Search PubMed⌕ Search

Biomedical subjects

J L Smith

Publications and source records attributed to J L Smith.

At least 145 records · Page 8Linked to original sources

Signals from dual gated TCD systems: curious observations and possible explanations.

Dual gated transcranial Doppler (TCD) systems have the potential to distinguish definitively embolic events in the cerebral circulation from artefact. While developing such a system, three curious observations were made and are the subject of this article. The first was that the velocity of propagation of an embolus in an artery as calculated from the ultrasonic Doppler shift was, in many cases, dramatically different from the velocity calculated using a time of flight method. The second observation was that the durations of embolic signals measured in two gates were often very different, despite the received gates being identical in length. The third observation was that some emboli detected in the deeper gate did not appear in the more superficial gate, and, more surprisingly (as has recently been reported by another group), some emboli that had not previously appeared in the deeper gate appeared in the more superficial gate. It is hypothesised that all of these effects can be explained in terms of the geometry of the middle cerebral artery in relation to the interrogating ultrasound beam. None of these effects should detract from the usefulness of TCD, but will need to be widely appreciated if dual gated measurements of cerebral emboli are to be interpreted correctly.

Cerebrovascular Circulation↗

Analysis of the frequency modulation present in Doppler ultrasound signals may allow differentiation between particulate and gaseous cerebral emboli.

The purpose of this study was to investigate the incidence of frequency modulation in Doppler signals from cerebral emboli and to seek possible explanations for its occurrence. Signals from 200 particulate emboli and 200 presumed gaseous emboli were studies. The Doppler signals were visualised in the time domain and were classified into three main types. Type I signals contained no modulation, type II signals showed gradual frequency changes and type III showed a rapid change evident in only a small percentage of the entire signal. Type I signals were observed from 71.5% of particulate emboli but only 19% of gaseous emboli (X(2)) = 111, p < 0.001). Type II signals were found in 28.5% of particulate emboli and 38% of gaseous emboli (X(2)) = 4.06, p < 0.05). The most surprising and significant finding was that 43% of gaseous embolic signals were categorised as type III signals compared with 0% of signals from particulate emboli (X(2)) = 109, p < 0.001). The finding that known particulate emboli appear never to produce rapid frequency modulation may provide a basis for differentiating between gaseous and particulate emboli.

Cerebral Arteries↗

Principles of chronic venous access: recommendations based on the Roswell Park experience.

At Roswell Park Cancer Institute, we have seen a dramatic increase in the need for long-term venous access. Chronic venous catheters are an indispensible part of the treatment provided to oncology patients. Cancer patients are often at higher risk for complications secondary to their underlying disease and treatments. These risks may be minimized by paying close attention to several important aspects of central line placement. These include matching individual patient needs with the access device most suited to those needs, a thorough preoperative assessment, and the safest and most appropriate operative approach for placement. Likewise, the prompt recognition and treatment of complications when they do occur is crucial to the care of these patients. In order to optimize the care of patients with long-term venous access devices, we have reviewed our experience of over 700 vascular access consultations and offer the following recommendations.

Bacterial Infections↗

Lecithin-cholesterol acyltransferase activity in normocholesterolaemic and hypercholesterolaemic roosters: modulation by lipid apheresis.

Lipid apheresis, a recently described procedure for the elimination of lipid but not apolipoproteins from plasma, was applied to normocholesterolaemic and hypercholesterolaemic roosters. Lipid apheresis resulted in an immediate reduction in plasma unesterified cholesterol concentration, which was sustained for 150 min. The reduction in unesterified cholesterol concentration was higher in the normocholesterolaemic animals than in the hypercholesterolaemic animals. Lipid apheresis induced changes in the ratio of plasma unesterified to total cholesterol in normocholesterolamic animals but not in hypercholesterolaemic animals. In hypercholesterolaemic animals, lecithin-cholesterol acyltransferase (LCAT) activity was not affected by lipid apheresis, whereas in normocholesterolaemic animals LCAT activity was acutely reduced for 150 min after lipid apheresis. Saturated LCAT kinetics occurred in the hypercholesterolaemic animals but not in the normocholesterolaemic animals. LCAT obeyed Michaelis-Menten kinetics. After lipid apheresis, there was a pool of unesterified cholesterol that was available as substrate for LCAT to a greater extent in hypercholesterolaemic animals than in normocholesterolaemic animals. These observations may have important implications for lipid apheresis as a treatment for atherosclerosis.

Animals↗

The effect of load carrying and floor contaminants on slip and fall parameters.

