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At least 433 records · Page 24Linked to original sources

PubMed2006

Binding assays with artificial tethered membranes using surface plasmon resonance.

Surface sensitive optical techniques based on surface plasmon resonance have become interesting for biosciences in the context of biorecognition and binding studies at functional surfaces. We use surface plasmon resonance spectroscopy (SPS) in combination with surface plasmon enhanced fluorescence spectroscopy (SPFS) for the characterization of interaction processes associated with biomembranes. The biological membrane is mimicked by a tethered membrane consisting of a planar lipid bilayer attached to a gold surface via a hydrophilic anchor peptide. The interaction between membrane-bound hydrophobic compounds and free hydrophilic molecules is monitored in real-time and with high sensitivity and selectivity by combined SPS/SPFS. In this review we shortly discuss the principles of surface plasmon resonance and its utilization in SPS and SPFS. A detailed description of the required instrumentation for combined SPS and SPFS is presented. Furthermore, we outline the design of a binding assay with a tethered bilayer and the procedure of the artificial membrane system built-up is delineated. We also present examples that demonstrate the potential of combined SPS/SPFS assays with artificial tethered membranes. The method provides insight into the interaction of integral membrane proteins with various hydrophilic ligands and the specific recognition of small lipophilic molecules by soluble proteins.

Amino Acid Sequence↗

PubMed2006

Systems cell biology based on high-content screening.

A new discipline of biology has emerged since 2004, which we call "systems cell biology" (SCB). Systems cell biology is the study of the living cell, the basic unit of life, an integrated and interacting network of genes, proteins, and myriad metabolic reactions that give rise to function. SCB takes advantage of high-content screening platforms, but delivers more detailed profiles of cellular systemic function, including the application of advanced reagents and informatics tools to sophisticated cellular models. Therefore, an SCB profile is a cellular systemic response as measured by a panel of reagents that quantify a specific set of biomarkers.

Animals↗

PubMed1998

Adrenergic and cholinergic inputs in preoptic area of rats interact for sleep-wake thermoregulation.

Isolated studies have shown that both norepinephrine and acetylcholine into the medial preoptico-anterior hypothalamic area tonically regulate sleep-wake and body temperature. A possible interaction between these neurotransmitters for the regulation of such functions has been investigated in this study. To study this interaction a combination of either prazosin and carbachol or, scopolamine and methoxamine was injected into the medial preoptico-anterior hypothalamic area and the effect on sleep, wake, and rectal temperature recorded simultaneously. The combination of chemicals were selected based on our previous studies where it was observed that each of the chemicals in a combination had opposite effects. It was observed that injection of the combination expressed a resultant summated effects of individual component chemicals when injected in isolation (observed in previous studies). Because effect of neither of the chemicals in the combination was dominant, the results suggest an interaction and integration of the adrenergic and cholinergic inputs in the medial preoptico-anterior hypothalamic area for the regulation of sleep-wakefulness and body temperature.

Acetylcholine↗

PubMed2001

Functional testing of putative oligopeptide permease (Opp) proteins of Borrelia burgdorferi: a complementation model in opp(-) Escherichia coli.

