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Biomedical subjects

M Carson

Publications and source records attributed to M Carson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Measurement of tissue viability using intravital microscopy and fluorescent nuclear dyes.

Intravital microscopy used with fluorescent vital stains provides the opportunity to measure the temporal and spatial extent of tissue injury following disease processes. However, this assumes that prolonged exposure to such dyes does not alter microvascular perfusion or cellular viability. To test this hypothesis, the extensor digitorum longus (EDL) muscle in 24 male Wistar rats, anesthetized with sodium pentobarbital (Somnotal, 65 mg/kg, ip), were prepared for microscopy. The EDL was either bathed continuously (n = 6) in Krebs solution containing bisbenzimide (5 micrograms/ml; labels nuclei of all cells) and ethidium bromide (5 micrograms/ml; labels nuclei of injured cells) or had dyes topically applied 1 hr (n = 4) and 4 hr (n = 4) following dissection of the muscle. Noxious stimuli (i.e., hypoxia:FiO2 of 8-10% (n = 3), 95% ethanol (n = 3), and 2 hr ischemia followed by 90 min reperfusion (n = 4) were used to test the ability of ethidium bromide, when used in conjunction with intravital microscopy, to differentiate injured tissue. Video recordings at the surface of the EDL muscle were made every 30 min for 5 hr from which the number of perfused capillaries was counted (NCper). The numbers of bisbenzimide- and ethidium bromide-labeled nuclei were counted at the surface of the muscle and at two to three additional locations within the muscle (to a maximum depth of approximately 120-160 microns). The average NCper (19.05 +/- 1.7) remained constant over 5 hr, while the number of nuclei stained by bisbenzimide increased linearly with time from an initial value of 1218 +/- 125.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Regulation of sex hormone-binding globulin production by isoflavonoids and patterns of isoflavonoid conjugation in HepG2 cell cultures.

The effect of the isoflavonoid phytoestrogens daidzein, equol, and genistein on sex hormone-binding globulin (SHBG) levels, SHBG mRNA transcript levels, and SHBG gene methylation was studied in HepG2 cell cultures by fluoroimmunometric SHBG assay and Northern and Southern hybridizations, respectively. The effect of 17 beta-estradiol on these parameters was studied as a control. The metabolism of isoflavonoids in HepG2 cells was determined by isotope dilution gas chromatography-mass spectrometry, after ion-exchange chromatography. Daidzein and equol increased SHBG levels in parallel intracellularly and extracellularly, whereas genistein increased SHBG levels only within the cells, resembling thus the effect of 17 beta-estradiol. The difference may originate from the fact that genistein has more hydroxyl groups than daidzein and equol. The regulation of SHBG production by phytoestrogens appears to occur at the post-transcriptional level. Firstly, daidzein, equol, or genistein did not have a clear effect on the steady-state SHBG mRNA levels. Secondly, no effect on SHBG gene methylation was observed by genistein. The findings applied also to 17 beta-estradiol. However, as the SHBG gene was more methylated in SHBG-negative MCF-7 cells than in SHBG-positive HepG2 cells, DNA methylation may play a role in the tissue-specific activation of this gene. The metabolism of isoflavonoids in HepG2 cells yielded mainly unconjugated and sulfated compounds. Similar metabolism in hepatocytes in vivo might retain their biological activity in tissues responsive to estrogens.

Carcinoma, Hepatocellular↗
PubMed1995

Regulation of sex hormone-binding globulin secretion and gene expression by cycloheximide in vitro.

