PubMed Health⌕ Search

Biomedical subjects

K Leonard

Publications and source records attributed to K Leonard.

At least 19 recordsLinked to original sources
PubMed1998

Consistent structure between bacterial and mitochondrial NADH:ubiquinone oxidoreductase (complex I).

Respiratory chains of bacteria and mitochondria contain closely related forms of the proton-pumping NADH:ubiquinone oxidoreductase (complex I). In bacteria the complex has a molecular mass of approximately 530 kDa and consists of 14 different subunits. The homologues of these 14 subunits together with some 27 additional subunits make up the mitochondrial complex, adding up to a molecular mass of approximately 1 MDa. We calculated three-dimensional models at medium resolution of isolated and negatively stained complex I particles from Eschericha coli and Neurospora crassa by electron microscopy using the random conical tilt reconstruction technique. Both the bacterial and the mitochondrial complexes are L-shaped molecules with an intrinsic membrane arm extending into the lipid bilayer and a peripheral arm protruding from the membrane. It is discussed whether the consistent length of the arms of both complexes has an implication for their function. The additional protein mass of the mitochondrial complex is distributed along both arms, but especially around the junction between the two arms and around the membrane arm. It appears that the structural framework of procaryotic complex I is stabilized in eucaryotes by this additional mass. A discrete location of additional protein in the peripheral arm of the mitochondrial complex is interpreted as being the possible position of two subunits with a specialized role in the biosynthesis of a yet unknown cofactor of complex I.

Bacterial Proteins↗
PubMed1997

Multimerization of the adenovirus DNA-binding protein is the driving force for ATP-independent DNA unwinding during strand displacement synthesis.

In contrast to other replication systems, adenovirus DNA replication does not require a DNA helicase to unwind the double-stranded template. Elongation is dependent on the adenovirus DNA-binding protein (DBP) which has helix-destabilizing properties. DBP binds cooperatively to single-stranded DNA (ssDNA) in a non-sequence-specific manner. The crystal structure of DBP shows that the protein has a C-terminal extension that hooks on to an adjacent monomer which results in the formation of long protein chains. We show that deletion of this C-terminal arm results in a monomeric protein. The mutant binds with a greatly reduced affinity to ssDNA. The deletion mutant still stimulates initiation of DNA replication like the intact DBP. This shows that a high affinity of DBP for ssDNA is not required for initiation. On a single-stranded template, elongation is also observed in the absence of DBP. Addition of DBP or the deletion mutant has no effect on elongation, although both proteins stimulate initiation on this template. Strand displacement synthesis on a double-stranded template is only observed in the presence of DBP. The mutant, however, does not support elongation on a double-stranded template. The unwinding activity of the mutant is highly reduced compared with intact DBP. These data suggest that protein chain formation by DBP and high affinity binding to the displaced strand drive the ATP-independent unwinding of the template during adenovirus DNA replication.

Adenosine Triphosphate↗
PubMed1997

TEM moiré patterns explain STM images of bacteriophage T5 tails.

A subtle combination of constant current and constant height modes in scanning tunnelling microscopy allowed the imaging of a non-flat uncoated biological specimen, namely the tail of the bacteriophage T5. In parallel, a reference three-dimensional structure of the T5 tail was calculated from cryo-transmission electron microscopy images, based on its helical symmetry. This three dimensional reconstruction was compared with scanning tunnelling microscopy data. The images of the tail obtained by transmission electron microscopy, as well as projections of the reconstructed model, show similar moiré patterns. Here we show that scanning tunnelling microscopy performed in an aqueous environment provides direct images which are remarkably similar to the projection of the three dimensional model obtained by transmission electron microscopy. We deduce that our scanning tunnelling microscopy images are the result of a transmission of electrons through the gap between the scanning tip and the conductive support across the biological specimen.

Image Processing, Computer-Assisted↗
PubMed1997

Electron microscopic distribution of mu opioid receptors on noradrenergic neurons of the locus coeruleus.