A study was conducted to provide set-up values of an important biomechanical parameter, heel velocity, for slip resistance testers. Four different floor surfaces (with or without oil contaminant) were prepared for ten subjects with each walking at a fixed velocity while carrying five different loads. Stride lengths were also measured to find whether a significant difference existed between a carrying method typically used in industry (two-handed carrying of a load in a tote box held directly in front of the body) and previously used load carrying techniques (backpack and front-back pack systems). A programmable slip resistance tester was used to measure dynamic coefficient of friction with conventional set-up values for heel velocity. The results showed that the conventional set-up value for heel velocity is valid for dry floors but is too low on oily floors because faster transfer of body weight was seen owing to decreased stride length. On oily floors, heel velocities of 60 to 140 cm/s would be recommended. An abnormal gait pattern, short stride length, was seen on oily floors or with heavy load carriage because subjects adjusted their stride length for a better stance.

Acceleration↗

Determination of hepatic acyl-coenzyme A-cholesterol acyltransferase activity in LPN hamsters: a model for cholesterol gallstone formation.

Acyl-coenzyme A-cholesterol acyltransferase (ACAT) catalyses the esterification of cholesterol with long-chain fatty acyl-coenzyme A derivatives and has been implicated in the development of cholesterol gallstones. In this study we have examined several key components of the hepatic ACAT assay in order to develop a reliable and sensitive ACAT assay for LPN hamsters, a breed of golden Syrian hamster which has been characterized recently by this laboratory as a particularly good model for studying the pathogenesis of cholesterol gallstones. The newly developed ACAT assays were subsequently used to examine whether hepatic ACAT activity is altered in this animal model. Important new methodological findings were: (i) ACAT activity displayed two pH optima, one at 7.0 when assayed using endogenous cholesterol as substrate, and the other at about pH 8.5-9.0 when assayed in the presence of exogenous cholesterol; (ii) ACAT activity increased markedly when exogenous cholesterol was delivered to ACAT in Tween 80 (125-fold) or hydroxypropyl-beta-cyclodextrin (200-fold) in contrast to the use of cholesterol/phosphatidylcholine liposomes (9-fold); (iii) the addition of dithiothreitol, but not reduced glutathione, to the assay mixture resulted in a marked decrease in ACAT activity. Using the optimal assay conditions (exogenous cholesterol added), hepatic ACAT activity was shown to be significantly reduced in hamsters fed a high sucrose lithogenic diet compared with controls (587+/-42 vs 737+/-44 pmol/min per mg; P=0.025). In contrast, ACAT activity measured using endogenous cholesterol as a substrate was greater in sucrose-fed hamsters compared with controls (22.3+/-2.5 vs 13.2+/-2.9 pmol/min per mg; P= 0.030). These results highlight the importance of using an ACAT activity assay which has been well characterized and supports the hypothesis that the pathogenesis of cholesterol gallstones in LPN hamsters is related to an altered hepatic cholesterol metabolism.

Animals↗

Effect of incorporation of cidofovir into DNA by human cytomegalovirus DNA polymerase on DNA elongation.

Cidofovir (CDV) (HPMPC) has potent in vitro and in vivo activity against human cytomegalovirus (HCMV), CDV diphosphate (CDVpp), the putative antiviral metabolite of CDV, is an inhibitor and an alternate substrate of HCMV DNA polymerase. CDV is incorporated with the correct complementation to dGMP in the template, and the incorporated CDV at the primer end is not excised by the 3'-to-5' exonuclease activity of HCMV DNA polymerase. The incorporation of a CDV molecule causes a decrease in the rate of DNA elongation for the addition of the second natural nucleotide from the singly incorporated CDV molecule. The reduction in the rate of DNA (36-mer) synthesis from an 18-mer by one incorporated CDV is 31% that of the control. However, the fidelity of HCMV DNA polymerase is maintained for the addition of the nucleotides following a single incorporated CDV molecule. The rate of DNA synthesis by HCMV DNA polymerase is drastically decreased after the incorporation of two consecutive CDV molecules; the incorporation of a third consecutive CDV molecule is not detectable. Incorporation of two CDV molecules separated by either one or two deoxynucleoside monophosphates (dAMP, dGMP, or dTMP) also drastically decreases the rate of DNA chain elongation by HCMV DNA polymerase. The rate of DNA synthesis decreases by 90% when a template which contains one internally incorporated CDV molecule is used. The inhibition by CDVpp of DNA synthesis by HCMV DNA polymerase and the inability of HCMV DNA polymerase to excise incorporated CDV from DNA may account for the potent and long-lasting anti-CMV activity of CDV.

