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At least 217 records · Page 12Linked to original sources

PubMed1999

New insights on flow-independent mechanisms of 99mTc-HMPAO retention in nervous tissue: in vitro study.

UNLABELLED: SPECT using 99mTc-hexamethyl propyleneamine oxime (HMPAO) mainly reflects regional cerebral blood flow, however metabolic abnormalities also affect the retention of 99mTc-HMPAO. METHODS: To rule out any flow factor, a test-tube model was used to evaluate the effects of metabolic alterations both on intracellular trapping of 99mTc-HMPAO and on extracellular glutamate and lactate dehydrogenase (LDH) outflow from rat brain slices. RESULTS: Under control conditions, slices took up 7.0%+/-1.4% of 99mTc-HMPAO contained in the medium, whereas prelabeled slices released 10.8%+/-2.6% of their radioactive content; glutamate and LDH outflow were 49.1+/-21.6 pmol/mg protein/ min and 4.8+/-0.9 U/L/mg protein/min, respectively. The control medium was altered by adding a metabolic poison (5 mmol/L azide), removing glucose and replacing O2 with N2 to mimic ischemia (in vitro ischemia) and replacing Krebs solution with hypotonic medium to evoke cell lysis. Both azide and in vitro ischemia induced a significant increase in 99mTc-HMPAO release (15.8%+/-3.3% and 18.3%+/-6.2%, respectively), without any modification in LDH efflux. However, only azide reduced the uptake of the tracer. Conversely, glutamate outflow was massive during in vitro ischemia and was far lower during azide treatment. Under hypotonic medium conditions, the release of 99mTc-HMPAO, glutamate and LDH were dramatically increased. Surprisingly, a two-fold increase of 99mTc-HMPAO uptake was also found. When 1 mmol/L glutathione was added to the medium, to convert native lipophilic 99mTc-HMPAO into hydrophilic derivatives, tracer uptake was inhibited both under control and hypotonic medium conditions. CONCLUSION: This study provides evidence that not only poisoning of the tissue but also in vitro ischemia induced a reduction of 99mTc-HMPAO retention. Moreover, we demonstrated that injuries causing cell membrane disruption led to hyperfixation of 99mTc-HMPAO.

Animals↗

PubMed2000

Role of ascorbic acid in the osteoclast formation: induction of osteoclast differentiation factor with formation of the extracellular collagen matrix.

Osteoclasts are bone-resorbing multinucleated cells. Tartrate-resistant acid phosphatase-positive (TRAP-positive) mononuclear and multinucleated cells, which are osteoclast-like cells (OCLs), were formed as a result of the coculture of mouse bone marrow cells and clonal stromal ST2 cells in the presence of 1alpha,25-dihydroxy-vitamin D3. Removal of ascorbic acid from the culture medium prevented the formation of TRAP-positive OCLs. Addition of ascorbic acid to the medium formed TRAP-positive OCLs, and the effect of ascorbic acid was dose-dependent. When we examined the level of messenger RNA (mRNA) for osteoclast differentiation factor (RANKL/ODF) in ST2 cells, we found that ascorbic acid caused an approximately 5-fold increase in the level of this mRNA. The half-life of the mRNA was unaffected by ascorbic acid. To characterize the mechanism of action of ascorbic acid, we investigated the relationship between formation of TRAP-positive OCLs and formation of the collagen matrix. Inhibitors of the formation of collagen triple helices blocked both the formation of TRAP-positive OCLs and the expression of the mRNA for RANKL/ODF in response to ascorbic acid. Our findings suggest that ascorbic acid might be essential for osteoclastogenesis and might induce the formation of TRAP-positive OCLs via induction of the synthesis of RANKL/ODF that is somehow mediated by the extracellular matrix.

Animals↗

PubMed2001

Unavailability of CD147 leads to selective erythrocyte trapping in the spleen.

