delta psi and the proton gradient delta pH, and also by valinomycin/K+, a potassium ionophore which disrupts delta psi. The inhibition of secretion by valinomycin/K+ was ameliorated by imposition of a pH gradient, the second component of the delta P, and selective depletion of delta pH by nigericin also blocked secretion. This indicates that, as in the secretion of beta-lactamase to the periplasm, HlyB/D-directed secretion requires delta P itself and not specifically one of its components. However, inhibition of HlyB/D-dependent secretion was only marked when CCCP, valinomycin/K+ or nigericin were present during the early stage of Actp secretion; at a later stage the secretion was not significantly inhibited. HlyB/D-dependent secretion appears therefore to share with conventional secretion across the cytoplasmic membrane an early requirement for delta P, but comprises in addition a late stage which does not require delta P, delta psi or delta pH. The translocation intermediate identified in the delta P-independent late stage of secretion was associated with the membrane fraction. Analysis of the protease accessibility of this intermediate in whole cells and spheroplasts showed that it was not in the periplasm, nor was it exposed on the cell surface or on the periplasmic faces of either the inner or outer membranes. This may reflect its close association with the inner membrane or a membrane translocation complex.

Amino Acid Sequence↗
PubMed1991

Phase II trial of indicine N-oxide in relapsed pediatric solid tumors. A report from the Childrens Cancer Study Group.

We used indicine N-oxide to treat 46 children with malignant solid tumors: 17 with osteosarcoma, 12 with neuroblastoma, 13 with a brain tumor, and 4 with other miscellaneous tumors. The efficacy and toxicity of the drug was assessed at the dose of 2000 mg/m2/day for five consecutive days. None of the 39 patients evaluable for response achieved a complete or partial response. Hepatotoxicity was experienced by 13 patients: 11 patients developed asymptomatic elevations of transaminases, 1 patient developed hyperbilirubinemia, and 1 developed ascites. Indicine N-oxide appears to be ineffective in the treatment of osteosarcoma, neuroblastoma, and pediatric brain tumors at this dose and schedule. Because higher doses are associated with an unacceptably high incidence of severe, irreversible hepatotoxicity, we do not recommend further study of this agent in pediatric solid tumors.

Adolescent↗
PubMed1991

Closely linked genetic loci required for swarm cell differentiation and multicellular migration by Proteus mirabilis.

The pathogenic bacterium Proteus mirabilis exhibits a form of multicellular behaviour called swarming migration. This involves the differentiation of vegetative cells at the colony margin into swarm cells which are long, aseptate, multinucleate, hyper-flagellated filaments able to undergo repeated cycles of co-ordinated population migration and consolidation (reversion to vegetative cells). Transposon mutagenesis of uropathogenic P. mirabilis strain U6450 with Tn5 generated 4860 chromosomal insertions and, of these, 75 (1.6%) caused visibly abnormal swarming behaviour, indicating that at least 45 genes are involved in directing motility, cell differentiation and multicellular behaviour. While about one fifth of the swarm-defective mutants lacked flagella and were non-motile non-swarming (NMNS) the majority were normally flagellated and motile but were unable to form swarm cells (motile non-swarming, MNS), or were motile and able to form swarm cells but displayed aberrant patterns of multicellular migration (dendritic swarming, DS) or consolidation (frequent and infrequent consolidation, FC and IC). Restriction enzyme mapping of representative mutant DNAs by Southern hybridization with transposon DNA probes identified eight different mutated genetic loci within the five phenotypic classes. Subsequent Southern analysis of large restriction fragments separated by pulsed-field electrophoresis showed that these eight mutated loci required for motility, cell differentiation and multicellular migration were clustered on a region of DNA spanning approximately 8% of the 4.2 mbp P. mirabilis chromosome. Further linkage analysis showed that the DS locus involved in the ordered migration of the swarm cell population mapped separately from two main clusters of swarm loci, one cluster containing, within 112 kbp, genetic determinants of motility (NMNS) and also differentiation into swarm cells (MNS1, MNS2), and a second within a neighbouring 95 kbp DNA sequence containing three loci involved in the control of consolidation (FC, IC1, IC2).

