Biomedical subjects
S M Lewis
Publications and source records attributed to S M Lewis.
Resolved conformational states of spin-labeled Ca-ATPase during the enzymatic cycle.
We have used frequency- and time-resolved electron paramagnetic resonance (EPR) to study the effects of substrate on the nanosecond conformational dynamics of the Ca-ATPase of sarcoplasmic reticulum, as detected by an iodoacetamide spin label (IASL) attached covalently to the enzyme. We confirm previous results [Coan, C. (1983) Biochemistry 22, 5826] showing that this probe is less rotationally mobile following the addition of nucleotides (ADP, AMPPNP, ATP) and that the shape of the spectrum suggests the presence of two components. We used two approaches to enhance EPR resolution in order to resolve the spectral components and their corresponding conformational states. First, to improve resolution in the frequency (spectral) domain, we used perdeuterated IASL, which results in narrower line widths. Digital spectral analysis resolves the EPR spectrum into two components, one that is indistinguishable from the spectrum observed in the absence of ligands and another that indicates more restricted probe motions, suggesting a distinct conformation of the labeled protein. Additions of substrate ligands appear to change only the mole fractions of the two components. The mole fraction of the restricted component (fR) was 0 in the absence of ligands, but increased to about 0.5 in the presence of saturating concentrations of AMPPNP and Ca2+. In general, ATP and its analogs increase fR, with larger effects observed in the presence of Ca. However, calcium has no effect by itself (fR = 0). Both monovanadate and decavanadate increase fR, but the formation of a covalent phosphoenzyme from inorganic phosphate (E2-P) had no effect (fR = 0).(ABSTRACT TRUNCATED AT 250 WORDS)
Oxidative DNA damage levels in rats fed low-fat, high-fat, or calorie-restricted diets.
Increased fat and caloric content of the diet has been associated with increased mammary tumor incidence. The dietary modulation of cellular redox state may be one mechanism behind this association. We have examined the effects of changes in dietary fat and caloric intake on the levels of 5-hydroxymethyluracil in DNA from rat liver and mammary gland. Female Fischer 344 rats, 40 days old, were maintained on 3% (low-fat), 5% (control), or 20% (high-fat) corn oil diets for 2 weeks. A fourth group of rats had the same daily fat intake as the control group, but total caloric intake was restricted by 40%. As a measure of oxidative DNA damage, 5-hydroxymethyluracil levels were measured in the DNA extracted from liver and mammary gland by gas chromatography-mass spectrometry. 5-Hydroxymethyluracil levels in the liver DNA of the low-fat, high-fat, and calorie-restricted groups were decreased relative to that of control, but the only significant decrease was in the calorie-restricted group (p less than 0.01). In the mammary gland DNA, statistically significant decreases in damage were found in each group relative to control (p less than 0.05). The relationship between fat in the diet and oxidative stress is thus complex. These results show that changes in dietary intake of both fat and calories can modulate oxidative DNA damage levels, and the effect of diet was more clearly evident in the DNA from mammary gland than in DNA from liver.
The selection of laboratory equipment.
The last few years have seen dramatic changes in laboratory haematology. Technological developments with increasing levels of automation have resulted in a plethora of new instruments. The choice of which to buy, often confused by conflicting advice and persuasive salesmanship, must be balanced by the need for cost containment. This calls for an understanding of the principles of selection to ensure that the system that is chosen will provide reliable results in the most efficient manner at the lowest possible cost. Most of the instruments which are currently available perform their intended tasks reasonably well albeit with some inter-instrument differences, usually due to different operating procedures and different methods of calibration. The task is to find a system that performs the required tests correctly and reliably, fits into the work requirements of the laboratory and is accepted by the staff who will operate it, whilst its cost both capital and recurrent, should be within the department's financial constraints. The selection of laboratory equipment is thus a complex and time consuming task.
The New Jersey health care delivery structure--a clarification.
