Biomedical subjects
S J Clark
Publications and source records attributed to S J Clark.
Validation of ambulatory monitors in special populations.
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The influence of ambulatory blood pressure monitoring on the design and interpretation of trials in hypertension.
OBJECTIVE: To describe the reproducibility of diastolic blood pressure (DBP) measurement by clinic and ambulatory monitoring and to evaluate the effects of this reproducibility on the design and interpretation of clinical trials in hypertension research. DESIGN: Prospective single-blind study of repeat measurement reproducibility of blood pressure recording. SETTING: Tertiary referral hospital hypertension clinic. PATIENTS: One hundred untreated mild-to-moderate hypertensive subjects taking 1 month of single-blind placebo. MAIN OUTCOME MEASURES: A single clinic measurement of DBP was poorly reproducible and the results for single DBP estimates taken out of a daytime ambulatory recording were similar. Average ambulatory DBP was much more reproducible, although this improvement depended upon the averaging of many measurements taken throughout the day. CONCLUSIONS: Ambulatory monitoring would decrease antihypertensive trial size by a factor of four or halve the size of detectable DBP difference between treatments. The use of poorly reproducible DBP measurements such as single clinic readings may have led to an underestimation of the risks of minor degrees of blood pressure elevation because of a 'regression dilution' bias. For single clinic DBP readings, we calculate this underestimation to be as much as 69%, and for average ambulatory DBP approximately 20%.
Effects of mild physical activity, atenolol and the combination on ambulatory blood pressure in hypertensive subjects.
OBJECTIVE: To evaluate whether beta-blocker treatment could enhance the effect of a mild physical training programme upon blood pressure. DESIGN AND METHODS: In 12 hypertensive subjects (mean age: 40.3 years) a prospective randomized Latin square-design trial was performed with three treatments: physical training and placebo tablets; atenolol 50 mg once a day and inactivity; and physical training and atenolol 50 mg once a day. RESULTS: Training significantly increased maximal ventilatory oxygen consumption (VO2MAX), and there was a decrease in ambulatory diastolic blood pressure (DBP) which did not reach statistical significance. Atenolol alone significantly reduced ambulatory systolic blood pressure (SBP) and DBP. Atenolol alone did not reduce VO2MAX. The combination of training and atenolol resulted in an increase in VO2MAX compared with atenolol alone, but no additional significant fall in blood pressure. CONCLUSIONS: Atenolol did not enhance the effect of physical training upon blood pressure and had little if any effect upon the training-induced increase in exercise tolerance.
High titers of cytopathic virus in plasma of patients with symptomatic primary HIV-1 infection.
BACKGROUND: Primary infection with the human immunodeficiency virus (HIV-1) frequently causes an acute, self-limited viral syndrome. To examine the relations among viral replication, the immune response of the host, and clinical illness during this initial phase of infection, we undertook a quantitative, molecular, and biologic analysis of infectious HIV-1 in the blood and plasma of three patients with symptomatic primary infection and of a sexual partner of one of them. METHODS: During an eight-week period of primary infection, HIV-1 was cultured frequently in dilutions of plasma and peripheral-blood mononuclear cells (PBMC), and levels of HIV-1 antigen and antibody were determined sequentially by enzyme-linked immunosorbent assay and immunoblotting. Replication-competent HIV-1 proviruses were cloned and characterized biologically. RESULTS: Six to 15 days after the onset of symptoms, high titers of infectious HIV-1 (from 10 to 10(3) tissue-culture-infective doses per milliliter of plasma) and viral p24 antigen were detected in the plasma of all three patients. These titers fell precipitously by day 27, and the decline coincided with an increase in the levels of antiviral antibodies and the resolution of symptoms. Sequential isolates of virus from plasma and PBMC obtained throughout the period of primary infection, as well as virus derived from two molecular proviral clones, were highly cytopathic for normal-donor PBMC and immortalized T cells, despite the marked reduction in the titers of virus in plasma. CONCLUSIONS: Primary, symptomatic HIV-1 infection is associated with high titers of cytopathic, replication-competent viral strains, and during such infection potential infectivity is enhanced. Effective control of HIV-1 replication during primary infection implies the activation of clinically important mechanisms of immune defense that merit further examination in relation to the development of antiviral therapy and vaccines.