Base Sequence↗
PubMed1991

In vitro activation of Escherichia coli prohaemolysin to the mature membrane-targeted toxin requires HlyC and a low molecular-weight cytosolic polypeptide.

The c. 110 kDa haemolysin toxin secreted by Escherichia coli and other pathogenic Gram-negative bacteria is synthesized as the non-toxic precursor, prohaemolysin (proHlyA), which is unable to target mammalian cell membranes until activated intracellularly by an unknown mechanism dependent upon the coexpressed c. 20 kDa protein, HlyC. We have established in vitro post-translational activation of proHlyA in membrane-depleted cell extract fractions from E. coli recombinant strains containing (separately) the proHlyA and HlyC proteins. In vitro activation was calcium-independent and effective over a pH range of 6 to 9 and at temperatures from 42 degrees C to 4 degrees C. HlyC cell extract was also able to activate proHlyA which had been secreted out of cells containing the export proteins HlyB and HlyD. Fractionation of HlyC cell extracts by sucrose gradient centrifugation and molecular weight chromatography revealed activating fractions as having a molecular mass of 40 kDa, suggesting that the HlyC activator is present physiologically in a multimeric form. Cell extracts containing activation-competent HlyC and proHlyA were inactive following dialysis, but activity was restored by complementation with a cell extract lacking both proteins. HlyC and proHlyA proteins which were overproduced separately from recombinant expression plasmids were inactive following purification, but activity could again be restored with a Hly-negative cell extract. These experiments demonstrated that HlyC is not sufficient for activation; an additional cellular factor is required. The cellular factor was found in enterobacteria but not other bacteria or eukaryotic cells. It was cytosolic, protease-sensitive, and behaved as a c. 10 kDa polypeptide in a number of assays including dialysis, sucrose gradient centrifugation, and gel filtration chromatography. Thus activation was possible in a defined in vitro reaction containing only purified proHlyA, HlyC, and the cellular factor. Kinetic studies in which the relative concentrations of the three components of proHlyA activation were varied suggested that neither HlyC nor the cellular factor acts as a conventional enzyme, with each participating in a finite number of activation events.

Bacterial Proteins↗
PubMed1991

Mutational analysis supports a role for multiple structural features in the C-terminal secretion signal of Escherichia coli haemolysin.

We have carried out an extensive mutational analysis of the C-terminal signal which targets the export of the 1024-residue haemolysin protein (HlyA) of Escherichia coli across both bacterial membranes into the surrounding medium. Over 60 variants of the HlyA C-terminal 53-amino-acid sequence were created by oligonucleotide-directed mutagenesis and fused to the HlyA N-terminal 830 residues. Transport of the HlyA derivatives by the HlyB/HlyD system was compared with the wild-type level and the data indicate that the HlyA C-terminal export signal lies within the last 48 amino acids and comprises three functional domains: an amphipathic, charged helix between residues 1,977 and R,996; a 13-amino-acid uncharged region from residue T,997 to S,1009; and an 8-amino-acid hydroxylated tail at the extreme C-terminus. Analogous features were found in the C-terminal sequences of an extended family of haemolysins, leukotoxins and proteases which are secreted by HlyB/HlyD-type translocators. In particular, all nine proteins which are secreted into the extracellular medium possess potential extended amphipathic helices. These results suggest a possible role for multiple regions of the HlyA C-terminal export signal in which the first two domains span the membranes and the third domain remains in the cytoplasm.

Amino Acid Sequence↗
PubMed1991

MRI: update on technology diffusion and acquisition.

