Biomedical subjects

K Leonard

Publications and source records attributed to K Leonard.

At least 55 records · Page 3Linked to original sources

PubMed1991

2D crystal forms of annexin IV on lipid monolayers.

Two-dimensional crystalline arrays of annexin IV were generated by interaction of the purified protein with a phospholipid monolayer. Image analysis of electron micrographs of the protein crystals, which diffracted to 3.5 nm respectively, revealed p6 and p3 symmetry. Annexin IV gave two crystal forms with unit cells of 18 x 18 nm and 28 x 28 nm. The former unit cell was similar to a previously described form of annexin VI. The implications of these observations are discussed.

Annexins↗

PubMed1991

Parent-child interactions in families with alcoholic fathers.

Adolescent offspring (N = 121) of alcoholic, depressed, and nondistressed fathers were observed during problem-solving discussions with their fathers, mothers, and with both parents together. Assessments were conducted when parents were and were not drinking alcohol. Nondistressed father-child dyads differed from both clinical samples in showing higher rates of congeniality and problem solving, whereas the impact of alcohol consumption on father-child, mother-child, or triadic interactions was not related to diagnostic status of father. Results are discussed in terms of the nonspecific effects of parental disturbance on family relationships and reasons for the absence of alcoholism specific effects on parent-child interaction.

Adolescent↗

PubMed1991

Experimental drinking procedures in the study of alcoholics and their families: a consideration of ethical issues.

In response to G. Koocher's (1991) expressed concerns regarding the use of experimental drinking procedures with alcoholic participants described in our earlier study (Jacob, Krahn, & Leonard, 1991), our discussion focuses on six major issues: risk-benefit analysis, safeguarding participants from harm, linking alcoholic participants with treatment, the use of monetary incentives, fostering the pathology of the alcoholic family, and relationships between alcoholism and adverse family effects. In presenting this material, we hope that readers will become more appreciative of the many implications that human use issues have for the science we pursue and for the welfare of the participants whom we wish to protect.

Adaptation, Psychological↗

PubMed1990

Ligand exclusion on acetylcholinesterase.

This paper examines covalent reactivity of AchE with respect to cationic and uncharged methylphosphonates and substrates in the absence and presence of cationic ligands selective for the active center and the peripheral anionic site. The organophosphorus inhibitors are enantiomeric alkyl methylphosphonothioates (1-5) containing cycloheptyl and isopropyl phosphono ester groups and S-methyl, S-n-pentyl, and S-[beta-(trimethylammonio)ethyl] leaving groups; these agents differ in their configuration about phosphorus and their steric, hydrophobic, and electrostatic characteristics. The synthetic substrates examined are acetylthiocholine, p-nitrophenyl acetate, and 7-acetoxy-4-methylcoumarin (7AMC). Antagonism of the methylphosphonothioate reaction by cationic ligands is strongly dependent on the nature of both the cation and the methylphosphonate but independent of the configuration about phosphorus. While all cations cause linear mixed inhibition of acetylthiocholine hydrolysis, there are observed a variety of inhibition patterns of 7AMC and p-nitrophenyl acetate hydrolysis that are distinctly nonlinear, as well as patterns in which the reciprocal plots intersect in the upper right quadrant. Strong antagonism of cationic (methylphosphonyl)thiocholines correlates very well with linear inhibition of acetylthiocholine. Ligands that cause only negligible antagonism of the uncharged methylphosphonates display nonlinear inhibition of uncharged substrates. These relationships, since they are most pronounced for peripheral site ligands and are strongly dependent on the charge carried by the reactant, suggest that the peripheral anionic site alters enzyme reactivity through an electrostatic interaction with the net negative active center. Such behavior indicates a potential role for the peripheral anionic site in conserving AchE catalytic efficiency within a narrow range of values.

Acetylcholinesterase↗

PubMed1990

A regular pattern of two types of 100-residue motif in the sequence of titin.

Titin is the largest polypeptide yet described (relative molecular mass approximately 3 x 10(6); refs 1, 2) and an abundant protein of striated muscle. Its molecules are string-like and in vivo span from the M to Z-lines. I-band regions of titin are thought to make elastic connections between the thick filament and the Z-line, thereby forming a third type of sarcomere filament. These would centre the A-band in the sarcomere and provide structural continuity in relaxed myofibrils. The A-band region of titin seems to be bound to the thick filament, where it has been proposed to act as a 'molecular ruler' regulating filament length and assembly. Here, we show that partial titin complementary DNAs encode a regular pattern of two types of 100-residue motif, each of which probably folds into a separate domain type. Such motifs are present in several evolutionarily divergent muscle proteins, all of which are likely to interact with myosin. One or both of the domain types is therefore likely to bind to myosin.