Studies of the protein function of Borrelia burgdorferi have been limited by a lack of tools for manipulating borrelial DNA. We devised a system to study the function of a B. burgdorferi oligopeptide permease (Opp) orthologue by complementation with Escherichia coli Opp proteins. The Opp system of E. coli has been extensively studied and has well defined substrate specificities. The system is of interest in B. burgdorferi because analysis of its genome has revealed little identifiable machinery for synthesis or transport of amino acids and only a single intact peptide transporter operon. As such, peptide uptake may play a major role in nutrition for the organism. Substrate specificity for ABC peptide transporters in other organisms is determined by their substrate binding protein. The B. burgdorferi Opp operon differs from the E. coli Opp operon in that it has three separate substrate binding proteins, OppA-1, -2 and -3. In addition, B. burgdorferi has two OppA orthologues, OppA-4 and -5, encoded on separate plasmids. The substrate binding proteins interact with integral membrane proteins, OppB and OppC, to transport peptides into the cell. The process is driven by two ATP binding proteins, OppD and OppF. Using opp-deleted E. coli mutants, we transformed cells with B. burgdorferi oppA-1, -2, -4 or -5 and E. coli oppBCDF. All of the B. burgdorferi OppA proteins are able to complement E. coli OppBCDF to form a functional Opp transport system capable of transporting peptides for nutritional use. Although there is overlap in substrate specificities, the substrate specificities for B. burgdorferi OppAs are not identical to that of E. coli OppA. Transport of toxic peptides by B. burgdorferi grown in nutrient-rich medium parallels borrelial OppA substrate specificity in the complementation system. Use of this complementation system will pave the way for more detailed studies of B. burgdorferi peptide transport than currently available tools for manipulating borrelial DNA will allow.

Amino Acid Sequence↗

PubMed1998

Structure, dynamics and composition of the lipid-protein interface. Perspectives from spin-labelling.

Implications of the data on lipid-protein interactions involving integral proteins that are obtained from EPR spectroscopy with spin-labelled lipids in membranes are reviewed. The lipid stoichiometry, selectivity and exchange dynamics at the lipid-protein interface can be determined, in addition to information on the configuration and rotational dynamics of the protein-associated lipid chains. These parameters, particularly the stoichiometry and selectivity, are directly related to the intramembranous structure and degree of oligomerisation of the integral protein, and conversely may be used to study the state of assembly of such proteins in the membrane. Insertion of proteins into membranes can be studied by analogous methods. Comparison with the results obtained from integral proteins helps to define the extent of membrane penetration and degree of transmembrane crossing that are relevant to protein translocation mechanisms.

Amino Acid Sequence↗

PubMed1997

Exocytotic release from neuronal cell bodies, dendrites and nerve terminals in sympathetic ganglia of the rat, and its differential regulation.

Stimulant-induced exocytosis has been demonstrated in sympathetic ganglia of the rat by in vitro incubation of excised ganglia in the presence of tannic acid, which stabilizes vesicle cores after their exocytotic release. Sites of exocytosis were observed along non-synaptic regions of the surfaces of neuron somata and dendrites, including regions of dendrosomatic and dendrodendritic apposition, as well as along the surfaces of nerve terminals About half the exocytoses associated with nerve terminals were parasynaptic or synaptic, and these appeared mostly to arise from the presynaptic terminal, but occasionally from the postsynaptic element. The results demonstrated that the neurons of sympathetic ganglia release materials intraganglionically in response to stimulation, that release from different parts of the neuron is subject to independent regulation, at least via cholinergic receptors, and that release is partly diffuse, potentially mediating autocrine or paracrine effects, and partly targeted toward other neurons, but that the latter mode is not necessarily, and not evidently, synaptic. Specifically, exocytosis from all locations increased significantly during incubation in modified Krebs' solution containing 56 nm potassium. Observation of the effects of cholinergic agonists (nicotine, carbachol, oxotremorine) and antagonists (atropine, AF-DX 116) showed that nicotinic and muscarinic excitation each, independently, increased the incidence of exocytosis from somata and dendrites. Exocytosis from nerve endings was not altered by nicotine, but was enhanced or, at high initial rates of exocytosis, decreased, by muscarinic stimulation. Evidence was obtained for muscarinic auto-inhibition of exocytosis from nerve terminals, occurring under basal incubation conditions, and for a muscarinic excitatory component of somatic exocytosis, elicitable by endogenous acetylcholine. The M2-selective muscarinic antagonist AF-DX 116 was found to modify the exocytotic response of the dendrites to oxotremorine, widening the range of its variation; this effect is consistent with recent evidence for the presence of M2-like muscarinic binding sites, in addition to M1-like binding, upon these dendrites [Ramcharan E. J. and Matthews M. R. (1996) Neuroscience 71, 797-832]. Over all conditions, disproportionately more sites of somatic and dendritic exocytosis were found to be located in regions of dendrosomatic and dendrodendritic apposition than would be expected from the relative extent of the neuronal surface occupied by these relationships. Such mechanisms of intraganglionic release may be expected to contribute to the regulation and integration of the behaviour of the various functionally distinctive populations of neurons in these ganglia, by autocrine, paracrine, and focal, neuroneuronal, routes of action. Similar phenomena of exocytotic soma-dendritic release might prove to subserve integrative neuroneuronal interactions more widely throughout the nervous system.

