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At least 1,189 records · Page 66Linked to original sources
PubMed2003

Association of Caspr/paranodin with tumour suppressor schwannomin/merlin and beta1 integrin in the central nervous system.

Caspr/paranodin is an essential neuronal component of paranodal axoglial junctions, associated with contactin/F3. Its short intracellular domain contains a conserved motif (GNP motif) capable of binding protein 4.1 domains [FERM domains (four point one, ezrin, radixin, moesin)]. Schwannomin/merlin is a tumour suppressor expressed in many cell types, including in neurons, the function and partners of which are still poorly characterized. We show that the FERM domain of schwannomin binds to the paranodin GNP motif in glutathione S-transferase (GST)-pull down assays and in transfected COS-7 cells. The two proteins co-immunoprecipitated in brain extracts. In addition, paranodin and schwannomin were associated with integrin beta1 in transfected cells and in brain homogenates. The presence of paranodin increased the association between integrin beta1 and schwannomin or its N-terminal domain, suggesting that the interactions between these proteins are interdependent. In jimpy mutant mice, which display a severe dysmyelination with deficient paranodal junctions, the interactions between paranodin, schwannomin and integrin beta1 were profoundly altered. Our results show that schwannomin and integrin beta1 can be associated with paranodin in the central nervous system. Since integrin beta1 and schwannomin do not appear to be enriched in paranodes they may be quantitatively minor partners of paranodin in these regions and/or be associated with paranodin at other locations.

Amino Acid Motifs↗
PubMed1977

Characterization of C-reactive protein and the complement subcomponent C1t as homologous proteins displaying cyclic pentameric symmetry (pentraxins).

Partial amino acid sequences of rabbit C-reactive protein, a peptide derived from human C-reactive protein by cyanogen bromide cleavage, and the C1t subcomponent of the human complement component C1 have been determined. Extensive sequence homology between these proteins establish their evolutionary relationships. In addition, examination of C-reactive proteins by negative-stain electron microscopy revealed that the protein is composed of five subunits arranged in cyclic symmetry. This structure is similar to that reported for both C1t and the amyloid P-component. The extensive structural relationship suggests similar or overlapping functions and the term pentraxin is proposed to describe these homologous proteins.

Amino Acid Sequence↗
PubMed1996

Formation of a ligand-binding site for the acetylcholine receptor in vitro.

Investigation of the mechanisms by which the subunits of ligand-gated ion channels fold and associate to form oligomers has been hampered by the lack of an in vitro system in which these reactions occur. We have established conditions in a rabbit reticulocyte translation system supplemented with canine pancreatic microsomes under which the alpha and delta subunits of the nicotinic acetylcholine receptor (AChR) fold and assemble to form a heterodimer with a cholinergic binding site comparable with that found in the intact AChR. Folding of the alpha subunit was followed by its ability to bind alpha-bungarotoxin. Folding efficiency was highly sensitive to changes in the redox potential of the translation medium and was favored by an oxidizing environment. Acquisition of the toxin binding conformation required N-linked core glycosylation but not oligosaccharide trimming, suggesting that oligosaccharide-dependent interaction of chaperones with the alpha subunit is not essential for correct subunit folding. The conformationally mature alpha subunit specifically associated with the delta subunit but not the beta subunit to form a heterodimer with a high affinity ligand-binding site. These data demonstrate, for the first time, correct folding and assembly of the AChR subunits in an in vitro system.

Animals↗
PubMed2002

Crystal structure of actinomycin D bound to the CTG triplet repeat sequences linked to neurological diseases.

The potent anticancer drug actinomycin D (ActD) acts by binding to DNA GpC sequences, thereby interfering with essential biological processes including replication, transcription and topoisomerase. Certain neurological diseases are correlated with expansion of (CTG)n trinucleotide sequences, which contain many contiguous GpC sites separated by a single base pair. In order to characterize the binding of ActD to CTG triplet repeat sequences, we carried out heat denaturation and CD analyses, which showed that adjacent GpC sequences flanking a T:T mismatch are preferred ActD-binding sites, and that ActD binding results in a conformational transition to A-type structure. The structural basis of the strong binding of ActD to neighboring GpC sites flanking a T:T mismatch was provided by the crystal structure of ActD bound to ATGCTGCAT, which contains a CTG triplet sequence. Binding of two ActD molecules to GCTGC causes a kink in the DNA helix. In addition, using a synthetic self-priming DNA model, 5'-(CAG)4(CTG)(16)-3', we observed that ActD can trap the cruciform or duplexes of (CTG)n and interfere with the expansion process of CTG triplet repeats as shown by gel electrophoretic expansion assay. Our results may provide the possible biological consequence of ActD bound to CTG triplet repeat sequences.