Amino Acid Sequence↗

PubMed1990

Identification and localization of high molecular weight proteins in insect flight and leg muscle.

Thick and thin filaments in asynchronous flight muscle overlap nearly completely and thick filaments are attached to the Z-disc by connecting filaments. We have raised antibodies against a fraction of Lethocerus flight muscle myofibrils containing Z-discs and associated filaments and also against a low ionic strength extract of myofibrils. Monoclonal antibodies were obtained to proteins of 800 kd (p800), 700 kd (p700), 400 kd (p400) and alpha-actinin. The positions of the proteins in Lethocerus flight and leg myofibrils were determined by immunofluorescence and electron microscopy. p800 is in connecting filaments of flight myofibrils and in A-bands of leg myofibrils. p700 is in Z-discs of flight myofibrils and an immunologically related protein, p500, is in leg muscle Z-discs. p400 is in M-lines of both flight and leg myofibrils. Preliminary DNA sequencing shows that p800 is related to vertebrate titin and nematode twitchin. Molecules of p800 could extend from the Z-disc a short way along thick filaments, forming a mechanical link between the two structures. All three high molecular weight proteins probably stabilize the structure of the myofibril.

Amino Acid Sequence↗

PubMed1990

Method for forming two-dimensional paracrystals of biological filaments on lipid monolayers.

A method is described for electron microscopic observation of two-dimensional paracrystals on unsupported lipid monolayers. The method uses a hydrophobic holey C-coated grid placed on a monolayer made positively charged by the inclusion of stearylamine (SA) and has been used to align scallop thin filaments and reconstituted actin/tropomyosin filaments to form paracrystals. The use of unsupported monolayers allows the paracrystals to be viewed in either negative stain or with cryoelectron microscopy. Those paracrystals in frozen hydrated specimens have better order than those with negative stain. It was found that varying the lipid composition between the less fluid distearolyphosphotidylcholine/SA and the more fluid egg yolk phosphotidylcholine/SA alters the size and order of the paracrystals, the more fluid system having smaller, more ordered paracrystalline domains. The advantage of the technique for studying actin/thin filaments is the ability to form large two-dimensional paracrystals under physiological conditions of [Mg2+] and pH.

Crystallization↗

PubMed1990

Ultrastructural localization of [125I]neurotensin binding sites to cholinergic neurons of the rat nucleus basalis magnocellularis.

The distribution of specifically-labeled neurotensin binding sites was examined in relation to that of cholinergic neurons in the rat nucleus basalis magnocellularis at both light and electron microscopic levels. Lightly prefixed forebrain slices were either labeled with [125I](Tyr3) neurotensin alone or processed for combined [125I]neurotensin radioautography and acetylcholinesterase histochemistry. In light microscopic radioautographs from 1-microns-thick sections taken from the surface of single-labeled slices, silver grains were found to be preferentially localized over perikarya and proximal processes of nucleus basalis cells. The label was distributed both throughout the cytoplasm and along the plasma membrane of magnocellular neurons all of which were found to be cholinesterase-positive in a double-labeled material. Probability circle analysis of silver grain distribution in electron microscopic radioautographs confirmed that the major fraction (80-89%) of specifically-labeled binding sites associated with cholinesterase-reactive cell bodies and dendrites was intraneuronal. These intraneuronal sites were mainly dispersed throughout the cytoplasm and are thus likely to represent receptors undergoing synthesis, transport and/or recycling. A proportion of the specific label was also localized over the nucleus, suggesting that neurotensin could modulate the expression of acetylcholine-related enzymes in the nucleus basalis. The remainder of the grains (11-20%) were classified as shared, i.e. overlied the plasma membrane of acetylcholinesterase-positive neuronal perikarya and dendrites. Extrapolation from light microscopic data, combined with the observation that shared grains were detected at several contact points along the plasma membrane of cells which also exhibited exclusive grains, made it possible to ascribe these membrane-associated receptors to the cholinergic neurons themselves rather than to abutting cellular profiles. Comparison of grain distribution with the frequency of occurrence of elements directly abutting the plasma membrane of neurotensin-labeled/cholinesterase-positive perikarya indicated that labeled cell surface receptors were more or less evenly distributed along the membrane as opposed to being concentrated opposite abutting axon terminals endowed or not with a visible junctional specialization. The low incidence of labeled binding sites found in close association with abutting axons makes it unlikely that only this sub-population of sites corresponds to functional receptors. On the contrary, the dispersion of labeled receptors seen here along the plasma membrane of cholinergic neurons suggests that neurotensin acts primarily in a paracrine mode to influence the magnocellular cholinergic system in the nucleus basalis.