Antiviral Agents↗

Oligoclonal populations of T and B cells in a case of angioimmunoblastic T-cell lymphoma predominantly infiltrated by T cells of the VB5.1 family.

AIMS: Immunohistological and molecular characterisation of a case of peripheral T-cell lymphoma (PTCL) of angioimmunoblastic T-cell lymphoma (AILD) type. METHODS: Frozen and paraffin wax sections of the diagnostic lymph node were stained with a panel of T- and B-cell lineage monoclonal antibodies. DNA was isolated from the paraffin wax embedded biopsy material for T-cell receptor (TCR) and immunoglobulin (Ig) PCR amplification, and resultant PCR products were cloned and sequenced. RESULTS: Immunohistological analysis of the presenting lymph node was consistent with an extensive infiltrate of pleomorphic CD3+CD8+ lymphocytes. Most (>80%) of these infiltrating CD3+ cells were also positive for the TCR VB5.1 gene family product, and were shown to be oligoclonal by TCRB PCR amplification and sequencing. Three oligoclones of B cells were also demonstrable by PCR amplification with Ig heavy chain primers and sequencing, a finding at variance with the diagnosis of AILD. CONCLUSIONS: These data demonstrate the complexity and heterogeneity of PTCL which require extensive histological examination and molecular characterisation.

Aged↗

Modification of IgH PCR clonal analysis by the addition of sucrose and cresol red directly to PCR reaction mixes.

Diagnostic immunoglobulin (Ig) polymerase chain reaction (PCR) clonality analyses need to be simple, reproducible, and rapid. Sucrose and cresol red (gel loading buffer reagents) were added to a routine IgH PCR reaction mix to obviate the need for adding gel loading buffer separately after PCR amplification. Not only did this decrease the time spent after PCR analysis but also gave similar or enhanced IgH PCR product intensity compared with normal IgH PCR conditions on polyacrylamide gel electrophoresis. This procedure was easily adapted to routine PCR analysis without the need for further manipulations or optimisation of the PCR reaction mix, and it increased the reproducibility and specificity of the IgH PCR products.

Clone Cells↗

Innovative system to improve use of patient education materials.

OBJECTIVE: To evaluate a new storage system for patient education materials. DESIGN: Anonymous surveys before and after implementation of new storage system. SETTING: Family medicine residency teaching centre. PARTICIPANTS: All nurses, staff doctors, and first- and second-year residents in the unit. INTERVENTIONS: Implementation of a new storage system for patient education materials, orientation of all health professionals in the unit to the new system, and periodic distribution of patient education newsletters. MAIN OUTCOME MEASURES: Self-reported use of patient education materials. RESULTS: Response rates were 73% (30 of 41 health professionals) in 1990 and 86% (36 of 42) in 1992. Responses to the first survey on use of 20 categories of patient education materials showed materials were seldom used by most respondents. Back Care, Nutrition, Diabetes, VD/Birth Control, and Pregnancy categories were the most frequently used. In the second survey, more respondents reported using these five categories of pamphlets. Rates of use varied only slightly for the remaining 15 categories. CONCLUSIONS: Health professionals reported more frequent use of certain patient education materials following implementation of a new storage system.

Abstracting and Indexing↗

Effect of exogenous cholesterol and dithiothreitol on the activity of human liver microsomal acyl-coenzyme A:cholesterol acyltransferase (ACAT).

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is the intracellular enzyme responsible for the esterification of cholesterol with long-chain fatty acyl-CoA derivatives and has been implicated in atherosclerosis and gallstone disease. The effects of exogenous cholesterol and dithiothreitol (DTT) on the ACAT activity of human liver microsomes have been determined. Pre-incubation of microsomes with exogenous cholesterol gave a marked stimulation of activity. Experiments with [3H]cholesterol and [14C]oleoyl-CoA indicated the time course of equilibration of exogenous with endogenous cholesterol as ACAT substrates, and showed that ACAT activity could be accurately measured using [3H]cholesterol/Tween 80, providing that the concentration of endogenous microsomal cholesterol was also determined. Pre-incubation of liver microsomes for 90 min in the presence of 2 mmol/l DTT and exogenous cholesterol/Tween 80 resulted in a 60% reduction in ACAT activity, compared with the corresponding activity when DTT was omitted. If microsomes were pre-incubated with DTT prior to the pre-incubation with exogenous cholesterol/Tween 80, an 85-90% reduction in ACAT activity occurred. In contrast, pre-incubation of microsomes with DTT in the absence of exogenous cholesterol/Tween 80 (only endogenous cholesterol present) resulted, initially in a stimulation of ACAT activity; on further pre-incubation, activity returned to control levels. These results indicate that the supply of cholesterol to the enzyme active site is an important factor in ACAT assays in vitro and that DTT has a major effect on this process, suggesting that these factors may be important in controlling ACAT activity in vivo.