The distribution of mu opioid receptors was examined by light and electron microscopic autoradiography in the locus coeruleus of the rat following in vitro labelling with the iodinated agonist [125I]FK-33824. At the light microscopic level, specific mu opioid binding sites were concentrated over the perikarya and dendrites of neurons that were tyrosine hydroxylase-immunopositive in adjacent sections. Accordingly, both the number of tyrosine hydroxylase-immunoreactive neurons and the density of labelled mu receptors decreased markedly throughout the rostrocaudal extent of the nucleus following treatment with the catecholaminergic neurotoxin 6-hydroxydopamine. By electron microscopy, specifically labelled receptors were detected both inside and on the surface of locus coeruleus neurons. Intracellular sites were found by resolution circle analysis to be highly concentrated within the endoplasmic reticulum and Golgi apparatus, suggesting that the ligand recognizes both glycosylated and preglycosylated forms of receptor. The remainder were found mainly over the cytoplasmic matrix or intracytoplasmic vesicles, and were attributed to newly synthesized or recycled receptors in transit. Cell surface receptors were present over both dendritic and perikaryal membranes of noradrenergic cells. These were most highly concentrated opposite abutting axon terminals, suggesting the existence of receptor 'hot spots' at sites of putative endogenous ligand release. However, only a small proportion of these sites was associated with synaptic specializations. Furthermore, an important contingent was detected opposite non-axonal elements, such as dendrites and glial cells, suggesting that mu opioid ligands act mainly parasynaptically on locus coeruleus neurons. Finally, approximately 5% of labelled receptors were associated with axoglial interfaces, indicating that a minor action of mu opioids in the locus may be presynaptic and/or glial.

Animals↗
PubMed1996

Global, behavioral and self ratings of interpersonal skills among adult children of alcoholic, divorced and control parents.

OBJECTIVE: The goal of this study is to evaluate interpersonal skills and the relationships among interpersonal skills in a nonclinical sample of adult children of alcoholic, divorced and control parents using a multimethod assessment strategy. METHOD: Subjects were prescreened to rule out parental psychopathology and maternal alcoholism. Subjects (N = 238, 50% male, 86% white) interacted with a same- or opposite-sex confederate in a series of videotaped roleplays, which were either unstructured or structured via discussion topics and acquaintance instructions. The videotapes were coded for (1) global or general impression ratings of subject and (2) four behavioral components of interpersonal skill: talk, gaze, smile and self-manipulation. Subjects also rated their own skills and anxiety during the roleplays. RESULTS: Male and female children of alcoholics were judged to be more anxious when interacting with an opposite-sex partner than with a same-sex partner. Children of divorce and controls were not influenced by the gender of their interaction partner. Children of alcoholics were also judged to be less socially skilled then children of divorce. There was some suggestion that children of alcoholics and divorce may not accurately perceive their own level of skill. CONCLUSIONS: The findings provide some support for interpersonal skills deficits among adult children of alcoholics, although the deficits were not pervasive or consistent across methods. Limitations of the findings and the overall study were discussed.

Adult↗
PubMed1994

Identification of a putative membrane-inserted segment in the alpha-toxin of Staphylococcus aureus.

To gain a fuller understanding of the regions of the Staphylococcus aureus alpha-toxin important in pore formation, we have used Förster dipole-dipole energy transfer to demonstrate that a central glycine-rich region of alpha-toxin (the so-called "hinge" region) inserts deeply into the bilayer on association of toxin with liposomes. Mutant alpha-toxins with unique cysteine (C) residues at positions 69 and 130 [Palmer, M., et al. (1993) J. Biol. Chem. 268, 11959) were reacted with the C-specific fluorophore acrylodan, which acted as an energy donor. The chosen acceptor was N-(7-nitrobenz-2-oxa-13- diazol-4-yl)-1,2-bis(hexadecanoyl)-sn-glycero-3-phosphoethanolamin e (NBD-PE). Measurement of the degree of donor quenching with increasing NBD-PE in the inner bilayer leaflet enables the distance of closest approach between donor and acceptor to be estimated. For toxin labeled with acrylodan at position 130 (in the hinge region), this distance is approximately 5 +/- 2 A, showing that the probe is close to the inner surface of the liposomes. A second probe labeled at position 69 (in the N-terminal domain) shows negligible energy transfer, indicating a distance of closest approach > 40 A. This implies that this N-terminal region remains "outside" the liposome. We propose a model in which the central region of the alpha-toxin inserts into the membrane and possibly participates in forming the wall of the pore.