Acetylcholinesterase↗

PubMed1990

Puzzling subunits of mitochondrial cytochrome reductase.

The ubiquinol-cytochrome c reductase complex, like the other proton-pumping respiratory complexes of mitochondria, is an assembly of many different subunits. However, only a few of these subunits participate directly in the electron transfer and proton translocation. The roles of the other subunits are largely unknown. We discuss here some intriguing features of two of these subunits.

Electron Transport Complex III↗

PubMed1990

Electron microscopic characterization of helical filaments formed by subunits I and II (core proteins) of ubiquinol: cytochrome c reductase from Neurospora mitochondria.

The isolated and water-soluble complex of subunits I and II (core proteins) of ubiquinone:cytochrome c reductase from Neurospora mitochondria forms filaments below pH 6.0. Three independent helical reconstructions of single filaments were compared with the 3-D reconstruction of the native enzyme. A model for the helix is proposed in which the core complex dimers are arranged radially with the face which is proximal to the membrane in the native enzyme on the outside of the helix. The dimension of the core complex dimer perpendicular to the helix axis (70 A) provides an independent estimate of the height of the core complex to that obtained previously from cytochrome reductase crystals. The results of STEM mass measurement and the helical model give a mass per repeating unit of 90 kDa, which would indicate that the monomeric core complex consists of one 45-kDa and one 50-kDa subunit.

Electron Transport Complex III↗

PubMed1989

Interaction of the pore-forming domain of colicin A with phospholipid vesicles.

The interaction of the 20-kDa pore-forming domain of colicin A with phospholipid vesicles was investigated by gel permeation chromatography, analytical centrifugation, and electron microscopy. Under the experimental conditions of this study, this peptide was found to interact only with vesicles containing negatively charged phospholipids. It forms a well-defined disklike complex with phosphatidylglycerols with a preference for those containing 12-14 atoms of carbon in their fatty acid chain. This complex has a diameter of 120 A and is about one bilayer thick. It contains nine molecules of peptide and is formed both at acidic pH (pH 5.0) and at neutral pH (pH 7.2).

Chromatography, Gel↗

PubMed1989

Sidedness of yeast plasma membrane vesicles and mechanisms of activation of the ATPase by detergents.

The binding of concanavalin A and of fluorescein 5'-isothiocyanate indicate similar amount of right-side-out and inside-out vesicles in plasma membrane vesicles from either glucose-starved or glucose-fermenting yeast cells. These vesicles contain low-activity and high-activity states of the ATPase, respectively. Unmasking of latent active sites can explain the limited ATPase activation (about 2-fold) produced by several detergents on both kinds of vesicles. On the other hand, lysophosphatidic acid (oleoyl) produces a 7-fold activation of the ATPase in vesicles from glucose-starved cells. This effect is accompanied by a change in Km of the enzyme and probably reflects a direct action of the detergent on the ATPase. A similar activation and Km change can be obtained by sonication of the vesicles, although in this case soybean phospholipids are required for maximal activity. Apparently the low-activity state of the yeast plasma membrane ATPase can be activated not only by glucose metabolism 'in vivo' (mechanism unknown) but also by some detergents and physical treatments 'in vitro'. Experiments with purified ATPase from glucose-starved cells also indicate that lysophosphatidic acid (oleoyl) specifically activates the enzyme. These results suggest a note of caution on considering the usual interpretation of the effects of detergents on membrane enzymes, which only take into account the unmasking of latent active sites.

Adenosine Triphosphatases↗

PubMed1989

Crystallization of p68 on lipid monolayers and as three-dimensional single crystals.

