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Biomedical subjects

J P Nolan

Publications and source records attributed to J P Nolan.

At least 19 recordsLinked to original sources
PubMed1998

Internal medicine in the current health care environment: a need for reaffirmation.

Managed care has substantially changed the environment of health care delivery for general internists and internist-subspecialists. In the current system, one may wonder whether detailed and thoughtful workups still have a role when the direction of medical practice increasingly prizes a high volume of brief encounters. However, the very forces that drive managed care make the role of internist in the care of adults even more central. The internist's unique training and clinical approach should lead to both medically effective and cost-effective health care for adults. This type of health care will be increasingly important as the U.S. population ages and an increasing number of Americans have chronic, multisystem disease. Over the past century, internal medicine has evolved from a consultative model to a discipline that encompasses total adult care, from prevention to diagnosis and treatment of acute and chronic illness and from outpatient care in the office to inpatient care in the intensive care unit. However, the leadership role of internists in the medical care of adults is now being threatened by family medicine and by fragmentation within internal medicine itself. Managed care organizations and the general public must be shown why internists are better able than family physicians to meet the health care needs of adults. Furthermore, as the marketplace becomes more competitive, the issue of when care given by a subspecialist is superior to that given by an internist has become more prominent. The rapidly developing "hospitalist" movement also threatens the traditional role of the internist as the caregiver for adults in health and disease. Given the historic flexibility of internal medicine and the assumption that appropriate roles can be defined for family physicians, subspecialists, and hospitalists, internists will continue to play a central role in providing the best care for adults in the new world of health care delivery.

Adult↗
PubMed1998

Modulation of GTPase activity of G proteins by fluid shear stress and phospholipid composition.

Mechanical forces arising from strain, pressure, and fluid shear stress are sensed by cells through an unidentified mechanoreceptor(s) coupled to intracellular signaling pathways. In vascular endothelial cells, fluid shear stress is transduced via pathway(s) involving heterotrimeric guanine nucleotide-binding proteins (G proteins) by molecular mechanisms that are unknown. In the present study, we investigated the activation of purified G proteins reconstituted into phospholipid vesicles. Vesicles containing G proteins were loaded with [gamma-32P]GTP and subjected to physiological levels of fluid shear stress in a cone-and-plate viscometer. Steady-state GTP hydrolysis was measured as an index of G protein function. Shear stress (0-30 dynes/cm2) activated G proteins in dose-dependent manner (0.48-4.6 pmol/min per microg of protein). Liposomes containing lysophosphatidylcholine (30 mol %) or treated with benzyl alcohol (40 mM), conditions that increase bilayer fluidity, exhibited 3- to 5-fold enhancement of basal GTPase activity. Conversely, incorporation of cholesterol (24 mol %) into liposomes reduced the activation of G proteins by shear. These results demonstrate the ability of the phospholipid bilayer to mediate the shear stress-induced activation of membrane-bound G proteins in the absence of protein receptors and that bilayer physical properties modulate this response.

Animals↗
PubMed1998

The emergence of flow cytometry for sensitive, real-time measurements of molecular interactions.

The analysis of macromolecular interactions is an essential element of biomedical research. Flow cytometry is uniquely capable of making sensitive and quantitative measurements of molecular interactions. These measurements can be made in real time with subsecond kinetic resolution using purified biomolecules or living cells. Combined with automated sample handling, these features make flow cytometry a versatile and robust technology for the analysis of molecular interactions.

Antigens, Surface↗
PubMed1997

Functional analysis of point mutations in human flap endonuclease-1 active site.