Bacterial Toxins↗
PubMed1994

Structure of the Aeromonas toxin proaerolysin in its water-soluble and membrane-channel states.

Aerolysin is chiefly responsible for the pathogenicity of Aeromonas hydrophila, a bacterium associated with diarrhoeal diseases and deep wound infections. Like many other microbial toxins, the protein changes in a multistep process from a completely water-soluble form to produce a transmembrane channel that destroys sensitive cells by breaking their permeability barriers. Here we describe the structure of proaerolysin determined by X-ray crystallography at 2.8 A resolution. The protoxin (M(r) 52,000) adopts a novel protein fold. Images of an aerolysin oligomer derived from electron microscopy have assisted in constructing a model of the membrane channel and have led to the proposal of a scheme to account for insertion of the protein into lipid bilayers to form ion channels.

Aeromonas↗
PubMed1993

Kettin, a large modular protein in the Z-disc of insect muscles.

Z-discs of insect flight muscle contain a large protein of 500-700 kDa. Monoclonal antibodies label an epitope in the molecule at the Z-disc in Drosophila and Lethocerus (waterbug). A partial cDNA of 1.6 kb from the Drosophila gene has been cloned and sequenced. The corresponding amino acid sequence has a modular structure composed of four conserved repeats of 95 amino acids homologous to immunoglobulin C2 domains (called class II domains in muscle proteins), separated by less conserved linker sequences of 35 amino acids. An expressed class II domain with flanking linker sequences binds to actin and alpha-actinin but not to myosin. Single molecules of the protein would be large enough to span the Z-disc. We suggest that the protein acts as scaffolding in the Z-disc and we call the protein kettin. The Ca2+ activated protease, calpain, disrupts the Z-disc of striated muscle, releasing alpha-actinin intact. Calpain digests kettin to a series of peptides of between 30 and 170 kDa which are released from the myofibril. Digestion of kettin may cause disintegration of the Z-disc and alpha-actinin release which lead to disassembly of the myofibril.

Amino Acid Sequence↗
PubMed1993

Phosphorylation of KSP motifs in the C-terminal region of titin in differentiating myoblasts.

Titin is a giant structural protein of striated muscle (M(r) approximately 3000 kDa) and single molecules span sarcomeres from the M- to Z-lines. We have cloned and sequenced the C-terminal region of the titin molecule, which is an integral part of M-lines and forms intimate contacts with the 165 and 190 kDa M-line proteins. In contrast to the regular motif patterns of the A-band portion of titin, the 5.7 kb of titin sequences from the M-line show a complex structure of immunoglobulin-C2 repeats, separated by unique interdomain insertion sequences. As a striking feature, one interdomain insertion comprises four KSP repeats analogous to the multi-phosphorylation repeats of neurofilament subunits H and M. In vitro phosphorylation assays with expressed titin KSP sequences detect high levels of titin KSP phosphorylating kinases in developing but not in differentiated muscle. Since this kinase activity can be depleted from myocyte extracts by antibodies against cdc2 kinase and p13suc1 beads, the titin KSP kinase is structurally related to cdc2 kinase. We suggest that titin C-terminal phosphorylation by SP-specific kinases is regulated during differentiation, and that this may control the assembly of M-line proteins into regular structures during myogenesis.

Amino Acid Sequence↗
PubMed1993

Titin antibodies in myasthenia gravis: identification of a major immunogenic region of titin.

Approximately 15% of patients with myasthenia gravis (MG) have thymus neoplasia. These MG with thymoma (MGT) patients show autoantibodies to striated muscle as well as autoantibodies to acetylcholine receptor. To characterize these thymoma-associated muscle antigens, we cloned a number of immunopositive cDNAs by immunoscreening muscle cDNA libraries with sera from MGT patients. Analysis of the isolated cDNAs show that all share a common sequence encoding a distinct region of the titin gene. We expressed this main immunogenic region (MIR) of titin in Escherichia coli, and determined autoantibody serum titers directed against the obtained recombinant antigen in a variety of patients. We could detect titin MIR autoantibodies in 97% of sera from MGT patients but not in control sera from healthy blood donors. Therefore, expressed titin from the MIR of the molecule is a sensitive marker antigen for evaluating the presence of thymoma in MG.