The role of protein synthesis in sex hormone-binding globulin (SHBG) secretion and gene expression was studied in HepG2 cell cultures. Inhibition of protein synthesis by cycloheximide suppressed SHBG levels. Triiodothyronine and estradiol increased SHBG production, and cycloheximide reduced their effects to an extent which correlated with the degree of suppression obtained with the drug alone. Insulin decreased SHBG production, and the effect of the treatment with insulin and cycloheximide together did not differ from that with cycloheximide alone. Cycloheximide did not, alone or with the hormones, decrease SHBG levels more markedly extra- than intracellularly. Therefore, cycloheximide does not impair the secretion of SHBG which is synthesized in the presence of the drug. In contrast to SHBG protein levels, cycloheximide increased SHBG mRNA levels. When the effect of cycloheximide on the rate of SHBG mRNA decay was tested, the drug was found to extend the half-life of SHBG mRNA. Of the hormones, insulin decreased and triiodothyronine modestly increased SHBG mRNA levels, whereas estradiol had no clear effect. Treatment with cycloheximide together with any of the hormones resulted in an increase in SHBG mRNA levels. We conclude that protein synthesis inhibition does not impair the secretion of SHBG produced under such conditions, but stabilizes SHBG mRNA by removing some hepatic protein species involved in the regulation of its degradation.

Blotting, Northern↗
PubMed1995

Single-stranded DNA arising at telomeres in cdc13 mutants may constitute a specific signal for the RAD9 checkpoint.

A cdc13 temperature-sensitive mutant of Saccharomyces cerevisiae arrests in the G2 phase of the cell cycle at the restrictive temperature as a result of DNA damage that activates the RAD9 checkpoint. The DNA lesions present after a failure of Cdc13p function appear to be located almost exclusively in telomere-proximal regions, on the basis of the profile of induced mitotic recombination. cdc13 rad9 cells dividing at the restrictive temperature contain single-stranded DNA corresponding to telomeric and telomere-proximal DNA sequences and eventually lose telomere-associated sequences. These results suggest that the CDC13 product functions in telomere metabolism, either in the replication of telomeric DNA or in protecting telomeres from the double-strand break repair system. Moreover, since cdc13 rad9 cells divide at a wild-type rate for several divisions at the restrictive temperature while cdc13 RAD9 cells arrest in G2, these results also suggest that single-stranded DNA may be a specific signal for the RAD9 checkpoint.

Base Sequence↗
PubMed1995

Regulation of production and secretion of sex hormone-binding globulin in HepG2 cell cultures by hormones and growth factors.

Regulation of the production and secretion of sex hormone-binding globulin (SHBG) was investigated in HepG2 cell cultures by measuring SHBG protein concentrations intra- and extracellularly and studying changes in SHBG messenger ribonucleic acid levels. Insulin (10 nmol/L), insulin-like growth factor-I (15 nmol/L), and epidermal growth factor (20 nmol/L) decreased SHBG levels in parallel both intra- and extracellularly. Ten nmol/L 17 beta-estradiol, 10 nmol/L testosterone, and 100 nmol/L to 1 mumol/L cortisol increased SHBG levels inside the cells, but did not increase its release into the culture medium. Two hundred and fifty to 500 nmol/L 17 beta-estradiol and 500 nmol/L to 1 mumol/L testosterone increased SHBG levels intra- and extracelularly, but relative to control values, the increase was considerably greater inside the cells. T3 (10 nmol/L) increased SHBG levels, but unlike the effect seen with steroids, the increase was equally evident within the cells and the medium. Northern hybridization showed that insulin decreased and 17 beta-estradiol and T3 increased SHBG messenger ribonucleic acid levels marginally. The variable secretion of SHBG is hypothesized to be due to the different effects of hormones and growth factors on either the glycan moiety of SHBG or the expression of the alternatively spliced transcripts of the SHBG gene.

Carcinoma, Hepatocellular↗
PubMed1995

Skeletal muscle injury induced by ischemia-reperfusion.