Two-dimensional crystals of p68, a Ca2+ -binding protein that has homology with members of the lipocortin/calpactin family, were obtained by interaction with a phospholipid monolayer. By measuring surface pressure at constant surface area, p68 was found to interact in a Ca2+ -dependent manner specifically with phosphatidylethanolamine, less so with phosphatidylserine and not at all with phosphatidylcholine. With dimyristoyl-phosphatidylethanolamine, two-dimensional crystalline arrays were formed. Image analysis of electron micrographs of these crystals, which diffracted to about 50 A, revealed p3 symmetry with a unit cell of about 178 A by 178 A; the protein densities showed a two-domain structure giving a cylindrical molecule of about 100 A by 35 A diameter packed as trimers. Three-dimensional microcrystals obtained without lipid or Ca2+ were suitable for electron microscopy and gave a tetragonal unit cell of about 256 A by 68 A. The implications of these observations on the structure and lipid specificity of p68 binding are discussed.

Annexin A6↗

PubMed1989

Chiral reactions of acetylcholinesterase probed with enantiomeric methylphosphonothioates. Noncovalent determinants of enzyme chirality.

Enantiomeric cycloheptyl- and isopropyl methylphosphonothioates containing uncharged and cationic leaving groups, and 3,3-dimethylbutyl methylphosphonyl thiocholines were synthesized, and their inhibition of acetylcholinesterase from Torpedo examined. Bimolecular inhibition constants spanned 10(1)-10(9) M-1.min-1, equilibrium dissociation constants 10(-3)-10(-7) M, and phosphonylation constants 1-300 min-1. A general but not absolute preference for the SP-enantiomer, in the range 170-4600 for cycloheptyl-, 0.6-150 for isopropyl-, and 30 for 3,3-dimethylbutyl methylphosphonothioates, varied with nature of the alkyl ester (-OR) and thioic leaving groups (-SR') surrounding phosphorus. While the overall bimolecular reaction constant showed no marked dependence on ionic strength of the medium, the microscopic kp and KD for the RP- but not SP-cycloheptyl methylphosphonyl thiocholine underwent marked reduction with decreases in ionic strength. This result unmasks the interplay between occupation of the active center and productivity of that occupation. These studies reveal that chiral reactions with acetylcholinesterase are dependent more on the nature of the groups surrounding the tetrahedral phosphorus than on the absolute configuration about the phosphorus atom and indicate that the active center comprises partially overlapping subsites that can accommodate the -OR and -SR' groups. The presence of neighboring subsites characterized by different steric, electrostatic, and hydrophobic properties permits a multiplicity of binding orientations, independent of chiral configuration, and which account for the large variation in chiral preference seen among organophosphonates containing different substituents.

Acetylcholinesterase↗

PubMed1989

Electron microscopic localization of neurotensin binding sites in the midbrain tegmentum of the rat. I. Ventral tegmental area and the interfascicular nucleus.

The distribution of specifically labeled neurotensin (NT) binding sites was examined by light and electron microscopic radioautography in the ventral tegmental area (VTA) and nucleus interfascicularis of the rat following incubation of lightly prefixed midbrain slices with the monoiodinated ligand, 125I-(Tyr3)-NT. Film radioautograms of whole 125I-NT-incubated slices exhibited intense NT displaceable binding throughout the VTA and interfascicular nucleus. In light microscopic radioautographs from 1-microns-thick sections taken from the surface of the slices, the label was found to be present both inside and outside neuronal perikarya. Probability circle analysis of silver grain distribution in electron microscopic radioautographs confirmed that a significant proportion (greater than 20%) of the specifically labeled binding sites was intraneuronal. The frequent association of these sites with profiles of rough endoplasmic reticulum or Golgi apparatus suggested that they corresponded in part to receptors undergoing synthesis and/or glycosylation. The remainder was associated with neuronal and/or glial plasma membranes, as attested by comparing the distribution of grains overlying apposed cellular elements with the distribution of hypothetical grains originating from randomly distributed membrane bound radioactive sources. Although the resolution of the technique did not make it possible to ascribe labeled membrane-bound receptors to either one of the apposed plasma membranes, their frequent association with interfaces involving the plasmalemma of perikarya and dendrites, together with the occurrence of silver grain alignments along the membrane of certain somata and dendrites suggested that a proportion of them was associated with the perikarya and dendrites of a subpopulation of ventral tegmental neurons. Interestingly, these perikaryal and dendritic receptors were not exclusively present on, or even concentrated opposite, abutting axon terminals but instead were more or less evenly distributed along the plasma membrane. Only an exceedingly small proportion was found to be associated with synaptic junctions. Such a low incidence makes it unlikely that only the synapse-linked binding sites correspond to functional receptors. On the contrary, the dispersion of labeled receptors seen here along the plasma membrane of presumptive dopamine neurons suggests that NT acts mainly in a paracrine or parasynaptic fashion in the ventral midbrain tegmentum.

