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Biomedical subjects

C A Charlton

Publications and source records attributed to C A Charlton.

At least 19 recordsLinked to original sources
PubMed2000

Neural cell adhesion molecule (NCAM) and myoblast fusion.

Considerable evidence points to an involvement of neural cell adhesion molecule (NCAM) in myoblast fusion. Changes in the level of NCAM expression, isoform specificity, and localization in muscle cells and tissues correspond to key morphogenetic events during muscle differentiation and repair. Furthermore, anti-NCAM antibodies have been shown by others to reduce the rate of myoblast fusion, whereas overexpression of NCAM cDNAs increases the rate of myoblast fusion compared to controls. In this study we have used a novel fusion assay based on intracistronic complementation of lacZ, in combination with fluorescent X-gal histochemistry and immunocytochemistry to assess levels of NCAM expression in individual muscle cells. Our results indicate that a substantial proportion of newly fused myoblasts have NCAM expression levels unchanged from the levels of the surrounding unfused population suggesting that increased expression of NCAM is not required for wild-type myoblasts to fuse. Moreover, pure populations of primary myoblasts isolated from mice homozygous null for NCAM and therefore lacking the molecule, when placed in differentiation medium, consistently fused to form contractile myotubes with kinetics equivalent to wild-type primary myoblasts. We conclude that the increase in expression of NCAM, although typically observed during myogenesis, is not essential to myoblast fusion to form myotubes.

Animals↗
PubMed1999

The phosphoprotein protein PEA-15 inhibits Fas- but increases TNF-R1-mediated caspase-8 activity and apoptosis.

We have characterized a phosphoprotein protein with a death effector domain that has a novel bifunctional role in programmed cell death. The 15-kDa phosphoprotein enriched in astrocytes (PEA-15) inhibits Fas-mediated apoptosis and increases tumor necrosis factor receptor-1 (TNF-R1)-mediated apoptosis in the same cell type in a ligand-dependent manner. Phosphorylation appears to play a role in its differential effects, since point mutations at one or both phosphorylation consensus sites within PEA-15 destroy its effect on Fas-mediated, but not TNF-R1-mediated, apoptosis. Furthermore, the differential effect is evident at the level of caspase-8 activity which is inhibited via Fas activation, but increased via TNF-R1 activation upon PEA-15 expression. These results show that PEA-15 provides a potential mechanism during development for distinguishing between diverse extracellular death-inducing signals that culminate either in apoptosis or in survival.

3T3 Cells↗
PubMed1997

Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation.

We present an approach for monitoring protein-protein interactions within intact eukaryotic cells, which should increase our understanding of the regulatory circuitry that controls the proliferation and differentiation of cells and how these processes go awry in disease states such as cancer. Chimeric proteins composed of proteins of interest fused to complementing beta-galactosidase (beta-gal) deletion mutants permit a novel analysis of protein complexes within cells. In this approach, the beta-gal activity resulting from the forced interaction of nonfunctional weakly complementing beta-gal peptides (Deltaalpha and Deltaomega) serves as a measure of the extent of interaction of the non-beta-gal portions of the chimeras. To test this application of lacZ intracistronic complementation, proteins that form a complex in the presence of rapamycin were used. These proteins, FRAP and FKBP12, were synthesized as fusion proteins with Deltaalpha and Deltaomega, respectively. Enzymatic beta-gal activity served to monitor the formation of the rapamycin-induced chimeric FRAP/FKBP12 protein complex in a time- and dose-dependent manner, as assessed by histochemical, biochemical, and fluorescence-activated cell sorting assays. This approach may prove to be a valuable adjunct to in vitro immunoprecipitation and crosslinking methods and in vivo yeast two-hybrid and fluorescence energy transfer systems. It may also allow a direct assessment of specific protein dimerization interactions in a biologically relevant context, localized in the cell compartments in which they occur, and in the milieu of competing proteins.

Animals↗
PubMed1997

Fusion competence of myoblasts rendered genetically null for N-cadherin in culture.

Myoblast fusion is essential to muscle tissue development yet remains poorly understood. N-cadherin, like other cell surface adhesion molecules, has been implicated by others in muscle formation based on its pattern of expression and on inhibition of myoblast aggregation and fusion by antibodies or peptide mimics. Mice rendered homozygous null for N-cadherin revealed the general importance of the molecule in early development, but did not test a role in skeletal myogenesis, since the embryos died before muscle formation. To test genetically the proposed role of N-cadherin in myoblast fusion, we successfully obtained N-cadherin null primary myoblasts in culture. Fusion of myoblasts expressing or lacking N-cadherin was found to be equivalent, both in vitro by intracistronic complementation of lacZ and in vivo by injection into the muscles of adult mice. An essential role for N-cadherin in mediating the effects of basic fibroblast growth factor was also excluded. These methods for obtaining genetically homozygous null somatic cells from adult tissues should have broad applications. Here, they demonstrate clearly that the putative fusion molecule, N-cadherin, is not essential for myoblast fusion.

Animals↗
PubMed1996

Spectrophotometric quantitation of tissue culture cell number in any medium.

We have developed a spectrophotometric assay for cell number in suspensions of tissue culture cells. For each cell type tested, absorbance between 650 and 800 nm is linearly dependent upon cell density over a 50-fold range and is independent of the color or composition of the medium in which cells are suspended. A standard curve of absorbance vs. cell density is used to estimate cell number with accuracy and reproducibility superior to hemacytometer counting and with speed and ease surpassing use of a Coulter counter. Less than 5000 cells are needed for this quantitation. The same cells that are counted can be maintained live in culture after the reading is taken, thus allowing the growth of cells to be measured within individual cultures over time. The assay should be readily extended to assays of cell number directly within microplate culture wells. The spectrophotometric assay described here is of significant use in all experiments requiring rapid, accurate measurements of cell number, including determinations of cell doubling time and equal plating of parallel cultures.