British hypertension protocol.
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Analysis of ambulatory blood pressure data.
Many statistical techniques can be used to describe different aspects of ambulatory records, but it is highly unlikely that any single parameter will suffice. A more realistic aim is for a combination of measures that combine statistical validity with physiological meaning and clinical usefulness. This will require compromises between conflicting demands, and only experience and research will ultimately determine the best summary statistics. Some of the available techniques are described here in terms of their value to research and to clinical practice, and an approach is proposed based on considerations of the physiology of blood pressure in free-living man.
On variables that affect estimates of the true sizes and densities of radioactively labeled cell nuclei.
Tritiated thymidine has been widely used as a nuclear marker of cell birth. The true diameters and packing densities (nuclei/microns 3) of such radioactively labeled nuclei cannot be measured directly from tissue sections. Here we show that existing stereological corrections cannot be applied to data from radioactively labeled nuclei. We empirically measured the number of silver grains exposed by nuclei containing tritiated thymidine. The nuclei were separated from the photographic emulsion by known thicknesses of fixed, embedded avian telencephalon. The results of this experiment were used to develop an equation that estimates the number of silver grains exposed by a cell nucleus of any given diameter, containing a given amount of radioactive label, and located at any given distance from the photographic emulsion. The equation also allows one to calculate the probability that a label-containing nucleus will be correctly classified as labeled. Simulations of the equation revealed that not all label-containing nuclei are correctly classified by using commonly employed identification procedures and that larger nuclei are less likely to be correctly classified than smaller nuclei, given the same amount of label. The equation can be used to modify one class of existing stereological equations so as to be applicable to measurements of radioactively labeled nuclei. Finally, we discuss the assumptions and limitations of this modification.
The dissociation of peripheral proteins from erythrocyte membranes brought about by p-mercuribenzenesulfonate.
The organic mercurial p-mercuribenzenesulfonate in 5 mM phosphate buffer (pH 8.0) solubilized ankyrin, bands 4.1 and 4.2, and glyceraldehyde-3-phosphate dehydrogenase from spectrin-depleted erythrocyte membranes. Glyceraldehyde-3-phosphate dehydrogenase was the protein most readily solubilized, being almost completely extracted by 0.5 mM reagent. The solubilization of ankyrin was similar to that of band 4.2, both showing maximal solubilization with 1.0 mM reagent. Band 4.1 was not appreciably solubilized below 2.5 mM p-mercuribenzenesulfonate. N-Ethylmaleimide did not itself solubilize proteins from ghosts or spectrin-depleted vesicles, and pretreatment at low temperature by 4 mM N-ethylmaleimide did not prevent subsequent solubilization by the mercurial. However, pretreatment at 37 degrees C with N-ethylmaleimide inhibited subsequent solubilization of ankyrin and band 4.2 by the mercurial and also resulted in the loss of binding of 1 mol mercurial per mol band 3. These data suggest that release of ankyrin and band 4.2 from the membrane by mercurial is linked to modification of band 3 by the reagent. After incubation of intact erythrocyte membranes with 0.1 M NaCl, treatment with p-mercuribenzenesulfonate selectively solubilized actin from the membranes. The resulting actin-depleted membranes did not vesiculate, but became spherical and lost their biconcave shape. Fragmentation was observed after subsequent removal of spectrin at low ionic strength.
Chromosome lesions which could be interpreted as "fragile sites" on the distal end of Xq.
