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At least 667 records · Page 37Linked to original sources
PubMed2000

Reduction of thymocyte numbers in transgenic mice expressing viral FLICE-inhibitory protein in a Fas-independent manner.

A viral FLIP (FLICE/caspase-8-Inhibitory Protein), equine herpesvirus type 2 E8 protein, has been shown to inhibit Death receptor-induced apoptosis by suppressing the activation of FLICE/caspase-8. We generated transgenic mice specifically expressing E8 in thymocytes under the control of lck-proximal promoter. Although E8-expressing thymocytes were resistant to Fas-mediated apoptosis, the total number of thymocytes in 4-8-week-old E8 transgenic mice was more than 3-fold less than that in control littermates. This reduction was also observed in E8 transgenic mice with a Fas-/- background suggesting the reduction to be independent of Fas. The thymocytes of the transgenic mice, however, could similarly respond to CD3-mediated stimulation, indicating that the reduction of thymocyte numbers might be independent of T cell receptor complex-mediated stimulation. Thus, the Death receptor-mediated signaling pathway is too complex to be regarded as only an executor for apoptosis.

Animals↗
PubMed1996

The use of null mutant mice to study complex learning and memory processes.

A number of neural substrates have been proposed to mediate complex learning and memory processes in mammalian organisms. One strategy for testing the involvement of a particular gene in learning and memory is to create a mouse line with a null mutation in that gene. Recently, embryonic stem cell-based gene-targeted homologous recombination techniques have been employed to create a number of such mutant mouse lines that do not express interesting candidate genes. These animals have been examined for impairments in several complex learning paradigms which are known to depend on the integrity of the hippocampus. In this review several complex learning and memory paradigms are described, the techniques to create null mutants are reviewed, and the results of recent studies with null mutants are described. Finally, the limitations for interpretation of behavioral data using null mutants are discussed.

Animals↗
PubMed2000

Recognition of preproteins by the isolated TOM complex of mitochondria.

A multisubunit complex in the mitochondrial outer membrane, the TOM complex, mediates targeting and membrane translocation of nuclear-encoded preproteins. We have isolated the TOM holo complex, containing the preprotein receptor components Tom70 and Tom20, and the TOM core complex, which lacks these receptors. The interaction of recombinant mitochondrial preproteins with both types of soluble TOM complex was analyzed. Preproteins bound efficiently in a specific manner to the isolated complexes in the absence of chaperones and lipids in a bilayer structure. Using fluorescence correlation spectroscopy, a dissociation constant in the nanomolar range was determined. The affinity was lower when the preprotein was stabilized in its folded conformation. Following the initial binding, the presequence was transferred into the translocation pore in a step that required unfolding of the mature part of the preprotein. This translocation step was also mediated by protease-treated TOM holo complex, which contains almost exclusively Tom40. Thus, the TOM core complex, consisting of Tom40, Tom22, Tom6 and Tom7, is a molecular machine that can recognize and partially translocate mitochondrial precursor proteins.

Binding Sites↗
PubMed2004

Transition metal half-sandwich complexes as redox mediators to glucose oxidase.

