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At least 721 records · Page 40Linked to original sources
PubMed2005

Interaction between hormonal signaling pathways in Drosophila melanogaster as revealed by genetic interaction between methoprene-tolerant and broad-complex.

Juvenile hormone (JH) regulates insect development by a poorly understood mechanism. Application of JH agonist insecticides to Drosophila melanogaster during the ecdysone-driven onset of metamorphosis results in lethality and specific morphogenetic defects, some of which resemble those in mutants of the ecdysone-regulated Broad-Complex (BR-C). The Methoprene-tolerant (Met) bHLH-PAS gene mediates JH action, and Met mutations protect against the lethality and defects. To explore relationships among these two genes and JH, double mutants were constructed between Met alleles and alleles of each of the BR-C complementation groups: broad (br), reduced bristles on palpus (rbp), and 2Bc. Defects in viability and oogenesis were consistently more severe in rbp Met or br Met double mutants than would be expected if these genes act independently. Additionally, complementation between BR-C mutant alleles often failed when MET was absent. Patterns of BRC protein accumulation during metamorphosis revealed essentially no difference between wild-type and Met-null individuals. JH agonist treatment did not block accumulation of BRC proteins. We propose that MET and BRC interact to control transcription of one or more downstream effector genes, which can be disrupted either by mutations in Met or BR-C or by application of JH/JH agonist, which alters MET interaction with BRC.

Alleles↗
PubMed1999

Identification of ribonucleoprotein (RNP)-specific protein interactions using a yeast RNP interaction trap assay (RITA).

We describe an adaptation of the yeast three-hybrid system that allows the reconstitution in vivo of tripartite (protein-RNA-protein) ribonucleoproteins (RNPs). To build and try this system that we called RNP interaction trap assay (RITA), we used as a model the autoantigenic Ro RNPs. hY RNAs bear distinct binding sites for Ro60 and La proteins, and Ro RNPs are thus physiologically tripartite (Ro60/hY RNA/La). Using recombinant La (rLa) and Ro60 (rRo60) proteins and recombinant hY RNAs (rhY) co-expressed in yeast, we found that RNPs made of rRo60/rhY/rLa were readily reassembled. Reconstitution of tripartite RNPs was critically dependent on the presence of an appropriate Ro60 binding site on the recombinant RNA. The RITA assay was further used to detect (rRo60/rhY RNP)-binding proteins from a HeLa cell cDNA library, allowing specific identification of La and of a novel Ro RNP-binding protein (RoBPI) in more than 70% of positive clones. RITA assay may complement already available two- and three-hybrid systems to characterize RNP-binding proteins by allowing the in vivo identification of interactions strictly dependent upon the simultaneous presence of a protein and of its cognate RNA.

Binding Sites↗
PubMed1986

Kinetics of protein-nucleic acid interactions: use of salt effects to probe mechanisms of interaction.

The kinetics of protein-nucleic acid interactions are discussed with particular emphasis on the effects of salt concentration and valence on the observed rate constants. A general review is given of the use of experimentally determined salt dependences of observed kinetic parameters as a tool to probe the mechanism of interaction. Quantitative analysis of these salt dependences, through the application of polyelectrolyte theory, can be used to distinguish reactions which occur in a single step from those reactions which involve distinct intermediates. For those rate constants which display a large salt dependence, in either the association or dissociation reaction, this is due to the high concentration of counterions (e.g., Na+) in the vicinity of the nucleic acid which are subsequently released (or bound in the case of dissociation) at some point before the rate limiting step of the reaction. A general discussion of other features which affect protein-nucleic acid kinetics, such as nucleic acid length and the ratio of nonspecific to specific DNA binding sites (in the case of sequence specific binding proteins), is also given. The available data on the nucleic acid binding kinetics of small ligands (ions, dyes, oligopeptides), nonspecific binding proteins (T4 gene 32 protein, fd gene 5 and Escherichia coli SSB), and sequence specific binding proteins (lac repressor, RNA polymerase, Eco RI restriction endonuclease) are discussed with emphasis on the interpretation of the experimentally determined salt dependences.

