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PubMed2004

NOE assignment with ARIA 2.0: the nuts and bolts.

The assignment of nuclear Overhauser effect (NOE) resonances is the crucial step in determining the three-dimensional structure of biomolecules from nuclear magnetic resonance (NMR) data. Our program, Ambiguous Restraints for Iterative Assignment (ARIA), treats Noe assignment as an integral part of the structure determination process. This chapter briefly outlines the method and discusses how to carry out a complete structure determination project with the new version 2.0 of ARIA. Two new features greatly streamline the procedure: a new graphical user interface (GUI) and the incorporation of the data model of the Collaborative Computing Project for the NMR community (CCPN). The GUI supports the user in setting up and managing a project. The CCPN data model facilitates data exchange with a great variety of other programs. We give practical guidelines for how to use ARIA and how to analyze results.

Calibration↗
PubMed1979

Effects of antiprostatic agents on 5alpha-dihydrotestosterone binding to rat hypophyseal and hypothalamic cytosol macromolecules.

The binding of 5alpha-dihydrotestosterone to the hypophyseal and hypothalamic cytosol macromolecules prepared from castrated male rats was observed. The effects of antiprostatic agents on 5alpha-dihydrotestosterone binding to both hypophyseal and hypothalamic cytosol macromolecules was examined. Cyproterone acetate and chlormadionone acetate showed the significant inhibiting effects on 5alpha-dihydrotestosterone binding to 7-8 S macromolecules of cytosol from both hypophysis and hypothalamus. SCH 13521 and AA 560 did also affect 5alpha-dihydrotestosterone binding to 7-8 S macromolecules of cytosol from both tissues.

Androgen Antagonists↗
PubMed2005

Smart polymeric carriers for enhanced intracellular delivery of therapeutic macromolecules.

Limited cytoplasmic delivery of enzyme-susceptible drugs remains a significant challenge facing the development of protein and nucleic acid therapies that act in intracellular compartments. Researchers have examined several approaches, including fusogenic proteins and protein transduction domains, to enhance the intracellular delivery of the therapeutic cargo. This review summarises efforts to develop 'smart' pH-sensitive and membrane-destabilising polymers that can shuttle therapeutic peptide, protein and nucleic acid molecules past the endosomal membrane into the cytoplasm of targeted cells. Several classes of 'smart' non-degradable polymeric carriers have been developed that have proved effective both in vitro and in vivo in enhancing the cytoplasmic delivery of a variety of therapeutic molecules.

Animals↗
PubMed1979

Studies on the association of cytochrome P-450 and NADPH-cytochrome c reductase during catalysis in a reconstituted hydroxylating system.

The interaction between cytochrome P-450 and NADPH-cytochrome c reductase during catalysis has been investigated with a reconstituted monooxygenase system composed of the two purified enzyme components and synthetic phospholipid. Steady state kinetic data are consistent with a scheme in which the formation of a binary complex between the two proteins precedes catalysis. The formation of this binary complex is described by a simple mass action equation. In agreement with this equation, the observed Vmax for benzphetamine N-demethylation was found to be directly proportional to the calculated concentration of the cytochrome P-450 . reductase complex. Furthermore, with appropriate reductase/cytochrome P-450 mole ratios, the Vmax could be shown to be linearly dependent on either the reductase or the cytochrome P-450 concentration alone. In contrast, the Km parameter is independent of the complex concentration, indicating that no change in the rate-limiting step has occurred. Thus a distinction should be made between a rate-limiting enzyme component and the rate-limiting step in this multienzyme system.

Animals↗
PubMed2003

Processing sequence annotation data using the Lua programming language.

The data processing language in a graphical software tool that manages sequence annotation data from genome databases should provide flexible functions for the tasks in molecular biology research. Among currently available languages we adopted the Lua programming language. It fulfills our requirements to perform computational tasks for sequence map layouts, i.e. the handling of data containers, symbolic reference to data, and a simple programming syntax. Upon importing a foreign file, the original data are first decomposed in the Lua language while maintaining the original data schema. The converted data are parsed by the Lua interpreter and the contents are stored in our data warehouse. Then, portions of annotations are selected and arranged into our catalog format to be depicted on the sequence map. Our sequence visualization program was successfully implemented, embedding the Lua language for processing of annotation data and layout script. The program is available at http://staff.aist.go.jp/yutaka.ueno/guppy/.