Antibiotics, Antineoplastic↗
PubMed1994

Rack-induced bonding in blue-copper proteins.

The unique spectroscopic properties of blue-copper centers, i.e. the strong charge-transfer band at approximately 600 nm and the narrow hyperfine coupling in the EPR spectrum, are reviewed. The concept of rack-induced bonding is summarized. The tertiary structure of the protein creates a performed chelating site with very little flexibility, the geometry of which is in conflict with that preferred by Cu2+. The structure of the metal site in azurin is discussed. It is shown that the three strong ligands, one thiolate S and two imidazole N, are in a configuration intermediate between those preferred by Cu2+ and Cu+. It is emphasized that cysteine is an obligatory component of a blue site, whereas the weak interaction with a methionine S is not necessary. The minimum rack energy is estimated to be 70 kJ.mol-1. It is pointed out that the high reduction potentials of blue-copper centers are a result of the protein-forced ligand-field-destabilized site structure. It is suggested that the potentials are tuned by variations in pi back bonding, and this is supported by a linear increase in delta LF (ligand field) with decreasing electron-transfer enthalpy. Site-directed mutagenesis has shown that large hydrophobic residues in the site increase the potential, whereas negative groups or water decrease it. It is also shown that the fine-tuning of the properties of the metal site by rack-induced bonding can alter the electron-transfer reorganization energy. Kinetic results with azurin mutants support a through-bond tunneling mechanism for intramolecular electron transfer in proteins. Finally, it is pointed out that the concept of rack-induced bonding is a universal principle of macromolecular structure/function relationships, which should be applied also to other systems.

Azurin↗
PubMed1973

Assembly of bacterial ribosomes.

I have not mentioned the remarkable progress made mainly by Fellner and his co-workers (86) in the elucidation of the primary structure of rRNA's and by Wittmann and his co-workers (87) in determining the structure of several ribosomal proteins. Such knowledge of primary structures is certainly the basis of complete understanding of the structure of the ribosome. With the current progress in technology, complete elucidation of the primary structure of all the ribosomal components is probably a matter of time. As indicated in this article, a rough approximation of the three-dimensional structure of ribosomes is likely to emerge soon. Although not mentioned in this article, studies of ribosomes from higher organisms are also progressing. We must, therefore, consider what further studies should be conducted and what kinds of questions we would like to solve. Some groups of investigators aim to elucidate the complete three-dimensional structure of ribosomes and to find out how these complex cell organelles function; they hope to determine the conformational changes of many of the component molecules within the ribosome structure in response to external macromolecules and cofactors engaged in protein synthesis. Such knowledge will also be important in enabling us to understand the regulation of translation of genetic messages. Other groups of investigators aim to elucidate the complex series of events which originate in the transcription of the more than 60 genes and culminate in the formation of the specific structure of the organelle. Complete reproduction in vitro of all the assembly events that occur in vivo should not be difficult to achieve in principle. It should then become possible to study in vitro any factor regulating the biogenesis of the organelle. Although we do not know whether such studies would reveal any new fundamental principle that governs the complex circuits of interconnected macromolecular interactions, the achievement of such a complete in vitro system would represent a necessary step in the comprehensive understanding of biogenesis of organelles, and eventually, of the more complex behavior and genesis of cells (89).

Bacillus↗
PubMed1991

DNA: a model compound for solution studies of macromolecules.

Well-defined, monodisperse, homologous series of oligonucleotides and DNA restriction fragments may now be produced and used as models of rigid and semirigid rodlike molecules in solution. Information from optical experiments on these model systems aids in the formulation and testing of theories of macromolecular dynamics in both dilute and concentrated solution.

Base Sequence↗
PubMed1997

Virion swelling is not required for cotranslational disassembly of cowpea chlorotic mottle virus in vitro.