Chromium and manganese half-sandwich complexes are evaluated as mediators to glucose oxidase (GOx) since they are of similar size to ferrocene derivatives (sandwich complexes) and contain a single pi-ligand for interaction with the enzyme co-factor. A series of seven amino derivatives of [(eta-C(6)H(6))Cr(CO)(3)] were investigated of which only [[eta-C(6)Me(4)(NH(2))(2)]Cr(CO)(3)] (7), with the lowest oxidation potential of +40 mV (versus SCE), was found to display reversible electrochemistry. Small catalytic currents were recorded in the presence of GOx and glucose when complex (7) was incorporated in a screen-printed carbon electrode. Manganese cyclopentadienyl (Cp) half-sandwich complexes were found to be more effective GOx mediators and comparable in efficacy to ferrocene derivatives. A mediator rate constant k(M) of 2.1 x 10(5)M(-1)s(-1) was determined for the water-soluble complex [(eta-MeC(5)H(4))Mn(NO)(CN)(2)]Na (11) compared to a range of 3 x 10(4) to 8 x 10(6)M(-1)s(-1) previously determined for ferrocenes under the same experimental conditions. beta-Cyclodextrin (beta-cd) was found to be helpful in solubilising hydrophobic complexes such as [(eta-MeC(5)H(4))Mn(NO)(S(2)CNMe(2))] (15) and the neutral oxidised form of [MeCpMn(NO)[(SCCN)(2)]]NEt(4) (14), either directly as an inclusion adduct or in situ during cyclic voltammetry. Screen-printed amperometric electrodes, containing a mediator and GOx immobilised in an organic conducting carbon layer, were useful in assessing the mediation ability of complex (15) where aqueous insolubility precluded any kinetic studies with GOx in solution. This work was briefly extended to other oxidoreductase enzymes apart from GOx. Thus, rotating ring-disk voltammetry demonstrated that the beta-cd complex of compound (15) is also a useful mediator to Horseradish peroxidase (HRP) since it displays an identical catalytic current to the ferrocene ethanolamine derivative (1) used in the MediSense ExacTech and Precision QID blood glucose biosensor electrodes.

Adsorption↗
PubMed2005

Human mitochondrial complex I assembly is mediated by NDUFAF1.

Complex I (NADH:ubiquinone oxidoreductase) is the largest multiprotein enzyme of the oxidative phosphorylation system. Its assembly in human cells is poorly understood and no proteins assisting this process have yet been described. A good candidate is NDUFAF1, the human homologue of Neurospora crassa complex I chaperone CIA30. Here, we demonstrate that NDUFAF1 is a mitochondrial protein that is involved in the complex I assembly process. Modulating the intramitochondrial amount of NDUFAF1 by knocking down its expression using RNA interference leads to a reduced amount and activity of complex I. NDUFAF1 is associated to two complexes of 600 and 700 kDa in size of which the relative distribution is altered in two complex I deficient patients. Analysis of NDUFAF1 expression in a conditional complex I assembly system shows that the 700 kDa complex may represent a key step in the complex I assembly process. Based on these data, we propose that NDUFAF1 is an important protein for the assembly/stability of complex I.

Cell Fractionation↗
PubMed1994

Binding of serum amyloid P component to heparin in human serum.

It has been proposed that the function of serum amyloid P component (SAP) may closely relate with its binding to polysaccharides, especially glycosaminoglycans. We employed a quantitative immunoelectrophoresis (QIE) method and a native polyacrylamide gel electrophoresis (PAGE) method to characterize the SAP-heparin binding in soluble state. The SAP-heparin binding showed positive cooperativity. The apparent numbers of heparin molecules bound to SAP varied with the calcium concentration with a ratio of 1:1 (SAP/heparin), a Kd of 2.06 x 10(-7) M at 0.1 mM CaCl2 and a ratio of 1:1.6 (SAP/heparin), a Kd of 3.91 x 10(-7) M at 2 mM CaCl2, when estimated by the QIE method. No binding between SAP and heparin was observed in the absence of calcium. Magnesium and barium failed to induce the formation of SAP-heparin complex. Furthermore, they showed inhibitory effects on the calcium-mediated complex formation. We propose that heparin might be a regulator to modulate the anticoagulant activity of SAP and a useful drug to prevent SAP deposition on amyloid deposits.

Barium↗
PubMed2005

Mim1, a protein required for the assembly of the TOM complex of mitochondria.

The translocase of the outer mitochondrial membrane (TOM complex) is the general entry site for newly synthesized proteins into mitochondria. This complex is essential for the formation and maintenance of mitochondria. Here, we report on the role of the integral outer membrane protein, Mim1 (mitochondrial import), in the biogenesis of mitochondria. Depletion of Mim1 abrogates assembly of the TOM complex and results in accumulation of Tom40, the principal constituent of the TOM complex, as a low-molecular-mass species. Like all mitochondrial beta-barrel proteins, the precursor of Tom40 is inserted into the outer membrane by the TOB complex. Mim1 is likely to be required for a step after this TOB-complex-mediated insertion. Mim1 is a constituent of neither the TOM complex nor the TOB complex; rather, it seems to be a subunit of another, as yet unidentified, complex. We conclude that Mim1 has a vital and specific function in the assembly of the TOM complex.

