PubMed Health⌕ Search

Biomedical subjects

R Craig

Publications and source records attributed to R Craig.

At least 73 records · Page 4Linked to original sources
PubMed1993

Three-dimensional reconstruction of caldesmon-containing smooth muscle thin filaments.

Caldesmon is known to inhibit actomyosin ATPase and filament sliding in vitro, and may play a role in modulating smooth muscle contraction as well as in diverse cellular processes including cytokinesis and exocytosis. However, the structural basis of caldesmon action has not previously been apparent. We have recorded electron microscope images of negatively stained thin filaments containing caldesmon and tropomyosin which were isolated from chicken gizzard smooth muscle in EGTA. Three-dimensional helical reconstructions of these filaments show actin monomers whose bilobed shape and connectivity are very similar to those previously seen in reconstructions of frozen-hydrated skeletal muscle thin filaments. In addition, a continuous thin strand of density follows the long-pitch actin helices, in contact with the inner domain of each actin monomer. Gizzard thin filaments treated with Ca2+/calmodulin, which dissociated caldesmon but not tropomyosin, have also been reconstructed. Under these conditions, reconstructions also reveal a bilobed actin monomer, as well as a continuous surface strand that appears to have moved to a position closer to the outer domain of actin. The strands seen in both EGTA- and Ca2+/calmodulin-treated filaments thus presumably represent tropomyosin. It appears that caldesmon can fix tropomyosin in a particular position on actin in the absence of calcium. An influence of caldesmon on tropomyosin position might, in principle, account for caldesmon's ability to modulate actomyosin interaction in both smooth muscles and non-muscle cells.

Actin Cytoskeleton↗
PubMed1993

A human lymphoid recombinant cell line with functional human immunodeficiency virus type 1 envelope.

Our goal has been to develop a safe and effective system that would allow us to explore the functions of the human immunodeficiency virus (HIV) envelope. We have generated a human lymphoid cell line (TF228.1.16) that stably expresses functional HIV envelope proteins on its cell surface, and therefore closely mimics the viral envelope and virus-infected cells. The TF228.1.16 line forms syncytia with human cells of the CD4+ phenotype and provides a facile virus-free cell-based assay for examining the mechanism of syncytia formation and for evaluating novel agents that may disrupt this process. The TF228.1.16 cells also provide an opportunity to present the HIV envelope proteins to the immune system in cellular form. In vitro immunization of human peripheral blood mononuclear cells (PBMC) and in vivo immunization of rhesus monkeys with this reagent results in the production of antibodies with neutralizing (anti-syncytia) activities. When the HIV envelope is expressed against the background of human lymphoid cells, it may exhibit immune protection with unique properties that have not yet been explored. Our results indicate that a virus-free cell system can play an important role in exploring the biology and function of HIV-envelope proteins without the interference of other viral components present in infected cells. This paper discusses these results, and examines the potential use of TF228.1.16 as a vaccine.

Animals↗
PubMed1992

Structure of the myosin filaments of relaxed and rigor vertebrate striated muscle studied by rapid freezing electron microscopy.

Rapid freezing followed by freeze-substitution has been used to study the ultrastructure of the myosin filaments of live and demembranated frog sartorius muscle in the states of relaxation and rigor. Electron microscopy of longitudinal sections of relaxed specimens showed greatly improved preservation of thick filament ultrastructure compared with conventional fixation. This was revealed by the appearance of a clear helical arrangement of myosin crossbridges along the filament surface and by a series of layer line reflections in computed Fourier transforms of sections, corresponding to the layer lines indexing on a 43 nm repeat in X-ray diffraction patterns of whole, living muscles. Filtered images of single myosin filaments were similar to those of negatively stained, isolated vertebrate filaments and consistent with a three-start helix. M-line and other non-myosin proteins were also very well preserved. Rigor specimens showed, in the region of overlapping myosin and actin filaments, periodicities corresponding to the 36, 24, 14.4 and 5.9 nm repeats detected in X-ray patterns of whole muscle in rigor; in the H-zone they showed a disordered array of crossbridges. Transverse sections, whose Fourier transforms extend to the (3, 0) reflection, supported the view, based on X-ray diffraction and conventional electron microscopy, that in the overlap zone of relaxed muscle most of the crossbridges are detached from the thin filaments while in rigor they are attached. We conclude that the rapid freezing technique preserves the molecular structure of the myofilaments closer to the in vivo state (as monitored by X-ray diffraction) than does normal fixation.

Animals↗
PubMed1992

Structure of the human DNA repair gene HAP1 and its localisation to chromosome 14q 11.2-12.