Computational Biology↗
PubMed1990

[Study of the structural organization of cytochrome P-450 oligomers using immobilized isoenzyme LM2].

Subunit interactions of highly purified hexameric cytochrome P-450 LM 2 has been studied using covalent binding of one of the six protomers to an insoluble matrix. Immobilized cytochrome was catalytically active in monooxygenase reactions and retained the spectral characteristics of cytochrome P-450 LM 2. High ionic strength, large scale pH changes and addition of guanidine chloride were without effect on the aggregation state of the immobilized hemoprotein. However, several detergents induced hexamer dissociation. The crucial role of hydrophobic forces in hexamer subunit interaction was demonstrated. Incubation of the immobilized cytochrome P-450 LM 2 with sonicated liposomes composed of various phospholipids did not result in oligomer dissociation and protein translocation from the matrix to the lipid phase, although the catalytic activity of the immobilized cytochrome significantly increased in the presence of liposomes. The data suggest that cytochrome P-450 LM 2 may be of hexameric structure in the native membranes.

Animals↗
PubMed1989

Mast cell chymase. A potent secretagogue for airway gland serous cells.

Submucosal glands are the major sources of airway secretions in most mammals. Mast cells are abundant in the environment of airway submucosal glands and are rich sources of secreted proteases. To investigate the hypothesis that mast cell proteases stimulate airway gland secretion, we studied the ability of the two major mast cell granule proteases, chymase and tryptase, to cause secretion of 35S-labeled macromolecules from a line of cultured bovine airway gland serous cells. Mast cell chymase and tryptase were purified from dog mastocytoma cells. Chymase markedly stimulated serous cell secretion in a concentration-dependent fashion with a threshold of 10(-10) M, whereas tryptase had no effect. The response to 10(-8) M chymase (1530 +/- 80% over base line) was approximately 10-fold higher than that evoked by other agonists such as histamine and isoproterenol. The predominant 35S-labeled macromolecule released by chymase was chondroitin sulfate proteoglycan, the glycoconjugate present in serous cell secretory granules. The response to chymase was non-cytotoxic and was blocked by active site inhibitors of chymase (soybean trypsin inhibitor and chymostatin) and by inhibitors of cellular energy metabolism (azide,2,4-dinitrophenol, dicumarol). Supernatant obtained by degranulation of mastocytoma cells caused a secretory response of comparable magnitude to that caused by chymase. These findings demonstrate that chymase, but not tryptase, is a potent secretagogue for airway gland serous cells, and they suggest a possible role for chymase-containing mast cells in the pathogenesis of airway hypersecretion.

Animals↗
PubMed1988

In vitro expression of the alpha and beta subunits of the Na,K-ATPase.

Our analysis of the cloned alpha 3 protein strongly suggests that this c-DNA represents a bona fide Na+,K+ ATPase isoform. Its similarity to alpha + may have made its detection in tissues by gel migration or immunoreactivity difficult. Expression of an enzymatically active alpha 3 beta Na+K+ATPase either in a completely in vitro system or in a heterologous tissue culture system will clearly establish the biochemical properties of this isoform. Development of alpha 3 specific immunochemical probes will allow a proper assessment of its in vivo expression.

Animals↗
PubMed1988

The structure of human complement component C7 and the C5b-7 complex.

The molecular architecture of human complement component C7 was elucidated at several structural levels. The complete primary structure of C7 was derived from the cDNA sequence of clones isolated from a human liver library. C7 is a mosaic protein that consists of 821 amino acids. The amino-terminal two-thirds of C7 has 23-30% homology with complement components C8 and C9. In addition, the carboxyl-terminal third contains four cysteine-rich segments that have overlapping internal homology. The protein is a single polypeptide chain with 28 disulfide bonds and is glycosylated at two sites. Virtually all the cysteines are found in small units of 35-77 amino acids that exhibit homology with those of various proteins including the low density lipoprotein receptor, epidermal growth factor precursor, thrombospondin, and blood coagulation factors IX and X. The secondary structural analysis, estimated by circular dichroism, suggested a high content of beta-sheet (38%) and beta-turns (24%). The tertiary structure, visualized by transmission electron microscopy, indicated a flexible elongated molecule with dimensions of 151 X 59 X 43 A. The quaternary structure of the C5b-7 complex bound to lipid vesicles was observed to be in the form of monomers or dimers. The monomer C5b-7 consists of a leaflet and a long flexible stalk, and the dimer has two leaflets linked through a supercoiled stalk. Membrane binding is mediated by the stalk part of the complexes. Using a radioiodinated photoreactive cross-linking reagent bound to the polar head group of phosphatidylethanolamine, the stalk part of the C5b-7 complex could be labeled preferentially, and it was found to consist mainly of C6 and C7. Thus, C7 plays a major role in bringing about the hydrophilic-amphiphilic transition during the formation of the membrane attack complex, and it serves as a membrane anchor for the C5b-7 complex.