Adhesive interactions with stromal cells and the extracellular matrix are essential for the differentiation and migration of hematopoietic progenitors. In the erythrocytic lineage, a number of adhesion molecules are expressed in the developing erythrocytes and are thought to play a role in the homing and maturation of erythrocytic progenitors. However, many of these molecules are lost during the final developmental stages leading to mature erythrocytes. One of the adhesion molecules that remains expressed in mature, circulating erythrocytes is CD147. This study shows that blockade of this molecule on the cell surface by treatment with F(ab')(2) fragments of anti-CD147 monoclonal antibody disrupts the circulation of erythrocytes, leading to their selective trapping in the spleen. Consequently, mice develop an anemia, and de novo, erythropoietin-mediated erythropoiesis in the spleen. In contrast, these changes were not seen in mice similarly treated with another antierythrocyte monoclonal antibody with a different specificity. These results suggest that the CD147 expressed on erythrocytes likely plays a critical role in the recirculation of mature erythrocytes from the spleen into the general circulation. (Blood. 2001;97:3984-3988)

Animals↗

PubMed2001

Extracellular superoxide production by Enterococcus faecalis requires demethylmenaquinone and is attenuated by functional terminal quinol oxidases.

The intestinal commensal bacterium, Enterococcus faecalis, is unusual among prokaryotic organisms in its ability to produce substantial extracellular superoxide. Transposon mutagenesis, allelic replacement, and electron spin resonance (ESR)-spin trapping showed that superoxide production and generation of derivative hydroxyl radical were dependent on membrane-associated demethylmenaquinone. Extracellular superoxide was generated through univalent reduction of oxygen by reduced demethylmenaquinone. Moreover, extracellular superoxide production was inhibited by exogenous haematin, an essential cofactor for cytochrome bd, and by fumarate, a substrate for fumarate reductase. As integral membrane quinol oxidases, cytochrome bd and fumarate reductase redox cycle demethylmenaquinone, and are necessary for aerobic and anaerobic respiration respectively. A rat model of intestinal colonization demonstrated that conditions exist in the mammalian intestinal tract that permit a mode of respiration for E. faecalis that results in the formation of hydroxyl radical. These results identify and characterize the mechanism by which E. faecalis generates extracellular free radicals.

Animals↗

PubMed1995

The association of pp125FAK, pp60Src, CDC42Hs and Rap1B with the cytoskeleton of aggregated platelets is a reversible process regulated by calcium.

The integrin alpha IIb beta 3-mediated redistribution of the tyrosine kinases pp125FAK and pp60Src and the small GTP-binding proteins CDC42Hs and Rap1B from the membrane skeleton to the cytoskeleton was found to be reversible: upon prolonged platelet aggregation (up to 15 min) induced by the thrombin-receptor activating peptide (TRAP) these signalling proteins dissociated from the cytoskeleton and reappeared in the membrane skeleton. Addition of the extracellular Ca2+ chelator EGTA and the intracellular Ca2+ chelator BAPTA/AM 30 s after TRAP allowed platelet aggregation and the association of pp125FAK, pp60Src, CDC42Hs and Rap1B with the cytoskeleton, but prevented their dissociation from the cytoskeleton. The results indicate that the prolonged elevation of cytosolic Ca2+ in stimulated platelets leads to the dissociation of signalling proteins from the cytoskeleton.

Amino Acid Sequence↗

PubMed2002

Cytosine deaminase and thymidine kinase gene therapy in a Dunning rat prostate tumour model: absence of bystander effects and characterisation of 5-fluorocytosine metabolism with 19F-NMR spectroscopy.

The rat prostate tumour cell line R3327 AT-1 was transfected with a gene coding for a fusion protein comprised of cytosine deaminase (CD from E. coli) and thymidine kinase (TK from Herpes simplex virus, HSV-1). The resulting AT-1/CDglyTK cell line was sensitive to the prodrug 5-fluorocytosine (IC(50) = 78 microM, 96-h incubation) via CD and to ganciclovir (GCV, IC(50) = 1 microM, 96 h) via TK. Subcutaneous tumours generated from 100% CDglyTK(+) cells responded well to 5-FC therapy (500 mg/kg, i.p., 14 daily treatments, four out of seven animals in remission) and to GCV therapy (30 mg/kg, i.p., 14 daily treatments, five of six animals in remission). However, experiments with mixtures of CDglyTK(+) and CDglyTK(-) cells showed low levels of connexins (intercellular gap junctions) and no bystander effect for nontransfected cells using either 5-FC or GCV therapy. Furthermore, (19)F-NMR spectroscopy showed that incubation of cultured CDglyTK(+) cells with 774 microM 5-FC for 16 h resulted in the following intracellular concentrations: 5-FC = 314 microM, 5-FU = 52 microM, cytotoxic fluoronucleotides = 163 microM; extracellular 5-FU reached only 6.4 microM. Thus, in this model system intracellular trapping of 5-FU (slow export) contributes to the failure of the CD/5-FC bystander effect via an extracellular route.