Recent SBME rules were developed from issues raised by a previous commissioner of health. SBME received comments on these issues at two public hearings. SBME drafted the rules to incorporate the substance and the principles of the state's Health Care Cost Reduction Act.
Single-operator comparison of early and mid-second-trimester amniocentesis.
We sought to determine whether early amniocentesis is a safe and acceptable method of genetic evaluation in early pregnancy. During the 54-month period from September 1986 to February 1991, 300 consecutive early second-trimester amniocenteses were performed transabdominally at 13-14 weeks' gestation and 567 consecutive mid-second-trimester transabdominal amniocenteses were performed at 16-18 weeks. Group assignment was nonrandomized, interoperator-dependent variables were eliminated, and analysis was performed in one cytogenetics laboratory. The median maternal age and indications for the procedure were similar in both groups. There were no significant differences between the early- and mid-second-trimester amniocenteses in failed sampling, ambiguous results, pregnancy loss from 4 weeks after the procedure to 28 weeks' gestation, preterm birth, or perinatal death rate. Pregnancy loss within 4 weeks of amniocentesis was more frequent in early- than in mid-second-trimester amniocenteses. We conclude that early amniocentesis is a safe and acceptable method of genetic evaluation.
Microsecond rotational dynamics of spin-labeled Ca-ATPase during enzymatic cycling initiated by photolysis of caged ATP.
We have measured the microsecond rotational motions of the sarcoplasmic reticulum (SR) Ca-ATPase as a function of enzyme-specific ligands, including those that induce active calcium transport. We labeled the Ca-ATPase with a maleimide spin probe and detected rotational dynamics using saturation-transfer electron paramagnetic resonance (ST-EPR). This probe's ST-EPR spectra have been shown to be sensitive to microsecond protein rotational motion, corresponding to large-scale protein rotations that should be affected by changes in the enzyme's shape, flexibility, protein-protein interactions (oligomeric state), and protein-lipid interactions. We found that the motions of the enzyme-nucleotide and the enzyme-nucleotide/Ca states are indistinguishable from the motions in the absence of ligands. Rotational mobility does decrease in response to the addition of DMSO, a solvent that inhibits Ca-ATPase activity and stabilizes the phosphoenzyme. However, the addition of phosphate to form phosphoenzyme, in the presence or absence of DMSO, does not change the motions significantly. During the steady state of active calcium transport, the microsecond rotational mobility is indistinguishable from that of the resting enzyme. In order to detect any transient changes in mobility that might not be detectable in the steady state and to improve the precision of steady-state measurements, we photolyzed caged ATP with a laser pulse in the presence of calcium and detected the ST-EPR response from the spin-labeled enzyme, with a time resolution of 1 s.(ABSTRACT TRUNCATED AT 250 WORDS)
Cutting and closing without recombination in V(D)J joining.
Open and shut junctions are rare (V(D)J joining products in which site-specific recognition, cleavage and re-ligation of joining signals has been uncoupled from recombination. Here, we investigate the relationship of opening and shutting to recombination in two ways. First, we have tested a series of substrates containing one or two joining signals in an in vivo assay. Opening and shutting can be readily observed in substrates that have only one consensus joining signal. Thus, unlike recombination, the majority of open and shut events do not require interactions between two canonical joining signals. Next we examined two-signal substrates to investigate the effect of signal proximity on the frequency of dual open and shut events. These experiments indicate that at least some of the time opening and shutting can be a two-signal transaction. Together these results point to two mechanistically related, but distinct origins for open and shut joining events. In one case, cutting and closing may occur without interaction between two signals. In the other, we suggest that interaction of a canonical signal with 'cryptic' signal-like elements whose sequence is extensively diverged from canonical signals, may bias the V(D)J recombination machinery towards opening and shutting rather than recombination. Open and shut operations could in this way provide a means whereby mistakes in target recognition by the V(D)J recombination machinery produce a non-recombinant outcome, avoiding deleterious chromosomal rearrangements in lymphoid tissues.