Human flap endonuclease-1 (hFEN-1) is highly homologous to human XPG, Saccharomyces cerevisiae RAD2 and S.cerevisiae RTH1 and shares structural and functional similarity with viral exonucleases such as T4 RNase H, T5 exonuclease and prokaryotic DNA polymerase 5'nucleases. Sequence alignment of 18 structure-specific nucleases revealed two conserved nuclease domains with seven conserved carboxyl residues and one positively charged residue. In a previous report, we showed that removal of the side chain of each individual acidic residue results in complete loss of flap endonuclease activity. Here we report a detailed analysis of substrate cleavage and binding of these mutant enzymes as well as of an additional site-directed mutation of a conserved acidic residue (E160). We found that the active mutant (R103A) has substrate binding and cleavage activity indistinguishable from the wild type enzyme. Of the inactive mutants, one (D181A) has substrate binding properties comparable to the wild type, while three others (D34A, D86A and E160A) bind with lower apparent affinity (2-, 9- and 18-fold reduced, respectively). The other mutants (D158A, D179A and D233A) have no detectable binding activity. We interpret the structural implications of these findings using the crystal structures of related enzymes with the flap endonuclease activity and propose that there are two metal ions (Mg2+or Mn2+) in hFEN enzyme. These two metal coordinated active sites are distinguishable but interrelated. One metal site is directly involved in nucleophile attack to the substrate phosphodiester bonds while the other may stabilize the structure for the DNA substrate binding. These two sites may be relatively close since some of carboxyl residues can serve as ligands for both sites.

Amino Acid Sequence↗
PubMed1997

True and false positive peaks in genomewide scans: applications of length-biased sampling to linkage mapping.

Disease-susceptibility loci are now being mapped via genomewide scans in which a linkage statistic is computed at each of a large number of markers. Such disease-susceptibility loci may be identified via a peak in the test statistic when the latter is plotted against the genetic map. In this paper we establish, by appealing to renewal theory, that true positive peaks are expected to be longer than false positive peaks. These results are verified by a realistic simulation of a genomewide linkage study based on the affected-sib-pair design. Since longer peaks are more likely to contain a gene of interest than are shorter peaks, these differences may aid in linkage mapping, justifying assignment of lower priority to shorter peaks. However, since these differences are generally small, statistics based on both peak length and height may not be much more powerful than those based on height alone. The results presented here also provide a theoretical framework for methods that use the length of shared haplotypes in populations to map disease genes.

Chromosome Mapping↗
PubMed1997

Analgesia after day-case knee arthroscopy: double-blind study of intra-articular tenoxicam, intra-articular bupivacaine and placebo.

Arthroscopy of the knee is performed regularly on a day-case basis. Intra-articular bupivacaine produces transient analgesia and reports of analgesia using intra-articular morphine have produced conflicting results. Non-steroidal anti-inflammatory drugs given systemically can provide effective analgesia for this procedure. In this study we attempted to determine if intra-articular tenoxicam provided useful analgesia after day-case arthroscopy. Sixty three ASA I-II patients were allocated randomly to one of three groups to receive 40 ml of a solution containing 0.9% saline (group Pla), 0.25% bupivacaine (group Bup) or tenoxicam 20 mg (group Ten). The injection was made into the knee joint at the end of surgery, 10 min before tourniquet deflation. Verbal rating and visual analogue pain scores (at rest and on knee flexion), use of analgesia, mobilization and disturbance by pain at home were recorded for the next 48 h. There were no differences between pain scores in any of the three groups when tested at rest or on movement. Less analgesia was used in the first 24 h by patients in the tenoxicam group but the difference in time to first analgesia was not statistically significant. Side effects and disturbance by pain were similar in all groups. The use of intra-articular tenoxicam 20 mg at the end of arthroscopy reduced oral analgesic requirements during the first day after operation but did not alter patients' perception of pain.

Adolescent↗
PubMed1997

Regulation of membrane stability and the acrosome reaction in mammalian sperm.