Cholesterol↗

Characterization and crystallization of the lumen side domain of the chloroplast Rieske iron-sulfur protein.

A soluble, 139-residue COOH-terminal polypeptide fragment of the Rieske iron-sulfur protein of the cytochrome b6f complex from spinach chloroplasts was obtained by limited proteolysis of the complex and a two-step chromatography purification protocol. The purified Rieske iron-sulfur protein fragment was characterized by: (i) a single NH2-terminal sequence, NH2-Phe-Val-Pro-Pro-Gly-Gly, starting with residue 41 of the intact Rieske protein; (ii) a single molecular weight species determined by mass spectrometry with a molecular weight of 14,620 +/- 2 without the [2Fe-2S] cluster; (iii) an optical absorbance spectrum with redox- and pH-dependent maxima and minima; and (iv) a reduced-oxidized optical difference spectrum characterized by DeltaepsilonmM = 3.8 mM-1 cm-1 for DeltaA at 394 versus 409 nm, which was used to determine the midpoint oxidation-reduction potential, which is +359 +/- 7 mV at 25 degrees C from pH 5.5-6.5, and +319 +/- 2 mV at pH 7, with an apparent pKox = 6.5 +/- 0.2 for the oxidized protein. The EPR spectrum measured at 17 K was characterized by the g values, gz = 2.03 and gy = 1.90, and a broad band centered at gx approximately 1.74, very similar or identical to those of the Rieske cluster in the b6f complex, implying that the environment of the [2Fe-2S] cluster is similar to that in the complex. Midpoint potential determination by low temperature EPR yielded a redox midpoint potential (Em) of +365-375 mV of the soluble Rieske fragment at pH 6 and 7 and an Em of +295-300 mV of the Rieske cluster in the cytochrome b6f complex at pH 6 and 7. The Em difference implies that the environment of the cluster in the soluble Rieske fragment is slightly more polar than that of the cluster in the intact complex. Single crystals of the Rieske polypeptide were obtained that are capable of x-ray diffraction to atomic resolution (<2.5 A), contain one molecule per asymmetric unit, a solvent content of approximately 30%, and belong to the triclinic space group P1 with cell dimensions, a = 29.1 A, b = 31.9 A, c = 35.8 A, alpha = 95.6 degrees, beta = 107.1 degrees, gamma = 117.3 degrees.

Amino Acid Sequence↗

Activation of Dictyostelium myosin light chain kinase A by phosphorylation of Thr166.

Phosphorylation of the regulatory light chain is an important mechanism for the activation of myosin in non-muscle cells. Unlike most myosin light chain kinases (MLCKs), MLCK-A from Dictyostelium is not activated by Ca2+/calmodulin. Autophosphorylation increases activity, but only to a low level, suggesting that there is an additional activation mechanism. Here, we show that MLCK-A is autophosphorylated on Thr289, which is C-terminal to the catalytic domain. Phosphorylation of MLCK-A increases in response to concanavalin A (conA) treatment of cells, which was previously shown to activate MLCK-A. However, a mutant kinase with an alanine at position 289 (T289A) is also phosphorylated in vivo, indicating that there is an additional phosphorylated residue. Based on comparisons with other protein kinases, we tested whether phosphorylation of Thr166 drives activation of MLCK-A. Our data indicate that phosphorylation of Thr289 occurs in vivo, but is not associated with conA-induced activation, whereas phosphorylation of Thr166 by some as yet unidentified kinase is associated with activation. Replacement of Thrl66 with glutamate results in a 12-fold increase in activity as compared with the wild-type enzyme, supporting the idea that phosphorylation of Thr166 increases MLCK-A activity.

Amino Acid Sequence↗

Myosin light chain kinase (MLCK) gene disruption in Dictyostelium: a role for MLCK-A in cytokinesis and evidence for multiple MLCKs.