Standardized hematology reports.
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Primary thrombocythaemia in pregnancy.
Nine pregnancies in six patients with primary thrombocythaemia are reported. Eight pregnancies resulted in the delivery of normal infants. One pregnancy ended in spontaneous abortion at 7 weeks gestation. One pregnancy was complicated by superficial thrombophlebitis and a postpartum haemorrhage. We suggest that pregnancy in patients with primary thrombocythaemia can have a favourable outcome, but requires close monitoring. Administration of aspirin during pregnancy may be of benefit.
Lauryl sulphate haemoglobin: a non-hazardous substitute for HiCN in haemoglobinometry.
The haemiglobincyanide (HiCN) method for measuring haemoglobin is used extensively worldwide; its advantage is the ready availability of a stable and internationally accepted reference standard/calibrator. However, its use may create a problem, especially in automated analysers, as the waste disposal of large volumes of reagent containing cyanide may constitute a potential toxic hazard. As an alternative, conversion of haemoglobin to a sulphate derivative by nontoxic sodium lauryl sulphate has been proposed and is available as a commercial product from Toa Medical Electronics Co. Ltd., Japan. This evaluation has shown it to be as reliable and reproducible as HiCN for measuring haemoglobin at all concentrations, by both spectrophotometry and automated analyser. Maximum absorbance is at 534 nm. Total conversion occurs almost instantaneously, and includes methaemoglobin but not sulphaemoglobin. HbF is also measured. The only disadvantage is that solutions of haemoglobin-sulphate are stable for only a few hours at room temperature and up to three weeks at 4 degrees C. Thus, for standardization and calibration of instruments it is necessary to use a sample of blood or lysate the haemoglobin value of which has been assigned initially by the HiCN method.
Comparability of haematological and biochemical parameters before and after apheresis of volunteer donors.
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Current concerns in haematology. 3: Blood count calibration.
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Intra-aortic balloon counterpulsation support in the high-risk cardiac patient undergoing urgent noncardiac surgery.
Patients with a recent myocardial infarction, congestive heart failure, sever angina, or uncorrected multivessel coronary artery disease are at increased risk of cardiac complications after major noncardiac surgery. Although invasive hemodynamic monitoring and preoperative optimization of cardiac status may lead to some reduction in the rate of perioperative cardiac events, the mortality from such events remains high. We report our experience with the use of perioperative intra-aortic balloon counterpulsation in eight patients with unstable coronary syndromes or severe coronary artery disease who underwent urgent noncardiac surgery. There were no perioperative cardiac events while the intra-aortic balloon pump (IABP) was in place. There were two postoperative cardiac events (non-fatal myocardial infarction, congestive heart failure) in the first postoperative week after the IABP was removed. One patient required emergent femoral thrombectomy as a result of intra-aortic balloon counterpulsation and subsequently died of a gastrointestinal hemorrhage. Intra-aortic balloon counterpulsation should be considered as an adjunct to maintain hemodynamic stability for the high-risk cardiac patient about to undergo urgent or emergent noncardiac surgery.
Cardiogenic oscillations of He and SF6 in expired gas in dogs.
Quantitative analysis of cardiogenic oscillations of He and SF6 during airway and venous loading demonstrated that both VA/Q and VA/VA inequalities were involved in the lung gas inhomogeneity producing cardiac oscillations in the expirogram. Both inequalities were coupled in such a manner that low VA/Q units had high VA/VA. The oscillations were modified in conducting airways where SF6 oscillations were attenuated more than He oscillations, probably by laminar Taylor dispersion.
Detection and functional assessment of accessory splenic tissue (splenunculi) with radiolabelled heat damaged autologous erythrocytes.