Amino Acid Sequence↗
PubMed1992

Drosophila projectin: relatedness to titin and twitchin and correlation with lethal(4) 102 CDa and bent-dominant mutants.

We have investigated projectin, a large protein of insect muscles, in Drosophila melanogaster. The 5.3 kilobases of coding sequence reported here contains Class I and Class II motifs characteristic of titin and twitchin, arranged in a three domain ... [II-I-I] [II-I-I] ... pattern. Two mutants mapped to the location of the projectin gene in the 102C subdivision of chromosome 4, lethal(4) 102 CDa and bent-Dominant, have DNA rearrangements within their projectin gene. The lethal(4) 102 CDa mutant has a 141 nucleotide insertion containing stop codons in all three reading frames within an exon sequence, showing that it cannot synthesize normal projectin. Both bent-Dominant and lethal(4) 102 CDa homozygotes die at the completion of embryogenesis because they are unable to escape the egg vitelline membrane. We propose that this hatching failure is due to muscle weakness caused by projectin defects.

Alleles↗
PubMed1992

Increased detection of HPV 16 virus in invasive, but not in early cervical cancers.

Human papilloma virus type 16 (HPV 16) DNA is found in about 50% of cervical squamous cell carcinomas (SCCs), and this association has raised the possibility of a causal role for HPV 16 in cervical carcinogenesis. We have tested this hypothesis by assaying a series of biopsies (n = 119) ranging from normal mucosa to infiltrating SCC with the PCR-technique for the presence of HPV 16 DNA. While HPV 16 DNA was detected in 50% of our cases with invasive SCC, the incidence of HPV 16-positive samples was about 10% in all other biopsies ranging from normal mucosa to cases of carcinoma in situ. HPV 16 therefore appears to be involved in late tumor promotion but not in early tumor development.

Base Sequence↗
PubMed1992

Sequential analysis of marital interactions involving alcoholic, depressed, and nondistressed men.

This study was based on the investigators' earlier work on alcoholism and the family and involved a comprehensive assessment of sequential patterns associated with the marital interactions of alcoholic, depressed, and nondistressed men. A total of 131 couples were assessed in a laboratory-based discussion of personally relevant problems. Videotaped observations were conducted during drinking and nondrinking sessions and were subsequently coded according to the Marital Interaction Coding System. Depressed couples were most distinguishable in terms of unique antecedent-consequent patterns, whereas alcoholic and nondistressed control couples were similar to one another and different from depressed couples. Results are discussed in regard to the extant literature on interpersonal theories of depression and alcoholism and in regard to future research needs.

Adolescent↗
PubMed1992

Interaction of tetrahydroaminoacridine with acetylcholinesterase and butyrylcholinesterase.

This paper examines inhibition of acetylcholinesterase (AchE) and butyrylcholinesterase (BuchE) by tetrahydroaminoacridine (THA), an acridine analog under consideration for palliative treatment of Alzheimer's dementia. THA causes linear mixed inhibition of AchE hydrolysis of acetylthiocholine, a cationic substrate (KI = 3.8 x 10(-9) M), and linear competitive inhibition of AchE hydrolysis of 7-acetoxy-4-methylcoumarin, an uncharged substrate (KI = 6.8 x 10(-9) M), and N-methyl-7-dimethylcarbamoxyquinolinium, a cationic carbamate (KI = 1.5 x 10(-8) M). Propidium association with AchE in the presence of saturating concentrations of THA is characterized by a dissociation constant of 7.7 +/- 0.7 x 10(-6) M, a value within 2-fold of the dissociation constant in the absence of THA. Association of THA with AchE is, therefore, not mutually exclusive with association of propidium at the peripheral anionic site. Moreover, THA causes dissociation of decidium complexes with AchE at concentrations compatible with a dissociation constant of 7.0 +/- 0.4 x 10(-9) M. Similar relationships were observed for THA inhibition of BuchE hydrolysis of butyrylthiocholine (KI = 2.5 x 10(-8) M) and dissociation of decidium complexes with BuchE (KD = 1.9 +/- 0.1 x 10(-8) M). These kinetic and equilibrium data uniformly indicate that THA associates with AchE and BuchE with high affinity and that the subsequent inhibition comes about through ligand association at the active center rather than at a peripheral site. The noncompetitive component of inhibition reflects association of THA with the acyl-enzyme intermediate, with subsequent effects on the rate of deacylation.