Animals↗

PubMedDate not supplied

Chemotactic activity present in liver extracellular matrix.

A selective pattern of metastasis, not accountable by a simple mechanical trapping mechanism, is exhibited by many primary tumors and appears to be controlled by properties of both the tumor cell and the host organ. This organotropism may be regulated, in part, by the migration of an invading tumor cell toward chemotactic factors present in the extracellular matrix which may be released as a result of proteolytic digestion. To test this hypothesis we have examined 4 M guanidine extracts of liver extracellular matrix, prepared by high salt extraction, for organ-specific chemotactic activity. The murine cell lines B16-L4b and M5076, which preferentially metastasize to the liver in an experimental metastasis model, demonstrated preferential motility toward the liver matrix extract while the lung-colonizing lines B16, B16-F10 and B16-BL6 did not. The liver specific chemotactic activity eluted as four fractions of Mr much less than 250,000, Mr approximately 245,000, Mr approximately 120,000 and Mr approximately 30,000 by gel filtration chromatography.

Animals↗

PubMed2003

[Tumor necrosis factor receptor superfamily 1A-associated periodic syndrome (TRAPS)].

PURPOSE: Tumor necrosis factor receptor superfamily 1A associated periodic syndrome (TRAPS) belongs to the group of hereditary fever syndromes, also called hereditary auto-inflammatory syndromes. CURRENT KNOWLEDGE AND KEY POINTS: The diagnosis of TRAPS should be evoked in presence of the following clinical signs, whatever the population of the affected patients. TRAPS acute inflammatory access, of 1 to 3 weeks' duration, is characterised by the presence of fever, abdominal pain, myalgias, various types of skin rash including erysepela-like erythema. Long term inflammatory response can lead to AA amyloidosis. Genetic testing will confirm the diagnosis when showing a mutation in the extracellular part of the TNFRSF1A receptor. Therapeutic management of TRAPS is not definitely established. Daily colchicine does not seem to prevent efficiently inflammatory attacks. Corticosteroids, in contrast can attenuate the intensity and diminish the duration of attacks. FUTURE PROSPECTS AND PROJECTS: The value of biological agents that inhibits TNF action is not yet completely determined in TRAPS. Mechanisms of the disease are not yet elucidated. In some families with specific mutations, a relative soluble TNF receptor deficiency has been found in the plasma. However this mechanism does not account for what is observed in other kindreds.

Abdominal Pain↗

PubMed2005

A conserved molecular motor drives cell invasion and gliding motility across malaria life cycle stages and other apicomplexan parasites.

Apicomplexan parasites constitute one of the most significant groups of pathogens infecting humans and animals. The liver stage sporozoites of Plasmodium spp. and tachyzoites of Toxoplasma gondii, the causative agents of malaria and toxoplasmosis, respectively, use a unique mode of locomotion termed gliding motility to invade host cells and cross cell substrates. This amoeboid-like movement uses a parasite adhesin from the thrombospondin-related anonymous protein (TRAP) family and a set of proteins linking the extracellular adhesin, via an actin-myosin motor, to the inner membrane complex. The Plasmodium blood stage merozoite, however, does not exhibit gliding motility. Here we show that homologues of the key proteins that make up the motor complex, including the recently identified glideosome-associated proteins 45 and 50 (GAP40 and GAP50), are present in P. falciparum merozoites and appear to function in erythrocyte invasion. Furthermore, we identify a merozoite TRAP homologue, termed MTRAP, a micronemal protein that shares key features with TRAP, including a thrombospondin repeat domain, a putative rhomboid-protease cleavage site, and a cytoplasmic tail that, in vitro, binds the actin-binding protein aldolase. Analysis of other parasite genomes shows that the components of this motor complex are conserved across diverse Apicomplexan genera. Conservation of the motor complex suggests that a common molecular mechanism underlies all Apicomplexan motility, which, given its unique properties, highlights a number of novel targets for drug intervention to treat major diseases of humans and livestock.