Animals↗

PubMed2001

Dynamic representations and generative models of brain function.

The main point made in this article is that the representational capacity and inherent function of any neuron, neuronal population or cortical area is dynamic and context-sensitive. This adaptive and contextual specialisation is mediated by functional integration or interactions among brain systems with a special emphasis on backwards or top-down connections. The critical notion is that neuronal responses, in any given cortical area, can represent different things at different times. Our argument is developed under the perspective of generative models of functional brain architectures, where higher-level systems provide a prediction of the inputs to lower-level regions. Conflict between the two is resolved by changes in the higher-level representations, driven by the resulting error in lower regions, until the mismatch is 'cancelled'. In this model the specialisation of any region is determined both by bottom-up driving inputs and by top-down predictions. Specialisation is therefore not an intrinsic property of any region but depends on both forward and backward connections with other areas. Because these other areas have access to the context in which the inputs are generated they are in a position to modulate the selectivity or specialisation of lower areas. The implications for 'classical' models (e.g., classical receptive fields in electrophysiology, classical specialisation in neuroimaging and connectionism in cognitive models) are severe and suggest these models provide incomplete accounts of real brain architectures. Generative models represent a far more plausible framework for understanding selective neurophysiological responses and how representations are constructed in the brain.

Brain Injuries↗

PubMed2000

The EPR study of LDL perturbed by alcohols with different molecular architecture.

In this work, the interaction of different isomers of lower aliphatic alcohols with LDL representing a complex macromolecular assembly is investigated in vitro. Emphasis is given to the comparison of the impact of molecular architecture of methanol, ethanol, propanol (n-, iso-) and butanol (n-, iso-, sec-, tert-) in perturbing the lipid-protein assembly. The geometrical characteristics as well as the lipophilicity of the respective alcohol are considered. The EPR method combined with the spin labeling of both the apoB and the lipid monolayer allowed parallel detection of changes provoked in both phases. In addition to the change in protein environment, the spectral decomposition of the experimental data revealed a decrease in lipid ordering with the increasing concentration of the alcohols. This phenomenon for aliphatic alcohols is linearly correlated with the equal volume occupation (EVO) of alcohol in LDL. The results support the molecular mechanism of alcohol action through its interference with the lipid-protein interactions in LDL, which could be applicable to the molecular mechanism of alcohol interaction with integral membrane proteins.

1-Propanol↗

PubMed2003

Synaptic gating: the potential to open closed doors.

Synaptic gating is normally thought to be a mechanism for excluding synaptic input, but three recent studies show how the resting membrane potential interacts with integrative properties to act as a permissive synaptic gate.

Animals↗

PubMed2003

Fibroblast biology in three-dimensional collagen matrices.

Research on fibroblast biology in three-dimensional collagen matrices offers new opportunities to understand the reciprocal and adaptive interactions that occur between cells and surrounding matrix in a tissue-like environment. Such interactions are integral to the regulation of connective tissue morphogenesis and dynamics that characterizes tissue homeostasis and wound repair. During fibroblast-collagen matrix remodeling, mechanical signals from the remodeled matrix feed back to modulate cell behavior in an iterative process. As mechanical loading (tension) within the matrix increases, the mechanisms used by cells to remodel the matrix change. Fibroblasts in matrices that are under tension or relaxed respond differently to growth factor stimulation, and switching between mechanically loaded and unloaded conditions influences whether cells acquire proliferative/biosynthetic active or quiescent/resting phenotypes.