Acetylcholinesterase↗
PubMed1991

Binding and internalization of iodinated neurotensin in neuronal cultures from embryonic mouse brain.

The binding and internalization of labeled neurotensin were studied by means of biochemical and light microscopic radioautography techniques in primary cultures of neurons from whole cerebral hemispheres of mouse embryos. Saturable, high affinity neurotensin binding was detected 5-7 days postplating in cells incubated with 0.1 nM 125I-Tyr3-neurotensin at 37 degrees C or 10 degrees C. The binding capacity at equilibrium was 3 times higher at 37 degrees C than at 10 degrees C. Moreover, whereas virtually all the radioactivity bound at 10 degrees C was membrane-bound (i.e. was readily washable by a hypertonic, high pH, NaCl solution), more than 70% of the radioactivity bound at 37 degrees C was intracellular (i.e. resisted the same treatment). Light microscopic radioautograms of whole cells revealed that approximately 16% of neurons were labeled with 125I-Tyr3-neurotensin at either 37 degrees C or 10 degrees C. The labeling was observed over cell bodies and processes, and the density of silver grains associated with perikarya, as compared to processes, was proportionally higher at 37 degrees C than at 10 degrees C. Semi-thin (1 micron thick) sections through cells incubated at 37 degrees C confirmed that a major fraction of the radioactivity was intracellular and showed that it was mainly confined to the cytoplasm. These results indicate that 125I-Tyr3-neurotensin binds to a distinct subset of primary cultured neurons and that a large proportion of the bound radioactivity undergoes rapid internalization in a temperature-dependent manner. It is proposed that this internalization is ligand-induced and that it may play a role in the modulation of central neurotensin receptor levels.

Animals↗
PubMed1991

Electron microscopic analysis of the peripheral and membrane parts of mitochondrial NADH dehydrogenase (complex I).

Two related forms of the respiratory chain NADH dehydrogenase (NADH:ubiquinone reductase or complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large form that consists of 25 subunits encoded by nuclear DNA and six to seven subunits encoded by mitochondrial DNA. Cells grown in the presence of chloramphenicol, however, make a smaller form comprising only 13 subunits, all encoded by nuclear DNA. When the large enzyme is dissected by chaotropic agents (such as NaBr), all those subunits of the large form that are missing in the small form can be isolated as a distinct, so-called hydrophobic fragment. The small enzyme and the hydrophobic fragment make up, with regard to their redox groups, subunit composition and function, two complementary parts of the large-form NADH dehydrogenase. Averaging of electron microscope images of single particles of the large enzyme was carried out, revealing an unusual L-shaped structure with two domains or "arms" arranged at right angles. The hydrophobic fragment obtained by the NaBr treatment corresponds in size and appearance to one of these arms. A three-dimensional reconstruction from images of negatively stained membrane crystals of the large-form NADH dehydrogenase shows a peripheral domain, protruding from the membrane, with weak unresolved density within the membrane. This peripheral domain was removed by washing the crystals in situ with 2 M-NaBr, exposing a large membrane-buried domain, which was reconstructed in three dimensions. A three-dimensional reconstruction of the small enzyme from negatively stained membrane crystals, also described here, shows only a peripheral domain. These results suggest that the membrane protruding arm of the large form corresponds to the small enzyme, whereas the arm lying within the membrane can be identified as the hydrophobic fragment. The two parts of NADH dehydrogenase that can be defined by the separate genetic origin of (most of) their subunits, their independent assembly, and their distinct contributions to the electron pathway can thus be assigned to the two arms of the L-shaped complex I.

Crystallography↗