DNA↗
PubMed1999

Immunohistochemical evidence for the brevican-tenascin-R interaction: colocalization in perineuronal nets suggests a physiological role for the interaction in the adult rat brain.

Brevican is one of the most abundant chondroitin sulfate proteoglycans in the adult rat brain. We have recently shown that the C-type lectin domain of brevican binds fibronectin type III domains 3-5 of tenascin-R. Here we report strong evidence for a physiological basis for this interaction. Substantial brevican immunoreactivity was detected in a number of nuclei and in the reticular formations throughout the midbrain and hindbrain, including, but not limited to, the deep cerebellar nuclei, the trapezoid body, the red nucleus, the oculomotor nucleus, the vestibular nucleus, the cochlear nucleus, the gigantocellular reticular nucleus, the motor trigeminal nucleus, and the lateral superior olive. Most of the brevican immunoreactivity exhibited pericellular and reticular staining patterns. In almost all of these sites, brevican immunoreactivity colocalized with that of tenascin-R, which was also substantially codistributed with versican, another member of the lectican family. Detailed analysis revealed that the pericellular staining of brevican resembled that in perineuronal nets in which tenascin-R has been localized. Immunoelectron microscopy identified brevican immunoreactivity in the intercellular spaces surrounding presynaptic boutons and on their surfaces, but not in the synaptic clefts or in their immediate vicinity, a distribution pattern consistent with perineuronal nets. Taken together, our results provide strong evidence that the previously reported interactions between brevican and tenascin-R may play a functional role within the perineuronal nets.

Age Factors↗
PubMed2000

Aryloxypropanolamine and catecholamine ligand interactions with the beta(1)-adrenergic receptor: evidence for interaction with distinct conformations of beta(1)-adrenergic receptors.

Pharmacological responses to aryloxypropanolamines were examined in cells expressing rat or human beta(1)-adrenergic receptors (ARs) using adenylyl cyclase assays. The aryloxypropanolamines CGP 12177 and LY 362884, originally developed as beta(3)-AR agonists, were found to stimulate the beta(1)-AR. Interestingly, both CGP 12177 and LY 362884 exhibited an anomalous biphasic effect on beta(1)-AR. Low concentrations of either CGP 12177 or LY 362884 potently blocked isoproterenol-induced stimulation of beta(1)-AR, whereas higher concentrations of these compounds stimulated the beta(1)-AR. The unusual interaction of these aryloxypropanolamine ligands with the beta(1)-AR was further characterized using beta-AR antagonists. Activation of beta(1)-AR by CGP 12177 or LY 362884 was observed to be significantly more resistant to blockade by beta-AR antagonists compared with activation by catecholamines. These results suggest that catecholamines and aryloxypropanolamines interact with distinct active conformations of the beta(1)-AR: a state that is responsive to catecholamines and is blocked with high affinity by CGP 12177 and LY 362884, and a novel state that is activated by aryloxypropanolamines but is resistant to blockade by standard beta-AR antagonists. Moreover, dependence of antagonist affinity on agonist structure is unprecedented, and its implications on the use of beta-AR agonists such as CGP 12177 in receptor classification are discussed.

Adenylyl Cyclases↗
PubMed1975

Proton magnetic relaxation studies of the interaction of D-xylose and xylitol with D-xylose isomerase. Characterization of metal-enzyme-substrate interactions.