We have created a strain of Dictyostelium that is deficient for the Ca2+/calmodulin-independent MLCK-A. This strain undergoes cytokinesis less efficiently than wild type, which results in an increased frequency of multinucleate cells when grown in suspension. The MLCK-A-cells are able, however, to undergo development and to cap crosslinked surface receptors, processes that require myosin heavy chain. Phosphorylated regulatory light chain (RLC) is still present in MLCK-A-cells, indicating that Dictyostelium has one or more additional protein kinases capable of phosphorylating RLC. Concanavalin A treatment was found to induce phosphorylation of essentially all of the RLC in wild-type cells, but RLC phosphorylation levels in MLCK-A-cells are unaffected by concanavalin A. Thus MLCK-A is regulated separately from the other MLCK(s) in the cell.

Animals↗

Automated routine HLA-B27 typing by flow cytometry.

An increasing demand for HLA-B27 typing as one of the tests used in the diagnosis of ankylosing spondylitis had led us to develop a rapid, automated, flow cytometric assay using whole blood and an HLA-B27 specific monoclonal antibody FD705. This article shows the data from 2093 samples tested during a 2 year period of routine HLA-B27 typing. 21.6% were clearly HLA-B27 positive whilst 73.2% were HLA-B27 negative, the remaining 5.2% required further testing before assignment of HLA-B27 status. Additional work was carried out on blood samples from individuals positive for the newly described subtype HLA-B2708.

Antibodies, Monoclonal↗

Structure and function of the glutamine phosphoribosylpyrophosphate amidotransferase glutamine site and communication with the phosphoribosylpyrophosphate site.

Glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase from Escherichia coli exhibits a basal PRPP-independent glutaminase activity having a kcat/Km that is 0.3% of fully active enzyme. Binding of PRPP activates the enzyme by a structural change that lowers the Km for glutamine 100-fold and couples glutamine hydrolysis to synthesis of 5-phosphoribosylamine. By analysis of the x-ray structure of the glutamine site containing bound 6-diazo-5-oxonorleucine, a glutamine affinity analog, and by site-directed mutagenesis we have identified residues important for glutamine binding, catalysis, and coupling with PRPP. Tyr74 is a key residue in the coupling between the sites for glutamine in the NH2-terminal domain and PRPP in the COOH-terminal domain. Arg73 and Asp127 have roles in glutamine binding. The x-ray structure indicates that there are no amino acid side chains sufficiently close to Cys1 to participate as a proton acceptor in formation of the thiolate needed for nucleophilic attack on the carboxamide of glutamine, nor as a general acid for amide nitrogen transfer. Based on the x-ray model of the glutamine site and analysis of a mutant enzyme we propose that the free NH2 terminus of Cys1 functions as the proton acceptor and donor. The results indicate that the side chain of Asn101 and the backbone nitrogen of Gly102 function to stabilize a tetrahedral oxyanion resulting from attack of Cys1 on the glutamine carboxamide. Cys1, Arg73, Asn101, Gly102, and Asp127 are conserved in the NH2-terminal domain of a subfamily of amidotransferases that includes asparagine synthetase, glucosamine 6-phosphate synthase, and glutamate synthase, implying a common function in the four enzymes. Tyr74, on the other hand, is conserved only in glutamine PRPP amidotransferase sequences consistent with a specific role in interdomain coupling. The catalytic framework of key glutamine site residues supports the assignment of glutamine PRPP amidotransferase to a recently described Ntn (NH2-terminal nucleophile) hydrolase family of enzymes.

Adenosine Monophosphate↗

Kinetic analysis of the interaction of cidofovir diphosphate with human cytomegalovirus DNA polymerase.

Cidofovir [CDV,(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine, HPMPC] is an acyclic cytosine nucleoside phosphonate analog with potent in vitro and in vivo activity against a broad spectrum of herpesviruses. CDV diphosphate (CDVpp), the putative antiviral metabolite of CDV, is a competitive inhibitor of dCTP and an alternate substrate for human cytomegalovirus (HCMV) DNA polymerase. HCMV DNA polymerase used a synthetic DNA primer-template with a Km value of 90 +/- 8 nM and incorporated dCTP approximately 42 times more efficiently than CDVpp. HCMV DNA polymerase also utilized a synthetic DNA primer containing a single molecule of CDV at the 3'-terminus. The Km value for this DNA primer-template was 165 +/- 42 nM and incorporation of dCTP was approximately 17 times more efficient than that of CDVpp. The slower rate of incorporation of CDVpp was due mostly to the higher Km value of CDVpp toward the enzyme-primer-template complexes. These data demonstrate that incorporation of a single CDV into DNA by HCMV DNA polymerase does not lead to chain termination.

Animals↗