OBJECTIVE: To study the temporal progression of injury in skeletal muscle after ischemia-reperfusion insult by means of intravital videomicroscopy and nuclear fluorescent dyes. DESIGN: A controlled study in an animal model. SETTING: A vascular research laboratory at a university-affiliated hospital. SUBJECTS: Eight male Wistar rats, for each of which the extensor digitorum longus muscle of the hind limb was exposed and prepared. INTERVENTIONS: Two hours of complete no-flow ischemia followed by 90 minutes of reperfusion in five of the rats; the other three rats acted as controls and underwent the same surgical procedure but not ischemia. During the reperfusion period the fluorescent vital dyes bisbenzimide, which permeates all cells, and ethidium bromide, which permeates cells with damaged membranes, were applied. Recordings to videotape were made with the intravital microscope very 15 minutes during the reperfusion period. MAIN OUTCOME MEASURES: The number of perfused capillaries crossing three straight lines on the video monitor were counted as a measure of microvascular dysfunction. An index of tissue injury was calculated as the ratio of the number of nuclei stained by ethidium bromide to the number stained by bisbenzimide (E/B). The number of stuck and rolling leukocytes and the velocity of the rolling leukocytes were determined in postcapillary venules. RESULTS: The mean number of perfused capillaries (and standard error of the mean) fell from 20.71 (1.64)/mm before ischemia to 11.69 (1.18)/mm during reperfusion in the experimental group but remained constant in the control group. In the experimental group E/B progressed from 0.43 (0.05) at the onset of reperfusion to 0.87 (0.03) at the end of reperfusion, the number of rolling leukocytes increased from a preischemia mean of 4.00 (1.90) to 14.80 (1.30)/1000 microns2, and the number of stuck leukocytes increased from 1.42 (0.20) to 9.20 (0.70)/1000 microns2. The velocity of the rolling leukocytes did not differ between the control and the experimental groups. CONCLUSIONS: Although microvascular perfusion decreased quickly to a constant level after 2 hours of noflow ischemia plus reperfusion, a progressive increase in tissue injury occurred, which may correlate with the number of stuck leukocytes.

Animals↗
PubMed1994

Structure of human factor D. A complement system protein at 2.0 A resolution.

Factor D, an essential enzyme for the activation of the alternative pathway of the complement system, belongs to the serine protease superfamily. The crystal structure of the enzyme was solved by a combination of multiple isomorphous replacement and molecular replacement methods. The present model was refined to an R-factor of 18.8% using 23,681 observed reflections between 7.5 and 2.0 A resolution, with a root-mean-square deviation from standard bond lengths of 0.016 A. The two non-crystallographically related molecules in the triclinic unit cell have distinctive active site conformations. The protein has the general structural fold of a serine protease, but there are several unique amino acid substitutions resulting in significant alterations in the critical loops responsible for catalysis and substrate specificity in serine proteases. Factor D is the first complement serine protease whose three-dimensional structure has been determined.

Amino Acid Sequence↗
PubMed1994

DNurbs: DNA modeled with NURBS.

DNurbs (dee-in-urbs) are a small number of bicubic patches wrapping each base pair in complementary DNA. NURBS, the nonuniform rational B-spline surfaces now popular in computer graphics, are employed for the patches. Control points for the surface patches are generated from the molecular surface based on canonical base pairs.

Base Sequence↗
PubMed1994

Opacification of Middlebrook agar as an aid in distinguishing Nocardia farcinica within the Nocardia asteroides complex.

Among 58 aerobic actinomycetes isolated from different sources and geographical locations, none of 23 Nocardia asteroides isolates, at 18 N. farcinica isolates, 1 of 5 N. otitidiscaviarum isolates, and 1 of 4 Rhodococcus species isolates opacified Middlebrook 7H10 medium. Within the N. asteroides complex, this characteristic, together with growth at 45 degrees C and resistance to each of erythromycin, cefotaxime, and tobramycin, provides a simple means of distinguishing N. farcinica from N. asteroides.

Actinomycetales↗
PubMed1994

Nurses' work: balancing personal and professional caregiving careers.