Amino Acid Sequence↗
PubMed1999

Increased mitogenicity of an alphabeta heterodimeric PDGF receptor complex correlates with lack of RasGAP binding.

The different platelet-derived growth factor (PDGF) isoforms cause activation of their alpha and beta protein tyrosine kinase receptors through dimerization. Homodimerization as well as heterodimerization of receptors occur. It has been shown previously that the heterodimeric receptor complex mediates a stronger mitogenic response than either of the homodimeric complexes. In this report, we show that in cells expressing both PDGF alpha- and beta-receptors, stimulation with PDGF-AB, which leads to preferential heterodimer formation, leads to a very low degree of phosphorylation of Tyr771 in the beta-receptor. In contrast, Tyr771 is phosphorylated in a homodimeric complex of beta-receptors. Phosphorylated Tyr771 is a binding site for RasGAP; an analogous site is not present in the alpha-receptor, which lacks the ability to associate with RasGAP. The lowered phosphorylation of Tyr771 in the heterodimeric receptor complex correlates with lowered association with RasGAP, as well as with a more efficient activation of Ras and MAP kinase, which is consistent with the increased mitogenicity elicited by PDGF-AB, compared to PDGF-AA or PDGF-BB.

Amino Acid Sequence↗
PubMed1993

Binding of human apolipoprotein E to synthetic amyloid beta peptide: isoform-specific effects and implications for late-onset Alzheimer disease.

Apolipoprotein E (apoE), a plasma apolipoprotein that plays a central role in lipoprotein metabolism, is localized in the senile plaques, congophilic angiopathy, and neurofibrillary tangles of Alzheimer disease. Late-onset familial and sporadic Alzheimer disease patients have an increased frequency of one of the three common apoE alleles, epsilon 4, suggesting apoE4 is associated with increased susceptibility to disease. To follow up on this suggestion, we compared the binding of synthetic amyloid beta (beta/A4) peptide to purified apoE4 and apoE3, the most common isoform. Both isoforms bound synthetic beta/A4 peptide, the primary constituent of the plaque and angiopathy, forming a complex that resisted dissociation by boiling in SDS. Oxygen-mediated complex formation was implicated because binding was increased in oxygenated buffer, reduced in nitrogen-purged buffer, and prevented by reduction with dithiothreitol or 2-mercaptoethanol. Binding of beta/A4 peptide was saturable at 10(-4) M peptide and required residues 12-28. Examination of apoE fragments revealed that residues 244-272 are critical for complex formation. Both oxidized apoE4 and apoE3 bound beta/A4 peptide; however, binding to apoE4 was observed in minutes, whereas binding to apoE3 required hours. In addition, apoE4 did not bind beta/A4 peptide at pH < 6.6, whereas apoE3 bound beta/A4 peptide from pH 7.6 to 4.6. Together these results indicate differences in the two isoforms in complexing with the beta/A4 peptide. Binding of beta/A4 peptide by oxidized apoE may determine the sequestration or targeting of either apoE or beta/A4 peptide, and isoform-specific differences in apoE binding or oxidation may be involved in the pathogenesis of the intra- and extracellular lesions of Alzheimer disease.

Aged↗
PubMed1996

Disulfide bond formation between the n-terminal region of p56LCK and the cytoplasmic domain of CD8 studied by electrospray ionization and matrix-assisted desorption/ionization time-of-flight mass spectrometry.