Over the past three years, magnetic resonance imaging (MRI) has become accepted as a valuable diagnostic tool, and its applications continue to expand. During this time, the number of units installed in the United States doubled. By 1990 about 2,000 MRI units were in place in the United States and nearly 20 percent of the MRI-installed base was mobile, according to a research study conducted by the Hadley Hart Group (Chicago) and Drew Consultants, Inc. (Concord, MA). With the introduction of the prospective payment system, many hospitals were hesitant to spend limited capital on new technology, such as MRI. At the same time, freestanding diagnostic imaging centers were on the rise. Some hospitals and entrepreneurs who foresaw the potential of MRI in health care pioneered its use in the clinical setting. Hospitals began to examine new partnership arrangements and alternative forms of financing, so that they too could offer MRI services. By the end of 1988, the majority of hospitals offering MRI services did not own their own unit and about 40 percent of the hospitals offering MRI services were in a mobile configuration according to the Hadley Hart Group. While the technology has been diffused into 100-bed hospitals via mobile service vendors in some parts of the country, many medium-sized and large hospitals also have entered the MRI services market in this fashion. In the larger hospitals, the patient demand or need for the service often would justify acquisition of MRI, but the expense of the technology, and in many areas restrictive state health planning policies, modified purchase of MRI systems by hospitals. Mobile service vendors offered hospitals a way to startup MRI services in a limited fashion without a major capital expenditure and its associated risk. As hospitals gain experience with mobile MRI and achieve or exceed their early utilization projections, administrators are reevaluating the need to expand services to a full-time fixed site. Early fixed-site MRI providers have been constantly upgrading their MRI capability while planning on adding more units. The technology itself has continued to improve, primarily through the implementation of new software that permits new techniques such as MR angiography (MRA) to be performed. Units are available in a wide price range, price usually reflecting both the field strength (0.5 tesla units cost less) as well as the additional capabilities beyond routine imaging (MRA, spectroscopy).(ABSTRACT TRUNCATED AT 400 WORDS)

Data Collection↗
PubMed1991

Bacterial swarming: an example of prokaryotic differentiation and multicellular behaviour.

Bacterial swarming involves the differentiation of vegetative cells into hyperflagellated swarm cells which undergo cycles of rapid and coordinated population migration across solid surfaces. Species capable of this simple form of developmental behaviour lie on the boundary between unicellular and multicellular organisms and provide processes for study which are not only of intrinsic interest but which are analogous to components of more complex eukaryotic systems. This review attempts to place current knowledge of bacterial swarming within the framework provided by more extensively studied forms of prokaryotic multicellular behaviour. It discusses the potential of swarming as a readily accessible model of differentiation and multicellular behaviour and describes evidence indicating that swarming differentiation plays an important role in bacterial virulence.

Bacterial Adhesion↗
PubMed1991

Descriptive analysis of interactions between co-workers and supported employees.

Varying types of interactions between workers without handicaps and supported employees were described. Results indicated that co-workers interacted extensively with persons who had mental retardation. Surprising findings included high levels of advocacy between co-workers and persons with severe or profound mental retardation and a relatively high percentage of co-workers assuming evaluation and training roles. Results were discussed in relation to the work behavior literature, which indicates that, in general, co-workers provide considerable natural support for employees across work environments.

Adaptation, Psychological↗
PubMed1991

Mutation of the p53 gene in a differentiated human thyroid carcinoma cell line, but not in primary thyroid tumours.

The p53 gene has been implicated as a tumour suppressor, with mutations occurring in many carcinomas, such as colon, breast and lung. We have sequenced exons 5, 7 and 8 containing conserved gene regions in the only available differentiated thyroid follicular carcinoma cell line and found a mutation at position 273, Arg----His, with no normal allele present. The same mutation was also present in DNA from the tumour of origin. However immunohistochemical analysis of 129 human thyroid tumours using a panel of p53 antibodies was unequivocally negative. Southern blotting in 20 cases failed to demonstrate any deletion or rearrangement, and direct genomic sequencing of 20 carcinomas showed normal DNA sequence for exons 5, 7 and 8. Thus p53 abnormalities may not be important in human thyroid carcinogenesis, in contrast to colon, breast and lung. However, the FTC 133 cell line was only established after 132 unsuccessful attempts with other differentiated thyroid follicular tumours. Since this line and the corresponding tumour of origin have a p53 mutation, we propose that p53 mutation may confer on thyroid follicular tumour cells the ability to grow in culture. This has potential applications for the future development of thyroid carcinoma cell lines.

Antibodies, Monoclonal↗