Actins↗

PubMed1989

Monitoring of the activation of receptor-operated calcium channels in human platelets.

Activation of receptor-operated calcium channels has been monitored by measurements of the quenching of the fluorescence of intracellularly trapped fura-2 by Mn entering from the extracellular medium. Release of calcium from intracellular stores was followed simultaneously by measurements of the ratio of the fluorescence excited at 340 and 380 nm. Thrombin, ADP, platelet-activating-factor (PAF) and collagen, all produced both release of calcium from the intracellular stores and uptake of Mn from the extracellular medium. The uptake of Mn, but not the increase of (Ca2+)i, was blocked by nickel. These results suggest the existence of plasma membrane calcium channels which can be activated by the different agonists tested here. The activation of calcium channels was very fast and transient with ADP and PAF, fast and maintained with thrombin, and delayed with collagen.

Adenosine Diphosphate↗

PubMed1998

Dissecting the mechanism of torso receptor activation.

Regulated activation of receptor tyrosine kinases depends both on the presence of the receptors at the cell surface and on the availability of their ligands. In Drosophila the torso (tor) tyrosine kinase receptor is distributed along the surface of the embryo but it is only activated at the poles by a diffusible extracellular ligand generated at each pole which is trapped by the receptor, thereby impeding further diffusion. However, it is not well understood how this signal is generated, although it is known to depend on the activity of many genes such as torso-like (tsl) and trunk (trk). To further investigate the mechanism involved in the local activation of the tor receptor we have altered the normal expression of the tsl protein by generating females in which the tsl gene is expressed in the oocyte under the control of the tor promoter rather than in the ovarian follicle cells. Analysis of the phenotypes generated by this hybrid gene and its interactions with mutations in other genes in the pathway has enabled us to further dissect the mechanism of tor receptor activation and to define more precisely the role of the different genes acting in this process.

Animals↗

PubMed2001

Volvox carteri as a model for studying the genetic and cytological control of morphogenesis.

The green alga Volvox carteri has a very simple and regular adult form that arises through a short sequence of well-defined morphogenetic steps. A mature gonidium (asexual reproductive cell) initiates a stereotyped sequence of rapid cleavage divisions that will produce all of the cells found later in an adult. A predictable subset of these divisions are asymmetric and result in production of a small set of germ cells in a precise spatial pattern. Throughout cleavage, all intracellular components are held in predictable spatial relationships by a cytoskeleton of unusually regular structure, while neighboring cells are also held in fixed spatial relationships by an extensive network of cytoplasmic bridges that form as a result of incomplete cytokinesis. As a result of these two orienting mechanisms combined, dividing cells are arranged around the anterior-posterior axis of the embryo with precise rotational symmetry. These relationships are maintained by the cytoplasmic bridge system when the embryo that was inside out at the end of cleavage turns right-side out in the gastrulation-like process of inversion. Inversion is driven by a cytoskeleton-mediated sequence of cell shape changes, cellular movements and coordinated contraction. Then, by the time the cytoplasmic bridges begin to break down shortly after inversion, a preliminary framework of extracellular matrix (ECM) has been formed. The ECM traps the cells and holds them in the rotational relationships that were established during cleavage, and that must be maintained in order for the adult to be able to swim. Transposon tagging is now being used to clone and characterize the genes regulating these morphogenetic processes.

Chlorophyta↗

PubMed1994

Twenty-five years of germinal centre physiology: implications for tolerance in the secondary B cell repertoire.