Masses of the complexes formed between the C-terminal region of CD8 alpha (CD8 alpha C) and the N-terminal region of p56lck (p56lckN) were measured by using Electrospray Ionization (ESI) and Matrix-Assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry (MS). The two peptides were incubated with various metal ions, Zn++, Cd++, Co++, Fe++ and Ca++. The complex formed from the incubation did not contain any metal ions. Instead, its mass suggested that the complex was formed by two disulfide bonds. The fact that the incubation of the complex with Dithiothreitol broke the complex confirmed into monomers that the complex was formed through disulfide bonds. The only disulfide-mediated complex formed was hetero-dimer (CD8 alpha C-p56lckN) and none of homo-dimers (CD8 alpha C-CD8 alpha C or p56lckN-p56lckN) were observed from ESI and MALDI-TOF mass spectrometry.

Amino Acid Sequence↗
PubMed1990

Neurotransmitter receptors mediating excitatory input to cells in the cat lateral geniculate nucleus. I. Lagged cells.

1. Synaptic mechanisms that might explain the functional properties of the recently discovered class of lagged cells in the dorsal lateral geniculate nucleus (LGN) were analyzed with electrophysiological and pharmacologic techniques. To study the type of excitatory amino acid (EAA) receptor that mediates visual responses of lagged cells, we recorded the response of single cells to a stationary flashing light spot before, during, and after microiontophoretic application of antagonists and agonists to EAA receptors. 2. The visual response of the lagged cells could be almost completely blocked by an antagonist to the N-methyl-D-aspartate (NMDA) receptor. The degree of suppression was dose dependent, and the average maximum degree of suppression for all the cells was 94%. NMDA enhanced the response, and this enhancement was antagonized by NMDA antagonists. A quisqualate/kainate receptor antagonist had no significant effect on the lagged cells. 3. These findings indicate that the visual response in lagged cells is dependent upon activation of NMDA receptors, which may directly result from activation of retinal inputs. 4. No pharmacologic difference was seen between lagged X- and Y-cells, or between lagged ON- and OFF-center cells. 5. gamma-Aminobutyric acid-A (GABA-A) receptor antagonists were used to study whether the characteristic lag of the visual response and the suppression of the initial transient response component of the lagged cells are dependent on geniculate inhibition. Beside enhancement of the visual response, the GABA antagonists strongly reduced the lag of the visual response, and an initial transient response component occurred instead of the initial suppression. The lag remained slightly longer than for nonlagged cells, and the peak firing rate of the transient was below values typical for nonlagged cells, indicating that the lagged cell properties are dependent on other factors beside GABA-A receptor-mediated inhibition. 6. The enhanced visual response during iontophoresis of GABA antagonists could be completely blocked by simultaneous iontophoresis of an NMDA-receptor antagonist. This gives further support to the hypothesis that the retinal input to these cells is mediated by NMDA receptors. 7. The NMDA-receptor/ionophore complex mediates excitatory postsynaptic potentials (EPSPs) characterized by slow rise and decay times and long duration. The ionophore is characterized by a voltage-dependent blockade that makes these receptors particularly sensitive to inhibitory input. The temporal interplay between the slow NMDA receptor-mediated EPSPs and the fast GABA receptor-mediated inhibitory postsynaptic potentials (IPSPs) may explain the characteristic response properties of the lagged cells.

Afferent Pathways↗
PubMed2006

Mast cell-mediated remodeling and fibrinolytic activity protect against fatal glomerulonephritis.

Mast cells are detrimental in several inflammatory diseases; however, their physiological roles are also increasingly recognized. Recent data suggest that mast cells may also be involved in renal diseases. We therefore used congenitally mast cell-deficient W/W(v) mice and normal +/+ littermates to assess their role in anti-glomerular basement membrane-induced glomerulonephritis. Following administration of anti-glomerular basement membrane Abs, W/W(v) mice exhibited increased mortality as compared with +/+ mice owing to rapid deterioration of renal function. Reconstitution of the mast cell population in W/W(v) mice restored protection. This was independent of activating FcgammaR, as protection was also obtained using mast cells deficient in FcRgamma. Comparative histological analysis of kidneys showed that deterioration of renal function was caused by the presence of thick layers of subendothelial glomerular deposits in W/W(v) mice, while +/+ mice or mast cell-reconstituted W/W(v) mice showed significantly less. Deposits appeared during the early phase of disease and persisted thereafter, and were accompanied by enhanced macrophage recruitment. Immunohistochemical analysis revealed increased amounts of fibrin and type I collagen in W/W(v) mice, which were also unable to maintain high tissue plasminogen activator and urinary-type plasminogen activator activity in urine in the heterologous phase of disease. Our results indicate that mast cells by their ability to mediate remodeling and repair functions are protective in immune complex-mediated glomerulonephritis.