Chromosome lesions which could be interpreted as "fragile sites" on the distal end of the long arm of the X chromosome were identified during a cytogenetic study of 160 mentally retarded adult males with no apparent cause of their mental retardation and one normal adult female with a family history of fra (X) syndrome. Peripheral blood samples were cultured in either M199 or RPMI 1640 medium with FUdR or BrdU. Metaphases were examined for chromosome lesions or fragile sites on the distal end of Xq and 3 distinct sites were observed: Xq26, Xq27.2, and Xq27.3. Other chromosome lesions at Xq28 were observed and interpreted as nonspecific telomeric structural changes. Chromosome lesions were observed in cells from 14 of the 161 individuals. These included: 5 patients with an Xq26 site, 2 with the recently reported Xq27.2 site, 4 with the Xq27.3 site (characteristic of the fra (X) syndrome), 2 with nonspecific telomeric structural changes, and one individual with 2 lesions (a nonspecific telomeric structural change and an Xq26 site). Additional research is necessary to determine the frequency and clinical significance, if any, of lesions occurring in this region of the X chromosome and to distinguish among heritable fragile sites, constitutive fragile sites, and nonspecific telomeric structural changes.
The two forms of the beta-subunit of initiation factor-2 from reticulocyte lysates arise from proteolytic degradation.
Dholakia and Wahba (J. Biol. Chem. (1987) 262, 10164-10170) have reported that preparations of purified initiation factor-2 (eIF-2) from rabbit reticulocytes contain two forms of the beta-subunit. These forms differ in their apparent molecular weights as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and are accordingly termed beta H (heavy, the slower-migrating species, apparent Mr = 54,300) and beta L (light, the faster-migrating species, apparent Mr = 53,100). We confirm that two forms of eIF-2 beta are present in such preparations, but present evidence that the beta L is generated from beta H during the isolation procedure. Crude reticulocyte lysates contain only the beta H species as judged from immunoblotting of reticulocyte proteins resolved by SDS-PAGE using an antiserum against eIF-2 beta. The beta L species appears after the ammonium sulphate fractionation step used early in the purification procedure, but is not apparent if a cocktail of proteinase inhibitors is included in the buffers used during the purification, indicating that it is a proteolytic degradation product generated during the isolation procedure. Cleveland mapping failed to reveal any differences between the two species. Both the beta H and the beta L forms are phosphorylated by casein kinase-2, and, as judged by one- and two-dimensional peptide mapping, at identical sites in each species. Since casein kinase-2 phosphorylates serine-2 in eIF-2 beta, the beta L form must still contain the N-terminal region and is presumably produced by limited proteolysis at the carboxyl terminus of the beta-subunit.
Casein kinase-2 phosphorylates serine-2 in the beta-subunit of initiation factor-2.
We have previously presented evidence which suggests that casein kinase-2 phosphorylates a serine residue near the N-terminus of the beta-subunit of the initiation factor eIF-2 (Clark, S.J. et al. Biochim. Biophys. Acta 968, 211-219). We now report further data which confirm that it is serine-2 which is phosphorylated by casein kinase-2. This data includes (1) the electrophoretic mobilities of the phosphopeptides produced by different cleavage techniques, (2) the amino acid composition of the principal phosphopeptide generated by treatment with cyanogen bromide and (3) the resistance of this phosphopeptide to Edman degradation.
Hormone ontogeny in the ovine fetus: XIX: The effect of a potent luteinizing hormone-releasing factor agonist on gonadotropin and testosterone release in the fetus and neonate.
To investigate further the role of the hypothalamic luteinizing hormone releasing factor (LRF) pulse generator and the pituitary LRF receptor in the regulation of gonadotropin secretion and gonadal steroidogenesis in the ovine (O) fetus and neonatal lamb, we measured the increment (the difference between the concentration of plasma LH at time 0 and peak LH) in oLH (delta oLH) and oFSH (delta oFSH) responses to a potent LRF agonist, D-Trp6Pro9NEt-LRF (LRF-A), after consecutive daily doses in 17 ovine fetuses (six females, 11 males) and in 15 neonatal lambs (six females, nine males). Seven of the lambs had been studied as fetuses. In addition, plasma concentrations of testosterone (T) and androstenedione (delta 4A) were measured in nine male fetuses. After a stimulatory response to the first dose of LRF-A, the mean delta oLH and delta oFSH responses in the 106- to 118-d gestation fetuses of both sexes were significantly suppressed by the fourth dose and in the neonatal lamb by the second dose. Suppression was sustained throughout the duration of LRF-A therapy which included the gestational interval when the fetal pituitary exhibits its greatest responsiveness to an acute dose of synthetic LRF. The duration of oLH and oFSH suppression after cessation of LRF-A therapy was studied by measuring the delta oLH and delta oFSH responses to LRF before and at intervals after LRF-A therapy. In the fetus, the delta oLH and delta oFSH responses remained significantly decreased 7-8 d after the agonist was discontinued.(ABSTRACT TRUNCATED AT 250 WORDS)
Bohr's principle of complementarity.