Forty full-time nurses who were also responsible for providing care to individuals in their private lives volunteered for a qualitative study of combined caregiving careers. Each respondent kept a diary of caregiving activities during two representative 24-hour periods, and was interviewed prior to, and after diary-recording. Most nurses experienced high levels of stress associated with caregiving in both their professional and private lives. In general, they were relatively satisfied with their lives in both spheres, and felt a slightly greater sense of control in their work lives than in their home lives. Regarding the tensions/conflicts they alluded to, the following themes emerged: an ethic of high expectation, feeling torn between two worlds, a sense of working in isolation, and working in overdrive. The rewards/benefits included remuneration, recognition and self-esteem, opportunities for personal growth, and opportunities for family growth.

Adaptation, Psychological↗
PubMed1994

Clinical audit of health visitor interventions in child accident prevention.

The Health of the Nation strategy has set a target for reducing the death rate from accidents among children aged under 15 by at least 33 per cent by 2005. Accidents are included because they present a major health issue. Maureen Carson and Rosemary Thompson describe the part played by health visitors in the Norwich Community Health Services strategy to cut child accident rates, and the important role of the hospital paediatric liaison health visitor.

Accident Prevention↗
PubMed1993

Long charge-rich alpha-helices in systemic autoantigens.

Autoimmune diseases are characterized by the presence of antibodies and T-cells targeting restricted sets of host proteins. This phenomenon may be due in part to greater non-specific immunogenicity for these proteins compared to others which are not autoantigenic. We used computer-based methods to analyze the sequenced human autoantigens for distinctive patterns of potential immunologic importance. Sequences longer than 27 residues predicted by these methods to form coiled-coil alpha-helices with a probability greater than 0.9 were detected in 40 of 109 (36.7%) of the known human autoantigens. These include many predominantly systemic disease-specific autoantigens not previously known to contain this structure. In comparison, 8.7% of human proteins in the Swissprot data base, and 1.1% of the proteins in the Brookhaven data base were found to contain such segments. These predicted coiled-coil alpha-helices are distinguished from most globular protein helices by greater length, higher charge content, and a heptad repeat multivalency. Coiled-coil segments correlate in part with known autoantibody epitopes and may contribute to autoantigenic potential. Systemic autoantigens generally are either basic or contain extended, multivalent, charge-rich segments such as coiled-coils.

Algorithms↗
PubMed1993

Three-dimensional structure of influenza A N9 neuraminidase and its complex with the inhibitor 2-deoxy 2,3-dehydro-N-acetyl neuraminic acid.

We present here the three-dimensional structure of neuraminidase (E.C. 3.2.1.18) from influenza virus A/Tern/Australia/G70c/75 (N9), determined by the method of multiple isomorphous replacement, and the structure of the neuraminidase complexed with an inhibitor, 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid (DANA). Native and inhibitor complex crystals are isomorphous and belong to space group I432 with unit cell dimensions of 183.78 A. The native enzyme structure and the inhibitor complex structure have been refined at 2.5 A and 2.8 A resolution, respectively, with crystallographic R-factor values of 0.193 for the native enzyme, and 0.179 for the inhibitor complex. The current enzyme model includes 387 amino acid residues which comprise the asymmetric unit. The root-mean-square deviation from ideal values is 0.013 A for bond lengths and 1.6 degree for bond angles. The neuraminidase (NA), as proteolytically cleaved from the virus, retains full enzymatic and antigenic activity, and is a box-shaped tetramer with edge lengths of 90 A and a maximal depth of 60 A. The NA tetramers are composed of crystallographically equivalent monomers related by circular 4-fold symmetry. Each monomer folds into six antiparallel beta-sheets of four strands. The secondary structure composition is 50% beta-sheet. The remaining 50% of the residues form 24 strand-connecting loops or turns. One of the loops contains a small alpha-helix. The structure of the complex of NA with DANA, a transition state analog, has enabled us to identify and characterize the site of enzyme catalysis. The center of mass of bound inhibitor is 32 A from the 4-fold axis of the tetramer, lodged at the end of a shallow crater of diameter 16 A with a depth of 8 to 10 A. There are 12 amino acid residues that directly bind DANA, with a further six conserved amino acids lining the active site pocket. The neuraminidase inhibitor complex provides a three-dimensional model which will be used to further the understanding of enzymatic hydrolysis and aid the design of specific, antineuraminidase antiviral compounds.