The interaction of D-xylose isomerase purified from two sources with Mn2+ and D-xylose or the competitive inhibitor xylitol has been examined by nuclear magnetic resonance. A greater paramagnetic effect of enzyme-bound Mn2+ on the alpha anomer of D-xylose than on the beta anomer was observed, providing independent evidence for the specificity of D-xylose isomerase for the alpha anomeric form of D-xylose. The exchange rate of alpha-D-xylose into the ternary complex, determined from the normalized paramagnetic contribution to the transverse relaxation rate (1/fT2p) of the carbon 1 proton of alpha-D-xylose, exceeds Vmax for the enzymatic reaction by 3 orders of magnitude. The amount of xylitol necessary to displace alpha-D-xylose from the substrate-enzyme-Mn2+ complex is consistent with the Km value for alpha-D-xylose and the inhibitor constant Ki for xylitol previously determined by the methods of enzyme kinetics. These results suggest that the NMR experiments observe complexes of D-xylose isomerase which are kinetically and thermodynamically competent to participate in catalysis. From the frequency dependence of the paramagnetic contribution to the longitudinal relaxation rate (1/T1p) of the carbon 1 proton of alpha-D-xylose, the correlation time (tauc) which modulates the dipolar interaction between enzyme-bound Mn2+ and alpha-D-xylose has been determined (5.1 x 1o(-10) s). From these observations a range of calculated distances between enzyme-bound Mn2+ and the carbon 1 proton of alpha-D-xylose (9.1 +/- 0.7 A) has been found. The enzyme-bound Mn2+ has comparable effects on the carbon 1, carbon 2, and carbon 5 protons of alpha-D-xylose, suggesting that these protons of the enzyme-bound substrate are equidistant from the bound Mn2+. A similar distance (9.4 +/- 0.7 A) between the enzyme-bound Mn2+ and the terminal methylene protons of xylitol, an analog of the open chain intermediate in the reaction, has been determined. The results of the present substrate relaxation and previous water relaxation studies suggest that two small ligands such as water molecules or a large portion of the protein intervene between the bound metal ion and the bound substrate in the active ternary complex.

Binding Sites↗
PubMed2005

Disrupted Schwann cell-axon interactions in peripheral nerves of mice with altered L1-integrin interactions.

The cell adhesion molecule L1 is important for peripheral nerve development. Mice lacking the 6th Ig domain of L1 (L1-6D mice) lose L1 homophilic binding and RGD dependent LI-integrin binding [Itoh,K., Cheng, L., Kamei, Y., Fushiki, S., Kamiguchi, H., Gutwein, P.,Stoeck, A., Arnold, B., Altevogt, P., Lemmon, V., 2004. Brain development in mice lacking Li-L homophilic adhesion. J. Cell Biol.165, 145-154]. We examined the ultrastructure of sciatic nerves from L1-6D at postnatal day 7 and 8 weeks. Unmyelinated axons frequently detached at the edge of Schwann cells, and naked axons were observed. Myelin was thinner in L1-6D and abnormal, multiple axons wrapped in a single myelin sheath were routinely observed. Previous work has shown that L1 on axons interacts with a heterophilic binding partner on Schwann cells to facilitate normal peripheral nerve formation. Taken together, it is likely that L1 on axons binds integrins on Schwann cells, resulting in interactions between axons and Schwann cells that are essential for ensheathment and myelination.

Animals↗
PubMed1991

Subunit interactions control protein phosphatase 2A. Effects of limited proteolysis, N-ethylmaleimide, and heparin on the interaction of the B subunit.

Protein phosphatase 2A consists of a heterotrimeric complex composed of a catalytic subunit (C) and two associated subunits (A and B). Limited tryptic digestion of the heterotrimeric ABC form resulted in the selective degradation of the Mr = 55,000 B subunit to a 48-kDa polypeptide. The cleavage sites were determined to be within a 3-7-kDa region of the COOH terminus. Proteolysis led to dissociation of the B subunit from the enzyme complex and correlated with an increase in cardiac myosin light chain, smooth muscle myosin light chain peptide, and Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) phosphatase activity. Purification of the digestion products and native gel electrophoresis indicated that dissociation of the B subunit was responsible for the increase in phosphatase activity. Kinetic analyses with several substrates revealed that dissociation of the B subunit resulted in a 2-7-fold increase in Vmax and a 1.6-5 fold increase in Km. Proteolytic dissociation of the B subunit increased the sensitivity of protein phosphatase 2A to inhibition by okadaic acid. Inhibition of the trypsinized enzyme was very similar to that observed for the purified AC form of protein phosphatase 2A. Incubation of the ABC complex with N-ethylmaleimide resulted in dissociation of the C subunit and generation of an AB complex. Selective release of the C subunit indicated that the B subunit interacts directly with the A subunit and that one or more free sulfhydryls are required to maintain the heterotrimeric structure of protein phosphatase 2A. Treatment of the enzyme with heparin resulted in an increase in specific activity that was due to the release of the B subunit from the complex. These results provide evidence that the B subunit binds directly to the A subunit to modulate enzyme activity and substrate specificity and that the COOH-terminal region of this protein is important for interaction with the AC complex. Dissociation of the B subunit by polyanionic substances related to heparin may represent a mechanism for regulating the activity of this enzyme.