Apurinic/apyrimidinic (AP) sites are pre-mutagenic DNA lesions which occur spontaneously and following exposure of cells to ionising radiation or chemical mutagens. HAP1 (Human AP endonuclease 1), the major enzyme in human cells initiating repair of AP sites, shows strong sequence homology to DNA repair enzymes from bacteria, Drosophila and other mammalian species. We have cloned the HAP1 gene and determined its complete nucleotide sequence. The site of transcription initiation has been mapped to 452 bp upstream of the ATG initiation codon in the genomic DNA. The HAP1 gene consists of five exons and is unusually small (less than 2.6 kb from transcription initiation site to polyadenylation sequence) with 54% of the protein coding region and the entire 3' untranslated region contained within a single exon. The first exon is non-coding. Regions of three exons show sequence homology to the E.coli xth (exonuclease III) gene. Using in situ hybridisation, the HAP1 gene has been localised to human chromosome 14q 11.2-12.

Base Sequence↗
PubMed1992

Electron microscopy of the actin-myosin head complex in the presence of ATP.

The structure of the actin-myosin head complex during the ATPase cycle has been studied by electron microscopy of negatively stained acto-heavy-meromyosin. In the absence of ATP, heavy meromyosin molecules generally showed a regular, angled appearance, with both heads attached to the actin filament. In the presence of ATP, attached molecules showed a less ordered structure, often with only one head attached. We conclude that configurations other than the rigor structure occur during the actomyosin cross-bridge cycle.

Actins↗
PubMed1992

Structural changes induced in scallop heavy meromyosin molecules by Ca2+ and ATP.

We have used physicochemical and ultrastructural methods to investigate the effects of Ca2+ and ATP on the structure of purified heavy meromyosin (HMM) from the striated adductor muscle of the scallop, a species with myosin-linked regulation. Using papain as a structural probe, we found that, in the presence of ATP, the head/tail junction was five times more susceptible to digestion at high levels of Ca2+ than at low levels. By HPLC gel filtration, two fractions of scallop HMM with different Stokes radii were detected in the presence of ATP at low Ca2+, while at high Ca2+ a single peak with the larger Stokes radius predominated. Electron microscopy of rotary-shadowed HMM suggested that molecules with the smaller Stokes radius had their heads bent back towards their tails, while those with the larger radius had heads pointing away from the tail. The number of molecules with their heads bent back decreased at high Ca2+ levels. The data also showed that in the absence of ATP or at high salt, HMM molecules behaved similarly to those in the presence of ATP at high Ca2+. These results suggest that scallop myosin heads can exist in two conformations (heads down towards the tail and heads up away from the tail) and that the equilibrium between these two conformations is altered by the concentrations of salt, ATP and Ca2+. However, the equilibrium between the two forms appears to be too slow to be involved in regulating contraction. The 'heads-down' configuration may instead be related to the inactive, folded (10S) form of scallop myosin and possibly involved in filament assembly during development.

Adenosine Triphosphate↗
PubMed1992

Behavioral effects of A71623, a highly selective CCK-A agonist tetrapeptide.

We studied the behavioral effects of a novel cholecystokinin tetrapeptide (CCK-4) analogue, A71623, with full agonist activity and high affinity and selectivity for the CCK-A receptor subtype relative to the CCK-B receptor. In tests for anorectic activity, A71623 was found to suppress 60-min intakes of a liquid diet in both deprived and sated rats, and the effects were blocked by a selective CCK-A antagonist, A70104. Compared with CCK-8, A71623 was found to have improved potency and duration of action; the most potent route of administration was intraperitoneal. A71623 also suppressed the intake of a liquid diet and a 0.2 M sucrose solution in lean and obese Zucker rats. In daily injection studies, the anorectic activity of CCK-8 diminished rapidly, whereas the suppressant effects of A71623 on food intakes and body weight gains persisted throughout the 11-day treatment period. Finally, A71623 reduced the spontaneous locomotor activity of rats at doses above those required to suppress intakes. These studies are the first to describe the behavioral effects of a potent and highly selective CCK-A receptor agonist.

Animals↗
PubMed1992

HIV-related cachexia: potential mechanisms and treatment.

Involuntary weight loss or wasting indicative of severe protein energy malnutrition is a frequent complication of acquired immune deficiency syndrome (AIDS). Malnutrition, with its associated adverse effects on immunocompetence, may contribute to the progression of AIDS itself. Since death from wasting is ultimately related to the magnitude of tissue depletion, restoration of body cell mass may enhance survival. The mechanism of weight loss in AIDS has not been clearly elucidated. The etiology is likely to be multifactorial, the result of interactions between decreased caloric intake, malabsorption, and alterations in energy expenditure secondary to hormonal and/or metabolic abnormalities. Although weight loss is occasionally reversible with treatment of underlying infections and/or easily identifiable and reversible causes, the majority of patients are not this fortunate. Enteral and parenteral nutrition, which are expensive, cumbersome, and potentially morbid, have been suggested by some as therapeutic options. Megestrol acetate, a synthetic, orally active progestational agent, has been reported to stimulate appetite and weight gain. Data regarding the use of megestrol acetate for the treatment of cachexia related to human immunodeficiency virus (HIV) infection demonstrate convincingly its effectiveness in treating many patients with HIV-related anorexia and cachexia.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Direct determination of myosin filament symmetry in scallop striated adductor muscle by rapid freezing and freeze substitution.