The mechanism by which virions of cowpea chlorotic mottle virus (CCMV) disassemble and allow for translation of the virion RNA is not well understood. Previous models have suggested that virion swelling is required to expose the virion RNA for translation in a process referred to as cotranslational disassembly (M. Brisco, R. Hull, and T. M. A. Wilson, Virology 148:210-217, 1986; J. W. Roenhorst, J. W. M. van Lent, and B. J. M. Verduin, Virology 164:91-98, 1988; J. W. Roenhorst, J. M. Verduin, and R. W. Goldbach, Virology 168:138-146, 1989). Previous work in our laboratory has identified point mutations in the CCMV coat protein which result in virions with altered swelling characteristics (J. Fox, F. G. Albert, J. Speir, and M. J. Young, Virology 227:229-233, 1997; J. M. Fox, X. Zhao, J. A. Speir, and M. J. Young, Virology 222:115-122, 1996). The wild-type and mutant CCMV virions were used to correlate virion swelling with the ability of virion RNA to be translated in a cell-free wheat germ extract. Mutant virions unable to swell (cpK42R) are as infectious as wild-type virions in vivo, and the levels of translated encapsidated virion RNA are similar to those of wild-type virions in vitro. Mutant virions capable of swelling but not of disassembling in vitro (cpR26C) are noninfectious and have severely reduced levels of translation of the encapsidated virion RNA in vitro. These studies suggest that virion swelling is not required for the cotranslational disassembly of CCMV. Additionally, the results indicate that there is a pH-dependent structural transition in the virion, other than swelling, that results in the RNA's being exposed for translation in vitro. An alternative model suggesting that cotranslational disassembly of CCMV involves presentation of the virion RNA through the virion fivefold axis is proposed.

Cell-Free System↗
PubMed2003

CD40-mediated up-regulation of Toll-like receptor 4-MD2 complex on the surface of murine dendritic cells.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns, which are non-self macromolecular components of pathogens that allow the innate-immune system to recognize infection. TLRs are expressed on macrophages and dendritic cells (DC). TLR stimulation or CD40 agonists can induce inflammatory cytokine secretion from macrophages and DC, and promote DC maturation. The regulation of TLR expression by inflammation has begun to be explored. Our studies have focused on the regulation of TLR4 surface expression on DC. TLR4, along with the adaptor molecule MD2, is involved in the recognition of lipopolysaccharide (LPS). CD40 stimulation via cross-linked anti-CD40 monoclonal antibody (mAb) up-regulates TLR4-MD2 surface expression on a DC cell line (DC2.4) and on ex vivo-cultured splenic DC. LPS treatment down-regulated surface TLR4-MD2 on DC2.4 cells, but if combined with anti-CD40 mAb, increased TLR4-MD2 expression was observed. The increased TLR4-MD2 surface expression by any treatment did not correlate with TLR4 mRNA levels. The functional consequence of increased TLR4-MD2 expression following LPS and anti-CD40 treatment was examined. Although CD40 prestimulation did slightly enhance interleukin-12p70 secretion after LPS restimulation, simultaneous anti-CD40 mAb and LPS treatment, which up-regulates TLR4-MD2 complex, does not restore DC responsiveness to subsequent LPS.

Adjuvants, Immunologic↗
PubMed1989

Ca2(+)-dependent proteolytic modification of the cAMP-dependent protein kinase in Drosophila wild-type and dunce memory mutants.

Two cAMP-dependent protein kinases with different activation constants were separated from Drosophila melanogaster head extracts. Both are only found in nervous tissue. The first cAMP-dependent kinase, with Mr = 190,000, has been already characterized as tetrameric Drosophila type II cAMP-dependent protein kinase R2C2. The second purified cAMP-dependent protein kinase, with Mr = 80,000, is dimeric in structure RPC, and the cAMP-concentration required for half maximal activation is 4 fold lower than for the type II kinase. The generation of RP can be stimulated in vitro by addition of exogenous calcium and is due to an endogenous Ca2(+)-dependent protease that selectively degrades the regulatory subunit. Extraction in the presence of various protease inhibitors does not affect the amounts of RP, suggesting that the observed quantitative change in RP occurs in vivo. The amounts of RP in the nervous tissue of the memory mutants dunce1 and dunce2, which have increased cAMP levels, are different from the amount of RP in wild-type flies. Also treatments of wild-type flies with drugs affecting cAMP-metabolism and acetylcholine levels led to amounts of RP different from untreated flies.