Binding Sites↗
PubMed1993

Typing of strains from a single-source outbreak of Pseudomonas pickettii.

Plasmid profiles, genome restriction fragment polymorphisms, carbohydrate oxidation-fermentation reactions, methylumbelliferyl substrate hydrolysis patterns, antimicrobial susceptibilities, and results obtained with the Biolog GN biochemical substrate kit were used to type 19 common-source, but mixed-biotype, outbreak strains and one epidemiologically distinct strain of Pseudomonas pickettii. Biotyping with conventional and methylumbelliferyl substrates failed to distinguish between strains. Plasmid profile testing was found to be inconsistent and not reproducible. The Biolog GN kit allowed greater strain differentiation than restriction fragment polymorphism did (12 biotypes versus 5 biotypes); antimicrobial susceptibility testing yielded 4 biotypes, and oxidation-fermentation tests gave 3 biotypes. Oxidation-fermentation results were consistent with restriction fragment polymorphs in all but 1 of the 20 strains tested. For ease of typing, comprehensive typeability, and reproducibility, oxidation-fermentation tests should be performed initially and followed if necessary by restriction fragment polymorph analysis for the elucidation of P. pickettii infection outbreaks.

Bacterial Typing Techniques↗
PubMed1992

Ultrastructure of spared dopamine terminals in caudate-putamen nuclei of adult rats neonatally treated with intranigral 6-hydroxydopamine.

Residual dopamine terminals in the dorsal striatum, caudate-putamen nuclei (CPN), of adult rats neonatally lesioned with 6-hydroxydopamine (6-OHDA) sustain a relatively high level of dopamine release. We examined whether there were morphological differences in the spared dopamine terminals that might correlate with this increased efficacy. Postnatal male rat pups from 50 litters were pretreated with desmethylimipramine (DMI) to protect from non-specific monoamine damage, then given unilateral intranigral injections of 6-OHDA or vehicle. Coronal sections through the CPN and substantia nigra of the surviving adult animals from each litter were co-processed for immunoautoradiographic or immunoperoxidase localization of the catecholamine synthesizing enzyme, tyrosine hydroxylase (TH). Quantitative ultrastructural analysis established that in animals showing maximal (greater than 90%) depletions in immunoautoradiographic labeling for TH, the number of TH-labeled axons in the CPN ipsilateral to the 6-OHDA injections was reduced to one third of the number seen in the contralateral, unlesioned hemisphere, or the CPN from vehicle-injected animals. The ultrastructural features of residual terminals ipsilateral to 6-OHDA lesions were morphologically similar to those of the contralateral side or in vehicle-injected animals. However, in comparison with controls, these TH-labeled terminals had significantly larger mean cross-sectional diameters. When subdivided into groups according to size, there were significantly fewer small (0.0-0.1 micron 2) and more large (0.41-0.50 micron 2) TH-immunoreactive profiles in lesioned versus unlesioned CPN. The remaining TH-labeled terminals ipsilateral to the 6-OHDA lesions also appeared to be more often in direct contact with unlabeled soma and proximal dendrites as opposed to dendritic spines in the unlesioned CPN. These results suggest that the enhanced activity of dopamine neurons innervating the CPN after nigral 6-OHDA lesions may contribute to changes in size and target of their terminals. Alternatively, the observed large size of remaining dopamine terminals may reflect selective vulnerability of smaller axons to 6-OHDA toxicity.

Aging↗