Chemically skinned, relaxed bundles of fibers from the striated adductor muscle of the scallop Placopecten magellanicus were rapidly frozen and freeze-substituted. In the electron microscope, ultrathin transverse sections of embedded specimens showed, in many cases, clear regularly organized projections (crossbridges) protruding from the backbones of the myosin filaments. In the majority of cases the number of projections was directly observed to be seven: this was confirmed by alignment and averaging of the images using correlation methods. The rotational power spectrum of the average image showed a strong peak at N = 7. Tilting of sections in the electron microscope showed that the long-pitch crossbridge helices were right-handed. These and other observations confirm directly the essential features of the low-resolution three-dimensional helical reconstruction of negatively stained scallop filaments calculated previously.

Animals↗
PubMed1991

Inhibition of lipoxygenase and prostaglandin endoperoxide synthase by anacardic acids.

C22:1 omega 5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 microM, respectively. Very similar inhibition was seen with the crude exudate, rich in omega 5-anacardic acids, from glandular trichomes of an arthropod-resistant strain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the omega 5-unsaturated compound. However, the dimethyl derivative of C22:1 omega 5-anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 microM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the omega 5-AnAs is discussed.

Aspirin↗
PubMed1991

A cystic fibrosis bronchial epithelial cell line: immortalization by adeno-12-SV40 infection.

An immortalized cell line was created from a primary culture of bronchial epithelia isolated from a patient with cystic fibrosis. The culture was transformed with a hybrid virus, adeno-12-SV40, which has been used successfully on a number of different human epithelial tissues. The transformed bronchial epithelial cells have the following characteristics. (1) Cyclic adenosine monophosphate (cAMP) is stimulated by beta-adrenergic agonists. (2) Outwardly rectifying Cl- channels are present on the apical cell membrane. These channels can be activated by depolarizing voltages but not by protein kinase A or C. (3) Keratin is present by immunofluorescence, and this is consistent with the epithelial origin of the cells. (4) The SV40 large T antigen is present as demonstrated by immunofluorescence. (5) Multiple karyotype analyses show modal chromosome number to be 80 to 90. There are an average of four chromosome 7 per cell. (6) The phenylalanine508 deletion in the gene coding for the cystic fibrosis transmembrane regulator is present on at least one chromosome. The cells can be grown in multiple passages, contain the abnormal regulation of the secretory Cl- channel, and should be an appropriate substrate for studies of the mutant cystic fibrosis transmembrane regulatory protein and its interaction with the Cl- channel.

Antigens, Viral, Tumor↗
PubMed1991

Characterization of two novel cholecystokinin tetrapeptide (30-33) analogues, A-71623 and A-70874, that exhibit high potency and selectivity for cholecystokinin-A receptors.

Based on their relative affinities for cholecystokinin octapeptide (26-33) (CCK-8), cholecystokinin tetrapeptide (30-33) (CCK-4), desulfated CCK-8, and gastrin, cholecystokinin (CCK) receptors have been classified as CCK-A (alimentary) and CCK-B (brain). Selective nonpeptide antagonists of CCK-A and CCK-B receptors, as well as highly selective CCK-A and CCK-B peptide agonists, have been described. We report here the characterization of two novel CCK-4-based peptides, A-71623 and A-70874. In radioligand binding assays, the IC50 values for A-71623 and A-70874 were 3.7 and 4.9 nM in guinea pig pancreas (CCK-A) and 4500 and 710 nM in cerebral cortex (CCK-B), respectively. Both were agonists in stimulating pancreatic amylase release, and their stimulatory effects were potently inhibited by the CCK-A antagonist L-364,718. A-71623 was a full agonist and A-70874 was a partial agonist (approximately 80%) in stimulating phosphoinositide breakdown in pancreas. Both peptides also were potent agonists in stimulating CCK-A receptors in the ileum. They were, however, weak and behaved as partial agonists in calcium studies in NCI-H345 cells, which possess CCK-B/gastrin receptors. In guinea pig gastric glands, the affinities of A-71623 and A-70874 for the CCK-B/gastrin receptor were 11 and 1.6 microM, respectively. These results demonstrate that A-71623 and A-70874 are potent and selective agonists at CCK-A receptors. The preferential interaction of these novel CCK-4 analogs with CCK-A receptors is in contrast to other CCK-4-based peptides, which are primarily selective for CCK-B receptors. In addition, A-71623 and A-70874 are the first two examples of potent CCK-A agonists that do not contain a tyrosine residue whose sulfation is required for potent CCK-A agonist activity of larger peptides.