Amino Acid Sequence↗
PubMedDate not supplied

[Hydrophobic interactions of serine proteases with low molecular compounds: role of the S'2-site in substrate activation and interaction with serpines].

An attempt is made to simulate the P1-P'2 site of the reactive centre of protein inhibitors of serine proteases (serpines). On the basis of data from literature structure requirements are formulated and compound 1,5 bis-dibenzyl-aminopentane is synthesized. It may simultaneously interact with S1- and S'2-sites of chymotrypsin and contains no bonds adequate to the hydrolytic centre of proteinase. The compound is studied for its effect on hydrolysis of low-molecular substrates and proteins by chymotrypsin. Results obtained are discussed as well as the possible role of the S'2-binding site in the substrate activation of serine proteinases and their interaction with serpines.

Binding Sites↗
PubMedDate not supplied

Molecular interactions between ribosomal proteins--a study of S4-S9 interaction.

The ribosomal proteins S4 and S9 were isolated from the 30S ribosomal subunit of Escherichia coli to greater than 95% purity and characterized in the reconstitution buffer. Neither of the proteins indicated any tendency to self associate at 3 degrees C in the concentration range studied. At higher temperatures (greater than 20 degrees C), protein S9 forms a significant amount of a soluble aggregate as seen from the sedimentation velocity and sedimentation equilibrium experiments. From an analysis of the solution mixture of S4 and S9 at 1:1.08 molar concentration ratio by sedimentation velocity experiment, an s20,w value of 1.77 +/- 0.02S was obtained. A fast moving component which accounts for approximately 20% of the mass was also observed. Increasing the concentration of S9 does not alter the observed s20w value significantly for that component which could be followed. A detailed analysis of the data obtained at 3 degrees C from sedimentation equilibrium experiments on mixtures of the proteins indicated that a species of molecular weight greater than either of the two proteins was present. The proteins were found to interact with a mean equilibrium constant of association of 3.66 +/- 2.39 x 10(4) M-1 and a Gibbs free energy of interaction, delta Go = -5.8 kcal/mole at 3 degrees C in TMKD buffer. This information helps in understanding the energetics of the 30S ribosomal subunits of E. coli.

Escherichia coli↗
PubMed1991

Cognate interactions between helper T cells and B cells. VI. TGF-beta inhibits B cell activation and antigen-specific, physical interactions between Th and B cells.

TGF-beta is a 25-kDa homodimeric protein that has been shown to have multiple roles in the regulation of lymphocyte activation. Previous studies have shown that TGF-beta is an inhibitor of numerous T and B lymphocyte activities. This study shows that TGF-beta is able to inhibit Th- and mitogen-induced murine B cell proliferation, as well as mitogen-induced B cell cycle entry and immunoglobulin secretion. Acridine orange analysis established that TGF-beta inhibits the LPS-induced B cell transition from G0 to G1A phase of the cell cycle. Evaluation of the effects of TGF-beta on the interactions between Th and B cells showed that TGF-beta inhibited antigen-specific Th-B cell physical interactions. Flow cytometric data showed that the ability of TGF-beta to interfere with the formation of Th-B cell conjugates was not due to decreased expression of IgD, IgM, class I, class II, LFA-1, or ICAM1 by the B cell. Taken together, these data establish that TGF-beta is able to act at multiple sites within the immune system.