Membrane destabilization is an essential step in the process of membrane fusion. In many cell types, exocytotic fusion may occur sporadically at microscopically localized sites on the surface of the cell, making it difficult to study the chemical and physical features of the membrane (or membranes) that promote fusion. In the sperm cell, exocytosis occurs synchronously at a distinct region on the sperm head. This localization of function makes the sperm cell a useful model to investigate the structural features of the bilayers that control membrane fusion. During sperm maturation, the anterior head membranes undergo a well-defined series of chemical, physical, and functional changes that are necessary to produce a fertile gamete. These changes include the addition of highly unsaturated phosphatidylcholine, a decrease in general membrane stability, and an increase in the ability to respond to physiological and pharmacological inducers of exocytosis. Concomitant addition of cholesterol and an actively maintained asymmetric transmembrane phospholipid distribution modulate these effects to stabilize the membrane of the mature sperm for storage. The environment of the female tract provides conditions that promote efflux of cholesterol from the sperm plasma membrane as well as the loss of membrane asymmetry. The cholesterol-poor, lipid-symmetric plasma membrane has a destabilized inner leaflet that facilitates membrane fusion upon binding of the sperm to the appropriate egg coat receptors. We summarize these features in a mechanistic model in which the sperm membrane contains destabilizing components to confer fusogenic potential as well as stabilizing components organized to maximize membrane integrity. This combination prevents premature fusion in the male tract. After deposition in the female tract and removal of the stabilizing components, followed by reorganization of the fusogenic components, the membrane becomes poised to fuse upon receipt of the final biological stimulus.

Acrosome↗
PubMed1997

New flow cytometric technologies for the 21st century.

The envelope that defines the limits within which flow cytometry was developed is being rapidly expanded. For example: detection sensitivity has been extended to single molecules, the size range of "particle" analysis now extends from DNA fragments to plankton (1,000.+ microns), cell and chromosome sorting rates are being increased dramatically by using inactivation procedures (50,000 per second versus 2,000 per second), rapid kinetic flow cytometry enables real-time analysis of molecular assembly and cell function in the sub-second time domain, the lifetime of a fluorochrome bound to a single cell can be measured with nsec precision, and classical karyotype information (cell to cell heterogeneity) can be determined in a flow based system. These frontiers have greatly expanded the range of new and exciting flow cytometric based biomedical applications. New enabling technologies have provided the means to measure DNA cleavage by the structure-specific nuclease, human Flap Endonuclease (FEN-1), in the 300 msec time frame. Phase sensitive measurements and fluorescence lifetime are proving to be major advances for understanding molecular environments that change with, for example, the process of apoptosis. The ability to detect single fluorescent molecules has been applied to the analysis of DNA fragments obtained from enzymatic digestion of lambda DNA. This technology is being used to rapidly and very accurately size DNA fragments for the human genome project. Optical chromosome selection is a faster, better, less complex approach to chromosome sorting. This method is based on the induction of specific damage to the DNA of selected chromosomes. Lastly, the miniaturization of a single cell fractionator has made it possible to perform single cell flow cytogenetics.

Chromosomes, Human↗
PubMed1996

Kinetic analysis of human flap endonuclease-1 by flow cytometry.

Human flap endonuclease-1 (FEN-1) is a structure-specific endonuclease and exonuclease which is essential for DNA replication and repair. We have cloned a human FEN-1 gene, overexpressed it in Escherichia coli, purified the recombinant protein to near homogeneity, and characterized its cleavage of a flap DNA structure using a novel analytical approach based on flow cytometry. With this approach, we were able to measure continuously the kinetics of DNA cleavage by FEN-1 and to separate experimentally the binding and catalysis functions of the enzyme. When the reaction was initiated by the addition of FEN-1, the cleavage kinetics were dependent on enzyme concentration and appeared to saturate at high concentrations. When enzyme and substrate were preincubated in the presence of EDTA and the reaction initiated by the addition of Mg2+, rapid kinetic flow cytometry measurements showed that cleavage is fast (t1/2 approximately 6 s, k = 0.10 s-1). Using the single-turnover kinetics as a measure of the amount of enzyme-substrate complex present, we estimated the Kd for the FEN-1-flap DNA substrate to be 7.5 nM in the absence of Mg2+ and the rate constant for dissociation of the enzyme-substrate complex to be 0.07 s-1. Computer fitting of the experimental data to a kinetic model confirms these estimates for the individual steps and suggests some interesting features of enzymology using a surface-bound substrate.

Base Sequence↗
PubMed1996

Essential amino acids for substrate binding and catalysis of human flap endonuclease 1.