Animals↗
PubMed1994

Urethral catheterisation--is anybody listening?

All house officers perform male urethral catheterisation and although complications may occur awareness of the potential problems varies considerably. 43 house officers from 12 different medical schools were interviewed to determine the amount of training that they received regarding this procedure as students and during their first pre-registration post. The results suggest that there is no planned approach to teaching urethral catheterisation which would be in the interests of both junior doctors and patients alike.

Education, Medical↗
PubMed1993

Penetration of Autographa californica nuclear polyhedrosis virus nucleocapsids into IPLB Sf 21 cells induces actin cable formation.

The budded form of Autographa californica nuclear polyhedrosis virus functionally enters cells by adsorptive endocytosis. During the period of virus uptake into Spodoptera frugiperda IPLB Sf21 cells (1 to 4 hr postinfection), filamentous F-actin cables, visualized by fluorescence microscopy, were formed within the cytoplasm of infected cells. Cable formation appeared to be a direct effect of viral inoculum in that the numbers of observed cables increased with an increase in multiplicity of infection, and cable formation was not dependent on protein synthesis. Cable induction was first apparent around 30 min postinfection, although uptake of virus into endocytic vesicles began much earlier. Chloroquine, a chemical that prevents release of nucleocapsids from endosomes, inhibited cable formation. Similarly, when virus adsorbed to the exterior of cells was exposed to neutralizing antibody, release of nucleocapsids from endosomes and subsequent cable induction were prevented. Careful observation of co-labeled F-actin cables and nucleocapsids revealed a single nucleocapsid at an end of most cables. These results suggested that nucleocapsids or nucleocapsid-associated proteins of budded virus induced the polymerization or bundling of actin following their penetration into the cytoplasm from endocytic vesicles.

Actins↗
PubMed1991

Sequential rearrangement and nuclear polymerization of actin in baculovirus-infected Spodoptera frugiperda cells.

Proper assembly of nucleocapsids of the baculovirus Autographa californica nuclear polyhedrosis virus is prevented by cytochalasin D, a drug that interferes with actin microfilament function. To investigate the involvement of microfilaments in A. californica nuclear polyhedrosis virus replication, a fluorescence microscopy study was conducted that correlated changes in distribution of microfilaments with events in the life cycle of the virus. Tetramethylrhodamine isothiocyanate-labeled phalloidin was used to label microfilaments, and monoclonal antibody was used to label p39, the major viral capsid protein. Three microfilament arrangements were found in infected cells. During uptake of virus, thick cables were formed. These were insensitive to cycloheximide, indicating that this configuration was a rearrangement of preexisting cellular actin mediated by a component of the viral inoculum. At the time of cell rounding and before viral DNA replication, ventral aggregates of actin were observed. These were sensitive to cycloheximide but not to aphidicolin, indicating that an early viral gene mediated this actin rearrangement. Ventral aggregates did not result from the rounding process itself. Uninfected cells prerounded with colchicine did not form ventral aggregates. Cells prerounded with colchicine and then infected did form aggregates. At the time of exponential production of progency virus, microfilaments were found in the nucleus surrounding the virogenic stroma. In this area (where nucleocapsid assembly is known to take place) microfilaments colocalized with p39. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblot analysis identified p39 among proteins retained on an f-actin affinity column. We postulate that microfilaments in the nucleus provide a scaffold to position capsids for proper assembly and filling with DNA.

Actin Cytoskeleton↗
PubMed1990

Indications for conservative management of acute scrotal pain in children.

Forty-three boys who presented with acute scrotal pain were studied retrospectively. Of these, 40 had torsion of scrotal contents. Torsion of testicular appendages, a self limiting condition, tended to present later than testicular torsion (P = 0.002). The testis was saved in all 11 cases of testicular torsion explored within 12h of the onset of pain but orchidectomy was required in four who presented later. In 11 cases, a torted testicular appendage diagnosed as a tender nodule was found on examining the upper pole of the testis. The remaining 14 cases of torted appendage were diagnosed at operation because testicular torsion could not be excluded due to scrotal swelling. Most (12) of this operated group were not seen until 3 days after the onset of pain. This study suggested that urgent operation was unnecessary in those in whom a tender nodule was found (pathognomonic of a torted appendage) and in those presenting after more than 24h of pain with scrotal erythema and oedema. The likely diagnosis in the latter case is a torted appendage and in cases of torsion of the testis there is no prospect of salvage at this stage. These guidelines were applied to a group of 20 boys studied prospectively. In eight, all with less than 24h of pain, immediate exploration was undertaken and testicular torsion was confirmed in six. The remaining 12 fulfilled the criteria for conservative management and were observed. All had normal testes at late review. Careful clinical assessment in boys with scrotal pain indicates which cases may be treated by non-operative management without fear of losing a salvageable testis.

Adolescent↗
PubMed1987

Renal trauma and the intravenous urogram.

A retrospective analysis of all patients with blunt abdominal trauma associated with haematuria admitted to one hospital (Royal United, Bath) in a 10-year period was conducted to establish the contribution of the intravenous urogram (IVU) in their management. Eighty-one case records were analysed. Of 35 IVUs performed in patients with microscopic (reagentstrip positive) haematuria, only one was abnormal. In contrast, 27 IVUs performed in patients with macroscopic (naked eye) haematuria revealed 17 major injuries and 5 previously unrecognized congenital abnormalities. It is concluded that an IVU is an unnecessary and non-contributory investigation in patients with microscopic haematuria and guidelines are suggested for the role of IVU in patients with blunt abdominal trauma associated with haematuria.

Abdominal Injuries↗