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1.
Acylphosphatase is one of the smallest enzymes known (about 98 amino acid residues). It is present in organs and tissues of vertebrate species as two isoenzymes sharing over 55% of sequence homology; these appear highly conserved in differing species. The two isoenzymes can be involved in a number of physiological processes, though their effective biological function is not still certain. The solution and crystal structures of different isoenzymes are known, revealing a close packed protein with a fold similar to that shown by other phosphate-bind ing proteins. The structural data, together with an extended site-directed mutagenesis investigation, led to the identification of the residues involved in enzyme catalysis. However, it appears unlikely that these residues are able to perform the full catalytic cycle: a substrate-assisted catalytic mechanism has therefore been proposed, in which the phosphate moiety of the substrate could act as a nucleophile activating the catalytic water molecule. Received 12 November 1996; accepted 27 November 1996  相似文献   

2.
Inhaled allergens from house dust, mites or animal danders activate human complement in vitro by engaging the C1-component through a non-antibody-dependent mechanism. These earlier findings are extended by showing that the allergenic components in extracts of Parietaria pollen are almost equally potent complement activators as those from house dust or mites. Spectroscopic evidence indicates that haemolytic complement consumption by the Parietaria allergens and their enzymic fragments is most likely related to post-translational side-chains comprising flavonoid derivatives. These adsorbed and/or peptide-bound tannin-like structures may also explain the exceptional stability of the high- and low-molecular mass allergenic components in Parietaria pollen extracts. Received 12 November 1996; received after revision 12 December 1996; accepted 17 December 1996  相似文献   

3.
Effects of serotonin (5-HT) on cerebral cortical neurons were examined by patch clamp techniques. 5-HT produced a variety of responses such as outward (19/73 patches/neurons), slow inward (15/73 patches/neurons), fast inward (8/73 patches/neurons), and mixed currents (initially fast inward deflection followed by an outward response: 2/73 patches/neurons), with a latency of 12 sec, 15 sec, 0 sec, and 0 sec respectively, at a holding potential of −60 mV in whole-cell patches. The fast inward currents were again evoked by a selective 5-HT3 receptor agonist, 1-(m-chlorophenyl)-biguanide hydrochloride (CPBG). In the cell-attached patch clamp configuration, 5-HT inside the patch pipette elicited single channel currents with slope conductances of 42 pS and 132 pS (4/42 patches/neurons). CPBG inside the patch pipette evoked inward single channel currents with a lower slope conductance of 41 pS (3/23 patches/neurons). In contrast, application of 5-HT or a 5-HT2 receptor agonist, α-methyl-5-hydroxytryptamine-maleate, outside the patch pipette induced outward single channel currents with a major slope conductance of 140 pS (8/30 patches/neurons) or 135 pS (6/20 patches/neurons), respectively. These results indicate that the outward and fast inward currents may be mediated respectively by the 5-HT2 receptor, which is coupled to a G-protein, and by the 5-HT3 receptor, which contains the non-selective cation channel, and that the mixed type may be caused by both the 5-HT2 and 5-HT3 receptors. Received 27 September 1996; received after revision 4 November 1996; accepted 7 November 1996  相似文献   

4.
Intermediate filament assembly: temperature sensitivity and polymorphism   总被引:6,自引:0,他引:6  
Intermediate filament (IF) proteins are encoded by a large multigene family and form polymers with a uniform diameter of approximately 10 nm. However, although the cytoplasmic representatives all confirm to a unit-type structural principle leading to the formation of extended coiled coils, it is becoming increasingly clear that subunit arrangements and physical properties vary among the different filaments. Thus, the intricate tissue-specific expression pattern of individual IF proteins (especially, their co-expression with other members of the IF protein family or with IF-associated proteins to form obligatory heteropolymers) points to distinct functions acquired during evolution relevant to cellular homeostasis in various tissues.  相似文献   

5.
An efficient Escherichia coli expression system for the production of a perchloric acid-soluble protein (PSP) has been constructed. Complementary DNA encoding PSP was inserted into an inducible bacterial expression vector pGEX-4T-1. After the plasmid introduced into E. coli was expressed by isopropyl 1-thio-β-D-galactopyranoside (IPTG), the recombinant product was purified by glutathione-Sepharose 4B affinity chromatography. The purified product showed the expected NH2-terminal sequence, but the translation inhibitory activity of this product was 10 times lower compared with that of authentic PSP isolated from rat liver. Received 8 October 1998; received after revision 6 November 1998; accepted 6 November 1998  相似文献   