Human flap endonuclease 1 (FEN-1) is a member of the structure-specific endonuclease family and is involved in DNA repair. Eight restrictively conserved amino acids in FEN-1 have been converted individually to an alanine to elucidate their roles in specific DNA substrate binding and catalysis. Flap endonuclease activity of the wild type and mutant enzymes was measured by kinetic flow cytometry. Mutants D34A, D86A, and D181A lost their cleavage activity completely but retained substrate binding ability, as measured by their ability to inhibit the wild type enzyme in a competition assay. This indicates that these amino acids contribute to integrity of the enzyme active site. Loss of both binding and cleavage competency for the flap substrate by mutants E156A, G231A, and D233A suggests that these amino acids are involved in substrate binding. Mutants R103A and D179A retained wild type-like enzyme activity.

Amino Acid Sequence↗
PubMed1995

A rapid mix flow cytometer with subsecond kinetic resolution.

Kinetic approaches are valuable tools for mechanistic studies of cell function. Flow cytometry is well suited to make sensitive kinetic measurements, but the time required to deliver mixed samples to the point of measurement (10-20 s in a conventional cytometer) limits analysis of rapidly occurring events. To address this limitation, we adapted a syringe-based stopped-flow rapid mixing device to a modified commercial flow cytometer to achieve mixing and measurement of sample in under 1 s. Because such screw-driven mixers are designed to deliver fluid at rates of microliters per millisecond and cytometers accept samples at microliters per second, the syringe mixer was modified with a screw to allow sample delivery at rates as low as 1.8 microliters/s. A custom-made nozzle holder featuring a fast-acting three-way sample delivery valve and a 1.5- microliters dead volume was designed for a Becton Dickinson FACS stream-in-air flow nozzle. Syringe motors and valves are computer controlled, as is the start signal for an adjustable time ramp. A stable sample stream can be established within the sheath stream in less than 1 s, enabling fluorescence measurements of microspheres with coefficients of variation of approximately 5%. Light scatter gating to select particles in the center of the laser beam enables fluorescence measurements at times of under 300 ms. Efficient mixing of reagents is demonstrated by the iodide quenching of microspheres surface labeled with fluorescein isothiocyanate (FITC). The instrument is capable of quantitatively proportioning cells and reagent, thereby allowing precise control of reagent concentration and dilution. Rapid kinetic measurements of intact cells are demonstrated by FITC-formyl peptide binding to cell surface receptors.

Flow Cytometry↗
PubMed1995

Characterization of N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide for the detection of zinc in living sperm cells.

Zinc stabilizes membranes and DNA and inhibits respiration in somatic cells. It is present in high concentrations in the male reproductive tract and may stabilize spermatozoa prior to fertilization. Herein, we evaluate N-(6-methoxy-8-quinolyl)-p-toluenesulfonamide (TSQ) for analysis of Zn2+ in phosphatidylcholine (PC) vesicles and living spermatozoa using spectrofluorometry and flow cytometry. TSQ:Zn fluorescence in decanol or PC vesicles was compared to that in aqueous buffer. Evaluation of cation specificity, kinetics of TSQ:Zn binding, quenching of TSQ by dithionite and Zn2+ chelation by D-penicillamine established that TSQ is more fluorescent in decanol or PC vesicles than in aqueous buffer, has a high affinity for lipid bilayers and is specific for Zn2+ compared to Mg2+ and Ca2+. Fluorescence measurement of vesicles with and without pretreatment with Zn2+ indicated that, in the absence of Zn2+, 90% of the residual TSQ fluorescence was destroyed by dithionite but > 50% was protected by the presence of Zn2+. When D-penicillamine was added the remaining fluorescence was quenched (T1/2 = 10 s) indicating that TSQ remains in/on the membrane. These results established that TSQ can be used to effectively evaluate Zn2+ in artificial membranes and sperm cells. Additional experiments will be necessary to explain the dynamics of TSQ:Zn:membrane interactions.

Aminoquinolines↗