Amino Acid Sequence↗
PubMed1982

Proton-translocating ATPase of chromaffin granule membranes.

Active transport of catecholamines into chromaffin granules is driven by the transmembrane pH gradient and membrane potential, created by an electrogenic proton-translocating ATPase in the granule membrane. The ATPase activity of highly purified chromaffin granule membranes is inhibited by a number of agents in common with mitochondrial ATPase, and also by antibodies raised against mitochondrial F1. Dichloromethane treatment of these membranes solubilizes an enzyme that is closely similar to mitochondrial F1, but distinguishable from it by its interaction with specific antisera and the inhibitor aurovertin. Chromaffin granule membranes contain a low-molecular-weight protein that reacts with dicyclohexylcarbodiimide; it can be extracted into chloroform-methanol, and is of higher electrophoretic mobility than the corresponding mitochondrial protein. Evidence is presented that this is a component of the proton-translocating ATPase complex.

Adenosine Triphosphatases↗
PubMed1984

Expression of acetylcholine receptor alpha-subunit mRNA during differentiation of the BC3H1 muscle cell line.

The accumulation of translatable acetylcholine receptor alpha-subunit mRNA was examined in the BC3H1 muscle cell line in response to serum and cell growth. Relative amounts of alpha-subunit mRNA were quantitated during differentiation by cell-free translation and immunoprecipitation with an alpha-subunit-specific monoclonal antibody. Logarithmically growing cells do not possess cell surface acetylcholine receptors; however, a significant amount of alpha-subunit mRNA is detectable in cells under these conditions. Furthermore, alpha-subunit is synthesized in growing undifferentiated cells at a rate similar to that of differentiated cultures. Following growth arrest of BC3H1 cells, surface receptors are induced to levels greater than 100-fold above that of growing cells. The relative level of translatable alpha-subunit mRNA in differentiated cells, however, is only approximately 4-fold greater than in growing cultures. Induction of alpha-subunit mRNA appears to be reversible since reinitiation of growth in quiescent differentiated BC3H1 cells results in a reduction in relative abundance of this mRNA species to levels comparable to that of undifferentiated cells and the concomitant loss of surface receptors. These results indicate that receptor expression during differentiation is regulated both post-translationally and at the level of receptor subunit mRNA accumulation.

Animals↗
PubMed1981

Membrane attack complex of complement. Evidence for its dimeric structure based on hybrid formation.

Molecular hybridization experiments provided new evidence for the dimeric nature of the membrane attack complex (MAC) of complement. Monomeric C5b-6, which constitutes the first intermediate complex in MAC formation, was prepared in two differentially labeled forms: biotin-125I-C5b-6 and 131I-C5b-6. Using a mixture of the differentially labeled C5b-6, the MAC was assembled on phospholipid vesicles upon addition of C7, C8, and C9. The assembled MAC containing biotin-125I and 131I was extracted from the vesicles with deoxycholate, purified, and exposed to avidin-Sepharose. Biotin-mediated binding of the MAC to avidin-Sepharose not only effected binding of 125I, but also of 131I, indicating that both radiolabels resided in the same molecular entity. When equimolar amounts of differentially labeled C5b-6 were available for MAC formation, 50% of MAC formed contained one molecule of each form. Theoretical analysis of the experimental data clearly favored the dimer structure over the structure of a higher oligomer. In contrast, fluid phase SC5b-9 was clearly monomeric on the basis of the same analysis. The electron microscopic appearance of the biotinated MAC hybrid closely resembled that of the characteristic membrane lesions of complement lysed cells. An avidin-ferritin conjugate attached itself to the ring-shaped portion of the biotinated MAC and not to its perpendicular structures, suggesting that C5b-6 is an integral part of the ring structure of the MAC.

Biotin↗