Animals↗

PubMed1988

Troponin of asynchronous flight muscle.

Troponin has been prepared from the asynchronous flight muscle of Lethocerus (water bug) taking special care to prevent proteolysis. The regulatory complex contained tropomyosin and troponin components. The troponin components were Tn-C (18,000 Mr), Tn-T (apparent Mr 53,000) and a heavy component, Tn-H (apparent Mr 80,000). The troponin was tightly bound to tropomyosin and could not be dissociated from it in non-denaturing conditions. A complex of Tn-T, Tn-H and tropomyosin inhibited actomyosin ATPase activity and the inhibition was relieved by Tn-C from vertebrate striated muscle in the presence of Ca2+. However, unlike vertebrate Tn-I, Tn-H by itself was not inhibitory. Monoclonal antibodies were obtained to Tn-T and Tn-H. Antibody to Tn-T was used to screen an expression library of Drosophila cDNA cloned in lambda phage. The sequence of cDNA coding for the protein was determined and hence the amino acid sequence. The Drosophila protein has a sequence similar to that of vertebrate skeletal and cardiac Tn-T. The sequence extends beyond the carboxyl end of the vertebrate sequences, and the last 40 residues are acidic. Part of the sequence of Drosophila Tn-T is homologous to the carboxyl end of the Drosophila myosin light chain MLC-2 and one anti-Tn-T antibody cross-reacted with the light chain. Lethocerus Tn-H is related to the large tropomyosins of Drosophila flight muscle, for which the amino acid sequence is known, since antibodies that recognize this component also recognize the large tropomyosins. Tn-H is easily digested by calpain, suggesting that part of the molecule has an extended configuration. Electron micrographs of negatively stained specimens showed that Lethocerus thin filaments have projections at about 39 nm intervals, which are not seen on thin filaments from vertebrate striated muscle and are probably due to the relatively large troponin complex. Decoration of the thin filaments with myosin subfragment-1 in rigor conditions appeared not to be affected by the troponin. The troponin of asynchronous flight muscle lacks the Tn-I component of vertebrate striated muscle. Tn-H occurs only in the flight muscle and may be involved in the activation of this muscle by stretch.

Amino Acid Sequence↗

PubMed1988

Three-dimensional reconstruction of maltoporin from electron microscopy and image processing.

Two dimensional crystals of maltoporin (or phage lambda receptor) were obtained by reconstitution of purified maltoporin trimers and Escherichia coli phospholipids by detergent dialysis. Two different trimer packing forms were observed. One was hexagonal (a = 7.8 nm) and one rectangular (a = 7.8 nm, b = 13.6 nm). In this paper we describe the three-dimensional structure of maltoporin, deduced from the study of the rectangular form by electron microscopy and image processing. At a resolution of approximately 2.5 nm, maltoporin trimers form aqueous channel triplets which appear to merge into a single outlet at the periplasmic surface of the outer membrane. The pore defined by maltoporin has a similar structure to that outlined by the matrix protein. From the results of functional studies by conductance measurement, it is concluded that the three channels defined by maltoporin act, contrary to those formed by the porin (OmpF protein), as a single conducting unit. A tentative outline of the maltoporin promoter is given. Maltoporin appears to be constituted by three different domains: a major rod-like domain spanning the membrane, a minor domain located near the periplasmic surface of the membrane and finally a central domain responsible for the splitting of the channel.

Bacterial Outer Membrane Proteins↗

PubMed1988

Alcoholism training in a family medicine residency.

The literature in alcoholism education for the health professions reveals that despite some strides in the development of training programs, both medical students and practicing physicians exhibit negative attitudes toward alcoholics that may adversely affect the care offered to these patients. This paper presents a program of alcoholism identification and management training for family practice residents which has been developed to supplement an existing drug abuse treatment program. Residents spend four half-day sessions engaged in seminars, outpatient experiences, and inpatient rotations under the direction of a clinical psychologist specializing in substance abuse and under the direction of the medical staff of a voluntary hospital well-known as a center of alcoholism treatment. Training activities include participation in group therapy sessions with alcoholic patients, interviewing patients in the early stages of rehabilitation, participation in an adolescent alcoholism treatment program and attendance in seminars on the prevention, identification, and treatment of alcoholism. Residents' evaluations of the experience reveal overall satisfaction with the program and their roles in its conduct, but they indicate a need for more active roles in the hands-on management of emergency cases.

Alcoholism↗