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K Leonard

Publications and source records attributed to K Leonard.

At least 19 recordsLinked to original sources
PubMed1999

Association of kettin with actin in the Z-disc of insect flight muscle.

The Z-discs of insect muscle contain kettin, a modular protein of 500-700 kDa. The Drosophila protein is made up of a chain of immunoglobulin (Ig) domains separated by linker sequences. Kettin differs from other modular muscle proteins of the Ig superfamily in binding to thin filaments rather than thick filaments. Kettin isolated from Lethocerus (waterbug) muscle is an elongated molecule 180 nm long, which binds to F-actin with high affinity (Kd=1.2 nM) and a stoichiometry of one Ig domain per actin protomer. Competition between kettin and tropomyosin for binding to actin excludes tropomyosin from the Z-disc. In contrast, kettin and alpha-actinin bind simultaneously to actin, which would reinforce the Z-disc lattice. In vitro, kettin promotes the antiparallel association of actin filaments, and a similar process may occur in the developing sarcomere: actin filaments interdigitate in an antiparallel fashion in the Z-disc with the N terminus of kettin within the Z-disc, and the C terminus some way outside. We propose a model for the association of kettin with actin in which the molecule follows the genetic helix of actin and Ig domains, separated by linker sequences, bind to each actin protomer.

Actins↗
PubMed1999

Structure of the insect troponin complex.

Isolated troponin-tropomyosin complex from Lethocerus indicus asynchronous flight muscle forms paracrystals on a positively charged lipid monolayer. Single particle analysis was carried out on individual complexes selected from electron micrographs of negatively stained paracrystals. By a combination of correlation and classification techniques, different average projections of the object were obtained. An initial three-dimensional model was calculated by determining the Euler angles for the different views using a common line approach. This starting model was then used as a reference for the further three-dimensional refinement of the model using the original data set. The refined model of the troponin complex has a diameter of approximately 90 A and a volume corresponding with a molecular mass of about 120 kDa for the globular domain. The resolution of the reconstruction was determined to be 32 A using the differential phase residual method and 26 A using the Fourier shell correlation criterion.

Animals↗
PubMed1999

The glutamine-rich domain of the Drosophila GAGA factor is necessary for amyloid fibre formation in vitro, but not for chromatin remodelling.

The Drosophila GAGA factor binds specifically to the sequence GAGAG, and synergises with nucleosome remodelling factor to remodel chromatin in vitro. It consists of an N-terminal domain (POZ/BTB) which mediates protein-protein interactions, a central region which contains the DNA-binding domain, and a C-terminal glutamine-rich region. It is shown that the glutamine-rich region is responsible for the formation of fibres in vitro which, on the basis of their tinctorial properties and CD spectra, may be classified as amyloid fibres. A large structural change, probably resulting in beta-sheet structure, is observed upon fibre formation. Mutants containing the central region, either alone or together with the glutamine-rich region, are largely lacking in secondary structure but they bind specifically to the cognate DNA and are able to remodel chromatin in vitro. Consequently, neither the N-terminal domain nor the C-terminal glutamine-rich regions of the GAGA factor are necessary for chromatin remodelling in vitro.

Amyloid↗
PubMed1998

Redox components and structure of the respiratory NADH:ubiquinone oxidoreductase (complex I).

The proton-pumping NADH:ubiquinone oxidoreductase is the first complex in the respiratory chains of many purple bacteria and of mitochondria of most eucaryotes. The bacterial complex consists of 14 different subunits. The mitochondrial complex contains at least 29 additional proteins that do not directly participate in electron transfer and proton translocation. We analysed electron micrographs of isolated and negatively stained complex I particles from Escherichia coli and Neurospora crassa and obtained three-dimensional models of both complexes at medium resolution. Both have the same L-shaped overall structure with a peripheral arm protruding into the aqueous phase and a membrane arm extending into the membrane. The two arms of the bacterial complex are only slightly shorter than those of the mitochondrial complex although the protein mass of the former is only half of that of the latter. The presence of a novel redox group in the membrane arm of the complex is discussed. This group has been detected in the N. crassa complex by means of UV-visible spectroscopy. After reduction with an excess of NADH and reoxidation by the lactate dehydrogenase reaction, a reduced-minus-oxidized difference spectrum was obtained that cannot be attributed to the known cofactors flavin mononucleotide (FMN) and the FeS clusters N1, N2, N3 and N4. Due to its positive midpoint potential the novel group is believed to transfer electrons from the FeS clusters to ubiquinone. Its role in proton translocation is discussed.

Computer Simulation↗
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↗
PubMed1998

A comparison of forensic and nonforensic malingerers: a prototypical analysis of explanatory models.

Explanatory models of malingering strive to understand the primary motivation underlying attempts to feign. Rogers, Sewell, and Goldstein (1994) provided empirical support for the conceptualization of pathogenic, criminological, and adaptational models. In the current study, a prototypical analysis of 221 forensic experts results in a slightly refined formulation: the adaptational models was decomposed into its two broad dimensions (cost-benefit analysis and adversarial setting). An important findings is that the factor structure for the explanatory models remained stable when applied to both forensic and nonforensic cases. As a first investigation, significant differences were observed in prototypical cases of malingering by the category of referral (forensic or nonforensic) and by type of feigning (mental disorders, cognitive impairment, and medical syndromes). Surprisingly, the feigning of medical syndromes appeared to play a relatively prominent role in both forensic and nonforensic cases and to be influenced by the apparent adversarial context of the assessment. Finally, important gender differences were observed, especially with nonforensic prototypical cases of malingering.

Adaptation, Psychological↗
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↗