Animals↗

PubMed2000

The role of the oocyte in folliculogenesis.

Novel regulatory proteins have been identified within oocytes that are crucially involved in folliculogenesis. One of the most exciting oocyte signaling molecules is a novel member of the transforming growth factor beta (TGF-beta) superfamily, growth differentiation factor 9 (GDF-9). Loss-of-function studies have established that GDF-9 is obligatory for proper folliculogenesis and fertility in female mice. The current challenges are to understand how oocyte morphogens regulate folliculogenesis and how their actions and interactions are integrated into the overall processes of physiology and pathophysiology. Who would have thought that oocyte morphogens would be so crucial for reproduction?

Animals↗

PubMed2001

Mitochondrial DNA in human malignancy.

Alterations in expression of mitochondrial DNA (mtDNA)-encoded polypeptides required for oxidative phosphorylation and cellular ATP generation may be a general characteristic of cancer cells. Mitochondrial DNA has been proposed to be involved in carcinogenesis because of high susceptibility to mutations and limited repair mechanisms in comparison to nuclear DNA. Since mtDNA lacks introns, it has been suggested that most mutations will occur in coding sequences and subsequent accumulation of mutations may lead to tumor formation. The mitochondrial genome is dependent upon the nuclear genome for transcription, translation, replication and repair, but precise mechanisms for how the two genomes interact and integrate with each other are poorly understood. In solid tumors, elevated expression of mtDNA-encoded subunits of the mitochondrial electron respiratory chain may reflect mitochondrial adaptation to perturbations in cellular energy requirements. In this paper, we review mitochondrial genomic aberrations reported in solid tumors of the breast, colon, stomach, liver, kidney, bladder, head/neck and lung as well as for hematologic diseases such as leukemia, myelodysplastic syndrome and lymphoma. We include data for elevated expression of mtDNA-encoded electron respiratory chain subunits in breast, colon and liver cancers and also the mutations reported in cancers of the colon, stomach, bladder, head/neck and lung. Finally, we examine the role of reactive oxygen species (ROS) generated by mitochondria in the process of carcinogenesis.

Breast Neoplasms↗

PubMed2001

Split luciferase as an optical probe for detecting protein-protein interactions in mammalian cells based on protein splicing.

We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp. PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin served to monitor the interaction between IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity. This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system.

Animals↗

PubMed2001

Structural properties of docosahexaenoyl phospholipid bilayers investigated by solid-state 2H NMR spectroscopy.

Polyunsaturated lipids in cellular membranes are known to play key roles in such diverse biological processes as vision, neuronal signaling, and apoptosis. One hypothesis is that polyunsaturated lipids are involved in second messenger functions in biological signaling. Another current hypothesis affirms that the functional role of polyunsaturated lipids relies on their ability to modulate physical properties of the lipid bilayer. The present research has employed solid-state 2H NMR spectroscopy to acquire knowledge of the molecular organization and material properties of polyunsaturated lipid bilayers. We report measurements for a homologous series of mixed-chain phosphatidylcholines containing a perdeuterated, saturated acyl chain (n:0) at the sn-1 position, adjacent to docosahexaenoic acid (DHA, 22:6omega3) at the sn-2 position. Measurements have been performed on fluid (L(alpha))-state multilamellar dispersions as a function of temperature for saturated acyl chain lengths of n = 12, 14, 16, and 18 carbons. The saturated sn-1 chains are therefore used as an intrinsic probe with site-specific resolution of the polyunsaturated bilayer structure. The 2H NMR order parameters as a function of acyl position (order profiles) have been analyzed using a mean-torque potential model for the chain segments, and the results are discussed in comparison with the homologous series of disaturated lipid bilayers. At a given absolute temperature, as the sn-1 acyl length adjacent to the sn-2 DHA chain is greater, the order of the initial chain segments increases, whereas that of the end segments decreases, in marked contrast with the corresponding disaturated series. For the latter, the order of the end segments is practically constant with acyl length, thus revealing a universal chain packing profile. We find that the DHA-containing series, while more complex, is still characterized by a universal chain packing profile, which is shifted relative to the homologous saturated series. Moreover, we show how introduction of DHA chains translates the order profile along the saturated chains, making more disordered states accessible within the bilayer central region. As a result, the area per lipid headgroup is increased as compared to disaturated bilayers. The systematic analysis of the 2H NMR data provides a basis for studies of lipid interactions with integral membrane proteins, for instance in relation to characteristic biological functions of highly unsaturated lipid membranes.