Animals↗
PubMed1997

[Injectable bioerodible polymer with 5-fluorouracil used in filtration surgery in rabbits].

OBJECTIVE: A prospective randomized and placebo-controlled study was performed to evaluate the effect and toxicity of injectable bioerodible polymer with 5-fluorouracil (5-Fu) on the filtration surgery in rabbits. METHODS: Posterior lip sclerectomies were performed on both eyes of 23 rabbits. Injectable bioerodible polymer impregnated with 2.5% 5-Fu 10 mg was injected subconjunctivally in 16 eyes, and 2.5% 5-Fu 10 mg was injected subconjunctivally in 14 eyes every other day for three times in the first week and every two days for three times in the second week post-operatively. Another 16 eyes received the drug-free polymer after surgery and served as controls. RESULTS: The bleb in the eyes treated with drug-free polymer failed in the postoperative 7 days and the bleb in the eyes treated with 5-Fu injection failed in 20-30 days, while the bleb in the eyes treated with 5-Fu polymer remained successful throughout the experimental period. From 7 to 30 postoperative days, the rate of scleral fistula occlusion was 6.7% in the eyes treated with 5-Fu polymer, 41.7% in the eyes treated with 5-Fu injection and 100% in the eyes treated with drug-free polymer. Corneal epithelial defects appeared in 3 eyes treated with 5-Fu polymer, in 8 eyes treated with 5-Fu injection and in 2 eyes treated with drug-free polymer. CONCLUSION: Injectable bioerodible polymer with 5-Fu may significantly prevent bleb scarring and subsequent blockage of the filtering tract, enhance success rate of filtering surgery in rabbits and reduce the toxicity of 5-Fu. This drug delivery system has considerable value for clinical use.

Animals↗
PubMed2004

Photochemical drug and gene delivery.

The utilization of macromolecules in the therapy of cancer and other diseases is becoming increasingly relevant. Recent advances in molecular biology and biotechnology have made it possible to improve the targeting and design of cytotoxic agents or DNA complexes for clinical applications. In many cases, to achieve the desired biological effect of these macromolecules, internalization to the cell cytosol is crucial. Although new delivery systems have improved the cellular uptake of macromolecules, tissue penetration, cellular uptake and efficient transfer of the molecules into the cytosol of target cells are still fundamental obstacles. At an intracellular level, the greatest obstruction for cytosolic release of the therapeutic molecule is the membrane barrier of endocytic vesicles. Photochemical activation and delivery of drugs and genes are novel technologies to overcome these obstacles. The background theory to these technologies is described in this review and recent data in this field are discussed.

Animals↗
PubMed1989

Molecular cloning of cDNA encoding the p70 (Ku) lupus autoantigen.

The Ku (p70/p80) autoantigen consists of two phosphoproteins of molecular mass approximately 70,000 and 80,000 forming a macromolecular complex that binds DNA. Autoantibodies from a patient with systemic lupus erythematosus were used to isolate cDNA clones encoding the human approximately 70-kDa Ku antigen (p70) from a lambda gt11 expression library. The deduced amino acid sequence of p70 consisted of 609 amino acid residues and was confirmed by partial amino acid sequencing. The protein contains two acidic domains of 61 residues (31% Glu + Asp) and 19 residues (53% Glu + Asp) that are similar in size and charge to those found in a number of proteins involved in transcriptional activation. The 61-residue acidic region is rich in serine, raising the possibility that its charge might be modulated by phosphorylation. The predicted amino acid sequence also contains two regions with periodic repeats of either leucine alone, or leucine alternating with serine every seventh position. The latter repeat displays sequence and secondary structural similarities with the "leucine zipper" regions of the c-myc and v-myc oncogene products. The p70 antigen does not appear to have extensive sequence homology with the 80-kDa Ku autoantigen based on analysis of RNA blots and immunological criteria. A major antigenic determinant or determinants recognized by human autoantibodies is located near a leucine repeat on the carboxyl-terminal 190 amino acid residues of p70.