Twenty-five years ago, when I delivered the inaugural lecture of the Scandinavian Society for Immunology, my mind was filled with germinal centres because of the extraordinary antigen-capturing mechanism there, which depended on follicular dendritic cells trapping the antigen and holding it in an extracellular location for long periods of time, following which the germinal centre reaction developed around the antigen depot. It was apparent that germinal centres had much to do with immunological memory, but few functional details of their role were known. In the intervening period, a great deal of knowledge has accumulated about germinal centres, which have become known as the sites of extensive B cell proliferation, immunoglobulin variable region gene hypermutation, and selection of better antigen-binding variants leading to affinity maturation in the antibody response. Our laboratory's interest was reawakened when we came to examine the hypothesis that B lymphocytes destined to develop into memory B cells might pass through a 'second window' of tolerance susceptibility if they encountered antigen in the absence of T-cell help. We have constructed a model of tolerance dependent on the injection of soluble deaggregated antigen before or even up to 6 days after T cell-dependent challenge immunization. The model was intended to mimic what might happen if a B cell, developing correctly in a germinal centre, fortuitously mutates to acquire cross-reactivity to a soluble self antigen. We have shown that the surrogate self-antigen can profoundly impair the germinal centre process and virtually stop the emergence of high affinity memory B cells.

Animals↗

PubMed2000

Thrombin induces endothelin expression in arterial smooth muscle cells.

Thrombin has been shown to stimulate endothelin release by endothelial cells, but the ability of thrombin to induce endothelin in nonendothelial cells is less well-known. Incubation of rat aortic smooth muscle cells with thrombin resulted in a stimulation of preproendothelin-1 (preproET-1) mRNA expression. This induction of preproET-1 mRNA expression by thrombin was accompanied by the release of immunoreactive peptide ET-1 into the extracellular medium. The synthetic thrombin receptor activator peptide (TRAP) confirmed ligand-specific receptor action to induce preproET-1 mRNA. Nuclear run-on analysis revealed that the transcriptional rate of preproET-1 mRNA increases twofold after 1 h of incubation with thrombin. In cells treated with thrombin, the half-life of preproET-1 mRNA was identical to that in untreated control cells. These results demonstrated that thrombin regulates endothelin synthesis at a transcriptional level but does not influence mRNA stability. Inhibition of protein kinase C (PKC) with selective inhibitors (chelerythrine and bisindolylmaleimide I) before thrombin stimulation failed to significantly inhibit preproET-1 gene expression. Inhibition of mitogen-activated protein (MAP) kinase kinase and protein tyrosine kinase decreased preproET-1 mRNA expression in thrombin-stimulated smooth muscle cells. Furthermore, addition of an activator of peroxisome proliferator-activated receptors-alpha (PPARalpha), fenofibrate, prevented the preproET-1 gene induction in response to thrombin. These results demonstrated that thrombin-induced endothelin gene transcription involved MAP kinase kinase rather than the PKC cascade in smooth muscle cells, which was repressed by PPARalpha stimulation.

Animals↗

PubMed1998

Esterified cholesterol accumulation induced by aggregated LDL uptake in human vascular smooth muscle cells is reduced by HMG-CoA reductase inhibitors.

Vascular smooth muscle cell (VSMC) proliferation is a key event in the development of atherosclerotic lesions. VSMCs synthesize extracellular matrix, where low density lipoproteins (LDLs) are trapped and become aggregated (agLDL). The objective of this study was to investigate the cholesterol uptake and accumulation triggered by agLDL in comparison with native LDL (nLDL) on unstimulated and platelet-derived growth factor-stimulated human aortic VSMCs and the role of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors on these processes. Esterified cholesterol (EC) accumulation induced by agLDL in VSMCs was correlated with the degree of aggregation and concentration. The EC content of VSMCs treated with 100 microg/mL of agLDL (80% aggregated) increased approximately 70-fold over that in VSMCs incubated with the same concentration of nLDL. Whereas nLDL-derived EC was increased approximately twofold in platelet-derived growth factor-stimulated VSMCs, there was no effect of platelet-derived growth factor (10(-9) mol/L) on the uptake of agLDL. The 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor simvastatin (5 micromol/L) reduced EC accumulation derived from agLDL uptake by 58% and 35% in platelet-derived growth factor-stimulated and unstimulated VSMCs, respectively. This inhibition was overcome by geranylgeraniol (10 micromol/L) and partially by farnesol (10 micromol/L). Fluorescence microscopy of the cellular internalization of agLDL labeled with the fluorochrome 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine demonstrated that simvastatin reduces EC accumulation derived from agLDL by inhibiting its endocytosis and that the effect is completely reversed by geranygeraniol. These results indicate that agLDLs are rapidly internalized by human VSMCs and that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors modulate EC accumulation. These data suggest a possible mechanism by which statins could contribute to the passivation and stabilization of actively growing atherosclerotic lesions.