6.
The fluorescent dye Merocyanine 540 (MC540) is often used as a probe to monitor the molecular packing of phospholipids in the outer leaflet of biomembranes. In a previous study we showed that the increased staining of erythrocytes with a perturbed membrane structure was mainly due to an increase in the fluorescence yield of cell-bound MC540, rather than to an increase of the number of bound molecules. Erythrocytes and ghosts exposed to continuous fluxes of H2O2 exhibited pronounced lipid peroxidation. Further, red blood cells subjected to this form of oxidative stress also showed increased staining with MC540. It appeared that this was caused by a strong increase in binding of MC540, together with a slight red shift of the fluorescence emission maximum and a small increase in the fluorescence yield of bound MC540. The changed MC540 binding characteristics were not observed when lipid peroxidation was suppressed by the presence of the antioxidant BHT in the incubation medium. However, open ghosts exposed to H2O2 showed no increase of MC540 binding, excluding a direct involvement of lipid peroxidation. Measurement of fluorescence emission spectra and gel filtration studies showed that MC540 can bind to H2O2-exposed hemoglobin. Experiments with erythrocytes lysed in hypotonic medium after exposure to H2O2 revealed that peroxidation of lipids with H2O2 induced a non-specific permeabilization of the plasma membrane to MC540, thereby allowing MC540 to bind to the oxidatively denatured, more hydrophobic hemoglobin. These results indicate that conclusions about packing of phospholipids in the outer leaflet of the membrane based on increased MC540-staining should be drawn with care. Received 27 September 1996; received after revision 5 November 1996; accepted 27 November 1996  相似文献   

7.
E-selectin, exclusively expressed on activated endothelial cells, is a potential target for site-directed delivery of agents. We and others have shown that sialyl Lewisx-liposomes (sLex-liposomes) are recognized by E-selectin. We now report an approach employing sLex-liposomes to deliver antisense oligonucleotides (AS-ODNs) directed against the adhesion molecule ICAM-1 to activated vascular endothelial cells. ICAM-1 expression was analyzed at the protein level by immunofluorescence and a cell surface ELISA, and at the RNA level by RT-PCR. We have investigated two different AS-ODNs complementary to the 3′ untranslated region and the AUG translation initiation codon of ICAM-1 mRNA. Both inhibited protein expression, but did not influence the mRNA level, pointing to a hybridization of AS-ODNs with the mRNA in the cytoplasm. Our results demonstrate the feasibility of a novel approach for the delivery of agents to activated endothelial cells by glycoliposomes targeted to E-selectin. Received 16 October 2000; revised 29 November 2000; accepted 29 November 2000  相似文献   

8.
Starting from the concept that lipopolysaccharide (LPS)-associated hepatotoxicity involves the action of reactive oxygen species, the present study was conducted to test whether vitamin E, a lipophilic antioxidant, prevents LPS-induced hepatic microvascular dysfunction and liver injury. Fifty-two rats were divided into three groups and fed diets containing 0 (n=16), 75 (n=18) or 8000 mg (n=18) α-tocopherol acetate/kg food for four weeks. At 1 h and 6 h after intravenous LPS-exposure (10 mg/kg E. coli LPS) hepatic microvascular response and liver injury were assessed by the analysis of Kupffer cell phagocytic activity, leukocyte-endothelial cell interaction and nutritive sinusoidal perfusion (intravital fluorescence epi- illumination technique) as well as bile flow, serum liver enzyme activities and tissue histomorphology. In animals fed with 75 mg vitamin E/kg (standard diet), LPS caused hepatic Kupffer cell activation (increased phagocytic activity) and hepatic microvascular leukocyte activation, with stasis in sinusoids and adherence in postsinusoidal venules (1 h) followed by leukocytic infiltration into tissue (6 h) and progredient sinusoidal perfusion failure (6 h). Hepatic microvascular injury was accompanied by reduced bile flow and enhanced liver enzyme release. Vitamin E-enriched diet (8000 mg/kg) and even vitamin E-deficient diet did not significantly affect LPS-induced hepatic microvascular cell activation and perfusion failure. Thus, we conclude, that vitamin E is not effective to protect from endotoxin-induced hepatic microvascular dysfunction. Received 7 November 1996; received after revision 30 December 1996; accepted 20 January 1997  相似文献   

9.
The mitochondrial H+-ATP synthase is a primary hub of cellular homeostasis by providing the energy required to sustain cellular activity and regulating the production of signaling molecules that reprogram nuclear activity needed for adaption to changing cues. Herein, we summarize findings regarding the regulation of the activity of the H+-ATP synthase by its physiological inhibitor, the ATPase inhibitory factor 1 (IF1) and their functional role in cellular homeostasis. First, we outline the structure and the main molecular mechanisms that regulate the activity of the enzyme. Next, we describe the molecular biology of IF1 and summarize the regulation of IF1 expression and activity as an inhibitor of the H+-ATP synthase emphasizing the role of IF1 as a main driver of energy rewiring and cellular signaling in cancer. Findings in transgenic mice in vivo indicate that the overexpression of IF1 is sufficient to reprogram energy metabolism to an enhanced glycolysis and activate reactive oxygen species (ROS)-dependent signaling pathways that promote cell survival. These findings are placed in the context of mitohormesis, a program in which a mild mitochondrial stress triggers adaptive cytoprotective mechanisms that improve lifespan. In this regard, we emphasize the role played by the H+-ATP synthase in modulating signaling pathways that activate the mitohormetic response, namely ATP, ROS and target of rapamycin (TOR). Overall, we aim to highlight the relevant role of the H+-ATP synthase and of IF1 in cellular physiology and the need of additional studies to decipher their contributions to aging and age-related diseases.  相似文献   