Animals↗
PubMedDate not supplied

Protein import into mitochondria.

Most mitochondrial proteins are encoded by the nuclear genome and thus have to be imported into mitochondria from the cytosol. Protein translocation across and into the mitochondrial membranes is a multistep process facilitated by the coordinated action of at least four specialized translocation systems in the outer and inner membranes of mitochondria. The outer membrane contains one general translocase, the TOM complex, whereas three distinct translocases are located in the inner membrane, which facilitates translocation of different classes of preproteins. The TIM23 complex mediates import of matrix-targeted preproteins with N-terminal presequences, whereas hydrophobic preproteins with internal targeting signals are inserted into the inner membrane via the TIM22 complex. The OXA translocase mediates the insertion of preproteins from the matrix space into the inner membrane. This review focuses on the structural organization and function of the import machinery of the model organisms of Saccharomyces cerevisiae and Neurospora crassa. In addition, the molecular basis of a new human mitochondrial disorder is discussed, the Mohr-Tranebjaerg syndrome. This is the first known disease, which is caused by an impaired mitochondrial protein import machinery leading to progressive neurodegeneration.

Animals↗
PubMed1988

Fluorescence analysis of size distribution and mode of dye release from carboxyfluorescein-loaded vesicles: application to the study of complement-membrane interactions.

We wish to report a novel method for visualizing large unilamellar vesicles loaded with a fluorescent dye and for monitoring changes in the size distribution as well as state of aggregation of such dye-loaded liposomes. In addition, we demonstrate that this method can be used to distinguish between all-or-none release of dye and graded release of dye from individual vesicles. Using this technique, we have characterized complement-mediated release of carboxyfluorescein from large unilamellar vesicles and have found that C5-8 complexes mediate a graded release of dye while C5-9 complexes cause an all-or-none release. Furthermore, complement appears to preferentially attack the medium to larger-sized vesicles in our population of large unilamellar vesicles while smaller vesicles appear to be selectively spared.

Cholesterol↗
PubMed2002

Differentiation of the human monocyte cell line, U937, with dibutyryl cyclicAMP induces the expression of the inhibitory Fc receptor, FcgammaRIIb.

FC receptor for IgG receptor (Fcgamma) mediated activation of macrophages is essential for the clearance of immune complexes and control of inflammation. However, activated macrophages play an integral role in tissue destruction associated with autoimmune and inflammatory disease processes. Understanding the mechanisms which balance activating and inhibitory signals generated by immune complexes are therefore of critical importance to human disease. Here, we demonstrate that differentiation of the human monocytic U937 cell line to a macrophage phenotype with dibutyryl cyclicAMP induces both mRNA and protein expression of the inhibitory IgG receptor, FcgammaRIIb1. We further demonstrate that, following receptor aggregation, FcgammaRII transiently recruits the 5'-inositol phosphatase, SHIP. These data define a role for FcgammaRIIb in the modulation of immune complex mediated macrophage activation in a human model system.

Antigen-Antibody Complex↗
PubMed2001

A dominant role for Fc gamma receptors in antibody-dependent corneal inflammation.