Cells, Cultured↗

PubMed2001

The spatial control of Torso RTK activation: a C-terminal fragment of the Trunk protein acts as a signal for Torso receptor in the Drosophila embryo.

Regulated activation of receptor tyrosine kinases depends on both the presence of the receptors at the cell surface and on the availability of their ligands. In Drosophila, the torso tyrosine kinase receptor is distributed along the surface of the embryo but it is only activated at the poles by a diffusible extracellular ligand generated at each pole that is trapped by the receptor, thereby impeding further diffusion. Although it is known that this signal depends on the activity of several genes, such as torso-like and trunk, it is still unclear how is generated. The identification of the signal responsible for the torso receptor activation is an essential step towards understanding the mechanism that regulates the local restriction of torso signalling. Here we report that a fragment containing the carboxy-terminal 108 amino acids of the trunk protein retains trunk activity and is sufficient to activate torso signalling. We also show that this fragment bypasses the requirements for the other genes involved in the activation of the torso receptor. These results suggest that a cleaved form of the trunk protein acts as a signal for the torso receptor. We therefore propose that the restricted activation of the torso receptor is defined by the spatial control of the proteolytic processing of the trunk protein.

Animals↗

PubMed2002

Molecular mechanisms of gating and drug block of sodium channels.

Voltage-gated Na+ channels are composed of an alpha subunit of 260 kDa associated with beta subunits of 33-36 kDa. Alpha subunits have four homologous domains (I to IV) containing six transmembrane alpha helices (S1-S6). The S4 segments serve as voltage sensors and move outward to initiate activation. The S5 and S6 segments and the short membrane-associated loops between them form the pore. Fast inactivation is mediated by closure of an inactivation gate formed by the intracellular loop between domains III and IV. The 3-D structure of the inactivation gate has been determined bv NMR spectroscopy, revealing the conformation of the pore-blocking IFM motif. Peptide scorpion toxins that alter gating of Na+ channels bind to the extracellular ends of the IIS4 and IVS4 segments, trap them in either an activated or non-activated position, and thereby selectively alter channel activation or inactivation. Voltage sensor-trapping may be a general mechanism of toxin action on voltage-gated ion channels. Local anaesthetics block the pore of Na+ channels by binding to a receptor site in segment S6 in domains III and IV. Anticonvulsants and antiarrhythmic drugs also interact with this site. A high-affinity Na+ channel blocker has recently been developed with this site as its target. The emerging knowledge of the molecular mechanisms of Na+ channel gating and drug block may allow development of novel therapeutics for epilepsy, cardiac arrhythmia and persistent pain syndromes.

Animals↗

PubMed1997

Tenascin-C knockout mouse has no detectable tenascin-C protein.

A recent study by Mitrovic and Schachner (J Neurosci Res 42:710-717, 1995) reported the detection of a small amount of truncated tensacin-C (TN-C) in the nervous system of the TN-C knockout mice created by Saga et al. (Genes Dev 6:1821-1831, 1992). The authors suggested that the truncated protein might be responsible for the failure to detect any phenotypic abnormalities in the knockout mice. We have reexamined the knockout mice in our laboratories by Western blot and immunocytochemistry, and have not detected any full-length or truncated TN-C protein. In addition, we note that the construction of the knockout gene deleted the signal sequence, so if any residual truncated protein were produced it would be trapped in the cytoplasm, and therefore inaccessible to extracellular ligands or receptors. We therefore conclude that the TN-C knockout created by Saga et al. is a valid TN-C null.

Animals↗