Amino Acid Sequence↗
PubMed1989

Functional labeling of insulin receptor subunits in live cells. Alpha 2 beta 2 species is the major autophosphorylated form.

Both receptor subunits were functionally labeled in order to provide methods allowing, in live cells and in broken cell systems, concomitant evaluation of the insulin receptor dual function, hormone binding, and kinase activity. In cell-free systems, insulin receptors were labeled on their alpha-subunit with 125I-photoreactive insulin, and on their beta-subunit by autophosphorylation. Thereafter, phosphorylated receptors were separated from the complete set of receptors by means of anti-phosphotyrosine antibodies. Using this approach, a subpopulation of receptors was found which had bound insulin, but which were not phosphorylated. Under nonreducing conditions, receptors appeared in three oligomeric species identified as alpha 2 beta 2, alpha 2 beta, and alpha 2. Mainly the alpha 2 beta 2 receptor species was found to be phosphorylated while insulin was bound to alpha 2 beta 2, alpha 2 beta, and alpha 2 forms. In live cells, biosynthetic labeling of insulin receptors was used. Receptors were first labeled with [35S]methionine. Subsequently, the addition of insulin led to receptor autophosphorylation by virtue of the endogenous ATP pool. The total amount of [35S]methionine-labeled receptors was precipitated with antireceptor antibodies, whereas with anti-phosphotyrosine antibodies, only the phosphorylated receptors were isolated. Using this approach we made the two following key findings: (1) Both receptor species, alpha 2 beta 2 and alpha 2 beta, are present in live cells and in comparable amounts. This indicates that the alpha 2 beta form is not a degradation product of the alpha 2 beta 2 form artificially generated during receptor preparation. (2) The alpha 2 beta 2 species is the prevalently autophosphorylated form.

Affinity Labels↗
PubMed1988

ATP-dependent proteolysis of hemoglobin alpha chains in beta-thalassemic hemolysates is ubiquitin-dependent.

beta-Thalassemia is an inherited human disorder which is characterized by a deficient production of hemoglobin beta chains and an attendant accumulation of structurally normal alpha chains in the erythropoietic cells. The objective of this work is to understand the mechanism of intracellular proteolysis of these excess alpha chains. Dialyzed stroma-free hemolysates (32 mg/ml hemoglobin) of blood reticulocytes from four individuals with beta-thalassemia intermedia were incubated with human hemoglobin 3H-alpha chains (0.13 mg/ml) at 37 degrees C in a reaction mixture supporting protein degradation. In the presence of ATP and an ATP-generating system, the fraction of alpha chain 3H radioactivity made acid-soluble after 4 h ranged from 4 to 12% among the different hemolysates; in the absence of ATP or when hemolysates of normal human erythrocytes were used, only 1 to 2% of the 3H-alpha chains were degraded. It is likely that the ATP-dependent proteolysis of 3H-alpha chains in the beta-thalassemic hemolysates corresponds to the ATP-dependent turnover of newly synthesized soluble alpha chains in intact beta-thalassemic reticulocytes observed previously (Shaeffer, J. (1983) J. Biol. Chem. 258, 13172-13177) because of the following similarities between the two systems: (a) free 3H-alpha chains, but not 3H-labeled tetrameric hemoglobins, were readily degraded; (b) the rate of 3H-alpha chain proteolysis in the cell-free system was at least one-half of that observed for the turnover of newly synthesized alpha chains (t1/2 approximately 6 h) in intact cells; and (c) the ATP-dependent proteolytic activity of both systems was inhibited substantially by certain chemical agents (orthovanadate, N-ethylmaleimide, o-phenanthroline, and phenylmethylsulfonyl fluoride) but only slightly, if at all, by others (epsilon-aminocaproic acid and leupeptin). When excess human erythrocyte ubiquitin was added to the beta-thalassemic cell-free systems, a stimulation in ATP-dependent proteolysis of 3H-alpha chains ranging from 30 to 58% was observed. Conversely, addition of from 1.25 to 2.50 mg/ml affinity-purified rabbit antiubiquitin inhibited almost all (greater than 90%) of the ATP-dependent 3H-alpha chain proteolysis; in control experiments, antiubiquitin neutralized with excess ubiquitin inhibited only 13 to 30% of the total (including ubiquitin-stimulated) ATP-dependent proteolysis.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