Animals↗
PubMed1991

[The synchronizing interaction and mechanism of the formation of the common rhythm of the cardiac pacemaker. I. The modeling of the interaction of 2 pacemaker elements].

The main physiologic hypotheses in establishing a common rhythm of the heart sino-atrial node (SAN) have been considered. A mathematical model of SAN which takes into account the interaction between pace-makers in pairs has been proposed. A quantitative description of common rhythm establishing taking into account interaction in pairs has been obtained, the expressions for the period of common rhythm, a delay and the rate of stimulation distribution have been found. A comparison of calculated and experimental data is given.

Action Potentials↗
PubMed1989

Requirement for delivery of signals by physical interaction and soluble factors from accessory cells in the induction of receptor-mediated T cell proliferation. Effectiveness of IFN-gamma modulation of accessory cells for physical interaction with T cells.

We analyzed the mechanism by which accessory cells support the induction of the proliferation of human peripheral blood T cells by a monoclonal anti-CD3 antibody, OKT3. Cross-linking of T cell receptor/CD3 complex by anti-CD3 coupled to latex beads and the addition of IL-1 are not enough to induce the IL-2 production and proliferation of T cells extensively depleted of accessory cells, while the addition of both the culture supernatant of macrophages or a monoblastic cell line, U937 cells, and the paraformaldehyde-fixed macrophages or U937 cells which had been precultured with interferon-gamma before fixation into the culture of the T cells with anti-CD3-latex did induce the T cell proliferation. Lack of the addition of either one of these did not induce the response. These results indicate that the signal(s) delivered by soluble factors released from the accessory cells and that delivered by the physical interaction between accessory cells and T cells are both required for the induction of IL 2 production and proliferation of T cells by anti-CD3-latex. Importantly, the macrophages or U937 cells had to be cultured with Con A-stimulated lymphocyte culture supernatant or IFN-gamma prior to fixation with paraformaldehyde, suggesting that a molecule(s) inducible on accessory cells surface by IFN-gamma or other lymphokine is necessary for the effective accessory cell-T cell interaction to induce the T cell response. It was further revealed that the activity of the culture supernatant of accessory cells may be mediated synergistically by IL 1 and a certain other factor(s) and was actually shown to be replaced by the combined addition of rIL-1 and rIL-6 but not by rIL-1 alone. The experimental system described here will be very useful for dissecting the accessory functions for T cell activation.

Adjuvants, Immunologic↗
PubMed1988

Drug-protein interactions. On the protein-protein interaction induced by levamisole in vitro. A mechanism hypothesis.

In the present communication, a model is reported in order to explain the aggregation of albumin induced by levamisole in vitro. The hypothesis suggests that the process of polymerization of albumins may include ligand-protein interaction as a biochemical catalysis, and covalent protein-protein interactions by a mechanism of disulfide-sulfhydryl interchange by intramolecular or intermolecular reactions.

Albumins↗
PubMed1987

Interaction of insulin with its receptor. I. Possible role of a histidine-arginine interaction.

The interaction of beef and pork insulin with its receptors on rat liver plasma membranes has been studied as a function of pH in tris buffer. The dissociation binding constant decreased from 6.5 to 1.2 nM as the pH was increased from 6.8 to 7.8. Analysis indicated that this was the result of the deprotonation of a single residue with a pK'A of 7.62 at 20 degrees C. The enthalpy change associated with this deprotonation was estimated to be -7,500 cal/mol. On the basis of these parameters it is suggested that this group is a histidine residue on the surface of the insulin receptor. The positively charged group on the insulin molecule which interacts with this histidine was not either of the N-terminal residues, nor the lysine at position B-29; by elimination, it appears to be the B-22 arginine residue.

Animals↗
PubMed1988

Temperature and ionic strength dependence of the subunit interactions in vertebrate skeletal myosin. A comparison of the interaction between the alkali light and heavy chains of mammalian and avian myosin.