Although production of specific Ab is a critical element of host defense, the presence of Ab in tissues leads to formation of immune complexes, which can trigger a type III Arthus reaction. Our studies on a mouse model of river blindness showed that Ab production is essential for recruitment of neutrophils and eosinophils to the cornea and for development of corneal opacification. In the current study, we determined the relative contribution of complement and FcgammaR interactions in triggering immune complex-mediated corneal disease. FcgammaR(-/-) mice, C3(-/-) mice, and immunocompetent control (B6/129Sj) mice were immunized s.c. and injected intrastromally with Onchocerca volvulus Ags. Slit lamp examination showed that control mice, C3(-/-) mice, and control mice injected with cobra venom factor developed pronounced corneal opacification, whereas corneas of FcgammaR(-/-) mice remained completely clear. Furthermore, recruitment of neutrophils and eosinophils to the corneal stroma was significantly impaired in FcgammaR(-/-) mice, but not in C3(-/-) mice or cobra venom factor-treated mice. We therefore conclude that FcgammaR-mediated cell activation, rather than complement activation, is the dominant pathway of immune complex disease in the cornea. These findings demonstrate a novel role for FcgammaR interactions in mediating ocular inflammation.

Animals↗
PubMed2000

Agonist-triggered modulation of the activated and silent state of the vitamin D(3) receptor by interaction with co-repressors and co-activators.

The nuclear receptor for the hormone 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), VDR, regulates gene expression via a ternary complex with the retinoid X receptor (RXR) and a 1alpha, 25(OH)(2)D(3) response element (VDRE). This complex mediates transcriptional repression through interaction with co-repressor proteins, such as NCoR, and transactivation through agonist-triggered contacts with co-activator proteins, such as SRC-1. This study demonstrates that the interaction of the VDR with NCoR results in a preferential stabilization of the VDR in a non-agonistic conformation (silent state), whereas within a complex with SRC-1 VDR is in its agonistic conformation (activated state). Helix 12 of the ligand-binding domain of the VDR was found to be a critical sensor for the differential stabilization of the activated and silent state of the receptor. VDR agonists that showed similar sensitivity in inducing VDR-RXR-VDRE complex formation were found to mediate a different dose-dependent release of NCoR from these complexes, which correlates with their ability to stabilize the silent state of the VDR in the presence of NCoR. Interestingly, up to 50 % of all VDR-NCoR complexes were found to be stable even in the presence of saturating agonist concentrations. This was confirmed by a quenching effect of overexpressed NCoR on agonist-induced gene activity mediated by VDR-RXR heterodimers. Taken together, co-activator and co-repressor proteins antagonize each other in stabilizing the activated and silent state of the receptor and modulate in this way the sensitivity and potency of the transcriptional activation by the ligand-responsive transcription factor VDR.

Animals↗
PubMed2001

The dystrophin-glycoprotein complex, cellular signaling, and the regulation of cell survival in the muscular dystrophies.

Mutations of different components of the dystrophin-glycoprotein complex (DGC) cause muscular dystrophies that vary in terms of severity, age of onset, and selective involvement of muscle groups. Although the primary pathogenetic processes in the muscular dystrophies have clearly been identified as apoptotic and necrotic muscle cell death, the pathogenetic mechanisms that lead to cell death remain to be determined. Studies of components of the DGC in muscle and in nonmuscle tissues have revealed that the DGC is undoubtedly a multifunctional complex and a highly dynamic structure, in contrast to the unidimensional concept of the DGC as a mechanical component in the cell. Analysis of the DGC reveals compelling analogies to two other membrane-associated protein complexes, namely integrins and caveolins. Each of these complexes mediates signal transduction cascades in the cell, and disruption of each complex causes muscular dystrophies. The signal transduction cascades associated with the DGC, like those associated with integrins and caveolins, play important roles in cell survival signaling, cellular defense mechanisms, and regulation of the balance between cell survival and cell death. This review focuses on the functional components of the DGC, highlighting the evidence of their participation in cellular signaling processes important for cell survival. Elucidating the link between these functional components and the pathogenetic processes leading to cell death is the foremost challenge to understanding the mechanisms of disease expression in the muscular dystrophies due to defects in the DGC.

Animals↗