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The effects of p-mercuribenzenesulfonate on purified spectrin and actin.
The compound p-mercuribenzenefulfonate was found to affect the self-association behavior of both spectrin and actin. The reagent brings about the depolymerization of F-actin, as judged from the decrease in the fluorescence of an attached pyrene label, with a second-order rate constant an order of magnitude less than that for the disruption of isolated erythrocyte cytoskeletons. Therefore, it is unlikely that the depolymerization of actin is the rate-determining step in the mercurial-dependent disruption of the erythrocyte cytoskeleton. Low reagent concentrations caused an initial rapid dissociation of spectrin tetramers at a rate comparable with that of cytoskeleton disruption. Prolonged incubation, or higher reagent concentrations, resulted in subsequent aggregation of spectrin. The reagent also prevented the interaction between spectrin and actin, presumably through its depolymerization of actin and its effects on spectrin. The early event in the disruption of isolated erythrocyte cytoskeletons by p-mercuribenzenesulfonate thus appears to be the dissociation of spectrin oligomers. Subsequent depolymerization of actin brought about by the reagent then results in total disruption of the cytoskeleton.
Activation of rat T lymphocytes by anti-CD2 monoclonal antibodies.
Rat T cells and thymocytes were induced to proliferate by a pair of mAbs, MRC OX-54 and MRC OX-55, directed against rat CD2. Accessory cells were required but their role was not simply for crosslinking of the two mAbs, as neither MRC OX-54 nor MRC OX-55 alone, in the presence of a crosslinking second antibody, caused T cell mitogenesis. Nor could the phorbol ester PMA replace either antibody. The two mAbs recognized distinct epitopes on rat CD2; however, MRC OX-54 could partially block MRC OX-55 binding whereas the reverse situation was not seen. A further CD2 epitope was recognized by two mutually competitive mAbs, MRC OX-34 and MRC OX-53, which were not mitogenic. Neither MRC OX-34 nor MRC OX-53 affected the binding of MRC OX-54 or MRC OX-55, yet they prevented the mitogenic effect induced by these mAbs. The presence of mAbs against CD4 and the IL-2-R also abrogated this mitogenesis, whereas an anti-CD5 mAb augmented the CD2-induced proliferation.
Structure and phosphorylation of eukaryotic initiation factor 2. Casein kinase 2 and protein kinase C phosphorylate distinct but adjacent sites in the beta-subunit.
Eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes can be phosphorylated on its beta-subunit by two different protein kinases, protein kinase C and casein kinase 2. Phosphorylation by these kinases is additive, suggesting that they phosphorylate different sites (serine residues) in eIF-2 beta. Two-dimensional peptide mapping of the phosphopeptides generated from labelled eIF-2 beta by digestion with trypsin, cyanogen bromide or Staphylococcus aureus V8 proteinase showed that protein kinase C and casein kinase 2 phosphorylated distinct and different sites in this protein. This conclusion was supported by the results of analysis of the phosphopeptides on reverse-phase chromatography. Analysis of the phosphopeptides derived from eIF-2 beta labelled by both kinases together strongly suggested that the sites labelled by protein kinase C and casein kinase 2 are adjacent in the primary sequence. These data are discussed in the light of the present understanding of the sequence specificity of the kinases. Rat liver eIF-2 beta was also found to be a substrate for protein kinase C and casein kinase 2, which were again shown to label different serine residues.
Reversible true precocious puberty secondary to a congenital arachnoid cyst.
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