Deuterium↗

PubMed2004

Surface treatments and roughness properties of Ti-based biomaterials.

Nowadays, the use of implanted devices is a well-acknowledged practice in the field of orthopaedic and dental surgery. Scientific research and clinical experience suggest that the successful exploitation of these devices mainly depends on osseointegration, considered as both anatomical congruency and load-bearing capacity. Indeed, the osseointegration process is influenced by a wide range of factors: anatomical location, implant size and design, surgical procedure, loading effects, biological fluids, age and sex, and, in particular, surface characteristics. For this reason, several attempts have been aimed at modifying implant surface composition and morphology to optimise implant-to-bone contact and improve integration. Preliminary interactions between implanted materials and biological environment are deemed to be governed by the surface properties; they control the amount and quality of cell adhesion on the surface and, consequently, cell/tissue growth. Thus, surface properties govern new bone tissue formation and implant osseointegration. This paper reviews the state of art in the field of physical, chemical and biochemical treatments commonly used on Ti-based biomaterials for the production of biomedical devices. In particular, roughness characteristics due to physical and chemical techniques are investigated; the development of biologically active surfaces by means of biochemical functionalisation is also considered.

Adsorption↗

PubMed2005

A role for LEDGF/p75 in targeting HIV DNA integration.

HIV DNA integration is favored in active genes, but the underlying mechanism is unclear. Cellular lens epithelium-derived growth factor (LEDGF/p75) binds both chromosomal DNA and HIV integrase, and might therefore direct integration by a tethering interaction. We analyzed HIV integration in cells depleted for LEDGF/p75, and found that integration was (i) less frequent in transcription units, (ii) less frequent in genes regulated by LEDGF/p75 and (iii) more frequent in GC-rich DNA. LEDGF is thus the first example of a cellular protein controlling the location of HIV integration in human cells.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Applied systems biology and malaria.

One of the goals of systems-biology research is to discover networks and interactions by integrating diverse data sets. So far, systems-biology research has focused on model organisms, which are well characterized and therefore suited to testing new methods. Systems biology has great potential for use in the search for therapies for disease. Here, the potential of systems-biology approaches in the search for new drugs and vaccines to treat malaria is examined.

Animals↗

PubMed2023

Structural basis for stabilisation of the RAD51 nucleoprotein filament by BRCA2.

The BRCA2 tumour suppressor protein preserves genomic integrity via interactions with the DNA-strand exchange RAD51 protein in homology-directed repair. The RAD51-binding TR2 motif at the BRCA2 C-terminus is essential for protection and restart of stalled replication forks. Biochemical evidence shows that TR2 recognises filamentous RAD51, but existing models of TR2 binding to RAD51 lack a structural basis. Here we used cryo-electron microscopy and structure-guided mutagenesis to elucidate the mechanism of TR2 binding to nucleoprotein filaments of human RAD51. We find that TR2 binds across the protomer interface in the filament, acting as a brace for adjacent RAD51 molecules. TR2 targets an acidic-patch motif on human RAD51 that serves as a recruitment hub in fission yeast Rad51 for recombination mediators Rad52 and Rad55-Rad57. Our findings provide a structural rationale for RAD51 filament stabilisation by BRCA2 and reveal a common recruitment mechanism of recombination mediators to the RAD51 filament.

Humans↗