Amylases↗
PubMed1991

[Method for direct determination of the rotational symmetry of thick muscle filaments by digital image processing].

We present a method based on digital image processing techniques that permits to determine directly the rotational symmetry of muscle thick filaments. Electron micrographs of transverse sections of relaxed muscle rapidly frozen against a copper block cooled with liquid helium were digitized, aligned to give maximal cross correlation and averaged. The final averaged image permits to determine directly the number of projections on the backbone surface.

Animals↗
PubMed1990

Caldesmon and the structure of smooth muscle thin filaments: electron microscopy of isolated thin filaments.

Native and synthetic vertebrate smooth muscle thin filaments have been examined by electron microscopy in order to determine the arrangement of the regulatory protein caldesmon. In synthetic filaments of actin-caldesmon, long slender molecules were sometimes seen running along the thin filament, suggesting that caldesmon can associate with actin along its length, while at other times lateral projections were observed. In native filaments, containing actin, caldesmon and tropomyosin, we found no evidence for lateral projections extending from the filaments, suggesting that caldesmon does not act as a crosslinking protein in vivo. In contrast, elongated molecules were clearly seen following the long pitch actin helices. We suggest that these may represent an association of caldesmon and tropomyosin. Antibodies developed against an N-terminal fragment of caldesmon caused thin filaments to aggregate laterally into arrays displaying approximately 35-38 nm repeats; thin filament aggregates with this periodicity were obtained previously (Lehman et al., 1989) using antibodies to the C-terminal segment of caldesmon. These results suggest that both ends of caldesmon are closely associated with the shaft of the thin filament, supporting a model in which the elongated caldesmon molecule runs along the filament, possibly interacting with tropomyosin, following the long pitch actin helices.

Animals↗
PubMed1990

Waste treatment technologies from the Genesearch Group of companies.

Genesearch is a 10 year old research and development company of Australian scientists, specializing in microbiology and genetics. This research expertise has formed the basis of a number of microbial processes for waste treatment. In addition, a novel type of high yield fermenter for aerobic bacteria allows the economical production of high-potency bacterial preparations for waste treatment processes. A novel approach to the rapid biodegradation of polychlorinated biphenyls has been developed. Photochemical pretreatment partially dechlorinates the molecules, rendering them susceptible to complete and rapid digestion by wild-type soil bacteria. In the area of non-toxic waste, Genesearch has developed products for on-site treatment of, for example, grease-trap wastes and waste oil in ship bilges; and a large scale process for conversion of municipal grease wastes into protein-rich biomass. The prospects for novel biological waste treatment are improving, as public pressure grows, and as increasing government monitoring and penalties make inadequate waste disposal uneconomic.

Bacteria, Aerobic↗
PubMed1989

Inhibition of conformational change in smooth muscle myosin by a monoclonal antibody against the 17-kDa light chain.

Monoclonal antibodies against gizzard smooth muscle myosin were generated and characterized. One of these antibodies, designated MM-2, recognized the 17-kDa light chain and modulated the ATPase activities and hydrodynamic properties of smooth muscle myosin. Rotary shadowing electron microscopy showed that MM-2 binds 51 (+/- 25) A from the head-rod junction. The depression of Ca2+- and Mg2+-ATPase activities of myosin and Ca2+-ATPase activity of heavy meromyosin at low KCl concentration were abolished by MM-2. Viscosity measurement indicated that MM-2 inhibits the transition of 6 S myosin to 10 S myosin. While the rate of the production of subfragment-1 by papain proteolysis of 6 S myosin was inhibited by MM-2, the rate of proteolysis of the heavy chain of 10 S myosin was enhanced by MM-2 and reached the same rate as that of 6 S myosin plus MM-2. These results suggest that MM-2 inhibits the formation of 10 S myosin by binding to the 17-kDa light chain which is localized at the head-neck region of the myosin molecule. MM-2 increased the Vmax of actin-activated Mg2+-ATPase activities of both dephosphorylated myosin and dephosphorylated heavy meromyosin about 10- and 20-fold, respectively. MM-2 also activated the actin-activated Mg2+-ATPase activity of phosphorylated myosin at a low MgCl2 concentration and thus abolished the Mg2+-dependence of acto phosphorylated myosin ATPase activity. These results suggest that MM-2 inhibits the formation of 10 S myosin, and this results in the activation of actin-activated Mg2+-ATPase activity even in the absence of phosphorylation.

Adenosine Triphosphatases↗