The stability of the interaction of A1 in myosin and subfragment 1 isolated from fast-twitch mammalian and avian muscles with respect to temperature and ionic strength has been examined. This was done by determining the extent of exchange of the endogenous free A1 light chain into these proteins from the two species. Whereas the extent of exchange at 37 degrees C into mammalian S1, occurring after 60 min, is about 80% of the theoretically expected amount at physiological ionic conditions, the level of exchange observed with the avian S1 is significantly lower. However, close to the theoretical limit is observed for the avian S1 when exchange is done at 43 degrees C which is close to average avian body temperature. A similar dependence with temperature is observed in the case of exchanges into avian myosin. In the case of mammalian myosin, 50% of the theoretical exchange is observed at 37 degrees C under physiological ionic strength, whereas the level of exchange observed under these conditions with the avian protein is much lower in agreement with recent observations (Waller, G. S., and Lowey, S. (1985) J. Biol. Chem. 260, 14368-14373; Pastra-Landis, S. C., and Lowey, S. (1986) J. Biol. Chem. 261, 14811-14816). If, however, the exchanges are done at 43 degrees C in physiological ionic strength, significant extents of exchange can be observed in avian myosin. These results suggest that at physiological ionic and temperature conditions relevant for the source of myosin and S1 being investigated, the alkali light chains are in dynamic equilibrium between free and heavy chain associated states. Therefore, the failure to observe alkali light chain exchange in avian myosin at 37 degrees C appears to be related to the higher temperature stability of its interaction with the heavy chain.

Animals↗
PubMed1986

Interactions of the DNA polymerase and gene 4 protein of bacteriophage T7. Protein-protein and protein-DNA interactions involved in RNA-primed DNA synthesis.

Three proteins catalyze RNA-primed DNA synthesis on the lagging strand side of the replication fork of bacteriophage T7. Oligoribonucleotides are synthesized by T7 gene 4 protein, which also provides helicase activity. DNA synthesis is catalyzed by gene 5 protein of the phage, and processivity of DNA synthesis is conferred by Escherichia coli thioredoxin, a protein that is tightly associated with gene 5 protein. T7 DNA polymerase and gene 4 protein associate to form a complex that can be isolated by filtration through a molecular sieve. The complex is stable in 50 mM NaCl but is dissociated by 100 mM NaCl, a salt concentration that does not inhibit RNA-primed DNA synthesis. T7 DNA polymerase forms a stable complex with single-stranded M13 DNA at 50 mM NaCl as measured by gel filtration, and this complex requires 200 mM NaCl for dissociation, a salt concentration that inhibits RNA-primed DNA synthesis. Gene 4 protein alone does not bind to single-stranded DNA. In the presence of MgCl2 and dTTP or beta, gamma-methylene dTTP, a gene 4 protein-M13 DNA complex that is stable at 200 mM NaCl is formed. The affinity of DNA polymerase for both gene 4 protein and single-stranded DNA leads to the formation of a gene 4 protein-DNA polymerase-M13 DNA complex even in the absence of nucleoside triphosphates. However, the binding of each protein to DNA plays an important role in mediating the interaction of the proteins with each other. High concentrations of single-stranded DNA inhibit RNA-primed DNA synthesis by diluting the amount of proteins bound to each template and reducing the frequency of protein-protein interactions. Preincubation of gene 4 protein, DNA polymerase, and M13 DNA in the presence of dTTP forms protein-DNA complexes that most efficiently catalyze RNA-primed DNA synthesis in the presence of excess single-stranded competitor DNA.

DNA Replication↗
PubMed1986

Modelling the interaction of small organic molecules with biomacromolecules. I. Interaction of substituted pyridines with anti-3-azopyridine antibody.

An approach is presented for modelling the biological activity of organic molecules. This approach requires a consideration of the influence of all factors (topological, steric, hydrophobic, electronic) which determine the bioactivity. In this work, the interaction between substituted pyridines and antibodies generated by anti-3-azapyridine is studied. The stereoelectronic interactions are responsible for the reaction. Meta-positions to nitrogen are found to be the most probable positions for attack. The most likely reaction products are pi-complexes with charges transfer from the biomolecule to the pyridine derivatives followed by the formation of covalent-type bonds.

Antibodies↗