Eight patients (six with idiopathic thrombocytopenic purpura, one with immune pancytopenia, and one with autoimmune haemolytic anaemia), who had previously been splenectomized, were found to have splenunculi using radioactively labelled heat damaged autologous erythrocytes. In all patients the splenunculi were found to have significant 'splenic function'. However, there was a poor correlation between the size and the function of the splenunculi. Splenunculectomies were carried out in two patients. This led to a complete haematological remission in one patient and partial remission in the other. In three patients, where splenunculectomies could not be undertaken, the patients had to remain on significant doses of immunosuppressive therapy. In one patient complete remission was achieved using a course of immunosuppressive therapy alone and in the remaining two there was insufficient information to draw valid conclusions. Blood clearance kinetic studies of heat damaged erythrocytes were found to provide an accurate functional assessment of the splenunculi in all patients. However, the presence of a functional splenunculus was found to be the cause of disease relapse in only some patients. Therefore, the management of similar patients should perhaps be along the same lines as that at their initial presentation, that is, immunosuppressive therapy should be tried if they are not on any at the time of relapse, if they are already on it, then splenunculectomy should be done.
Standardization and harmonization of the blood count: the role of International Committee for Standardization in Haematology (ICSH).
Scientific principles of standardization were first applied in haematology in 1963 when the International Committee for Standardization in Haematology was established with a primary objective to improve the measurement of haemoglobin. Subsequently, ICSH has established Expert Panels on a wide range of haematological topics, including especially a Panel on Cytometry. The purpose of haematological standardization is to obtain precision, accuracy, specificity and harmonization of results between different laboratories in all countries and also between different instruments or methods in the same laboratory. To achieve these objectives ICSH sponsors collaborative studies by scientists from academic centres and from industry and uses a consensus procedure for establishing standards on the basis of the scientific data, followed by an educational programme to ensure that the standards are adopted worldwide. ICSH defines material standards and standardized methods. Material standards are classified as primary international standards, certified reference materials, secondary standards and calibrators. These must be distinguished from control preparations which are intended exclusively for quality control. Standardization of methods must also be considered at four levels: definitive, reference, selected and routine. Each has a place in practice but their roles must be clearly defined. ICSH has an established protocol for evaluation of automated blood cell counters. This defines the levels of precision and accuracy of instrument performance. It is also necessary to assess "clinical utility". The main requirement of the practising haematologist is clinical reliability and harmonization of results for comparability. One of the major functions of ICSH is to provide an interface for collaboration between the manufacturers who develop the instruments and the users in order to achieve this goal.
Dietary fiber for dogs: I. Effects of graded levels of dietary beet pulp on nutrient intake, digestibility, metabolizable energy and digesta mean retention time.
The optimal level of beet pulp (BP) inclusion in a meat-based dog diet and the effects of graded levels of dietary BP on fecal excretion responses and mean retention time of marked fiber in the gastrointestinal tract of the dog were evaluated using 30 female English Pointers assigned to isonitrogenous diets containing 0, 2.5, 5.0, 7.5, 10.0 or 12.5% BP (DM basis). Beet pulp replaced portions of dietary cornstarch. Digestibilities of DM and OM decreased by an average of 6% when comparing diets containing BP to the control diet, and quadratic and cubic responses were noted in digestibilities of fiber constituents (lower values at the 7.5 and 10.0% levels, higher values at the 2.5, 5.0 and 12.5% levels). Digestible energy (DE) and ME intakes (kcal/d) were not affected by treatment, but when expressed as a percentage of GE, values decreased (4.8% for DE; 6.2% for ME) linearly with increasing BP levels. Wet weight of feces increased (from 117 to 374 g/d) linearly as percentage of dietary BP increased. Frequency of defecation was higher (P less than .05) for dogs fed the diet containing 12.5% BP than for dogs fed the other diets (5.2 vs mean value of 2.8/24 h). Mean retention time of marked fiber decreased linearly (high value of 23.4 h for the 2.5% BP treatment, low value of 13.0 h for the 10.0% BP treatment) with increased level of BP. Beet pulp levels up to 7.5% of diet DM appear acceptable as a dietary fiber source in a meat-based canine diet.