10.
Metallothioneins I+II (MT-I+II) are antioxidant, neuroprotective factors. We previously showed that MT-I+II deficiency during experimental autoimmune encephalomyelitis (EAE) leads to increased disease incidence and clinical symptoms. Moreover, the inflammatory response of macrophages and T cells, oxidative stress, and apoptotic cell death during EAE were increased by MT-I+II deficiency. We now show for the first time that demyelination and axonal damage are significantly increased in MT-I+II deficient mice during EAE. Furthermore, oligodendroglial regeneration, growth cone formation, and tissue repair including expression of trophic factors were significantly reduced in MT-I+II-deficient mice during EAE. Accordingly, MT-I+II have protective and regenerative roles in the brain. Received 31 October 2002; received after revision 23 November 2002; accepted 26 November 2002 RID="*" ID="*"Corresponding author. M. Penkowa and C. Espejo contributed equally to this paper.  相似文献   

11.
Dietary long-chain polyunsaturated fatty acids are known to influence brain levels of the endocannabinoid anandamide in newborn pigs and mice. Furthermore, endocannabinoids were shown to control pup suckling and body weight in mice, and food intake in adult rodents. Here we determined the effect of maternal under-nutrition during gestation, lactation, or both, on body weight, and on the levels of endocannabinoids and expression of cannabinoid CB1 receptors and fatty acid amide hydrolase in the hypothalamus of rat pups at weaning (21 days old) or adult rats (4 months old). Maternal under-nutrition resulted in a striking decrease in body weight of weaning rats, paralleled by a decrease in the hypothalamic levels of the endocannabinoid anandamide, but not of 2-arachidonoylglycerol. No significant change in the hypothalamic expression of either cannabinoid CB1 receptors or fatty acid amide hydrolase mRNA was detected in any of the three groups of weaned pups. The decrease in pup body weight and hypothalamic anandamide levels was not observable in 4-month-old rats from any of the three groups. These data suggest that maternal under-nutrition causes a decrease in hypothalamic anandamide levels and loss of body weight, and confirm a crucial role for endocannabinoid signalling in neonatal development. Received 4 November 2002; received after revision 29 November 2002; accepted 16 December 2002 RID="*" ID="*"Corresponding author.  相似文献   

12.
13.
Adrenocorticotropin hormone (ACTH) receptor-like messenger RNA was localized in molluscan hemocytes and human peripheral blood mononuclear cells by in situ hybridization using a digoxigenin-labelled bovine complementary DNA probe. These findings suggest that the ACTH receptor gene has been highly conserved during evolution. Moreover, these data represent further support for a relationship between the immune and neuroendocrine systems in invertebrates, as documented in our previous studies [1]. Received 25 September 1997; received after revision 12 November 1997; accepted 12 November 1997  相似文献   

14.
OCT4 is considered a main regulator of embryonic stem cell pluripotency and self renewal capacity. It was shown that relevant OCT4 expression only occurs in cells of embryonic pluripotent nature. However, several recent publications claimed to have demonstrated OCT4 expression in human somatic tumor cells, human adult stem or progenitor cells and differentiated cells.We analysed 42 human tumor cell lines from 13 entities and human bone marrowderived mesenchymal stem cells (MSC). To validate OCT4 expression we used germ cell tumor (GCT) cell lines, derived xenografts and GCT samples. Analysis by RT-PCR, western blotting, immunocytochemistry and immunohistochemistry was performed. With exception of typical embryonal carcinoma cells, we did not observe reliable OCT4 expression in somatic tumor cell lines and MSC. We suggest that a high level of expression of the OCT4 protein together with its nuclear localization still remains a reliable and definitive feature of cells with embryonic pluripotent nature. Received 30 September 2008; received after revision 05 November 2008; accepted 10 November 2008  相似文献   

15.
To identify individual chromosomes of a frog karyotype by their fluorescence banding patterns, chromosomes were stained with actinomycin D and 4,6-diamidino-2-phenylindole (DAPI) after incorporation of BrdU during the late S-phase. The chromosomes of three Rana species which were selected for this study (R. ridibunda, R. lessonae and R. japonica) showed well-defined late replication bands. The fluorescence patterns obtained were the reverse of those produced by a 4Na-EDTA Giemsa-staining technique. Fluorescence patterns of the two water frog species (R. ridibunda and R. lessonae) were similar to each other, except for the different fluorescence of the centromeric heterochromatin, which gave extremely bright signals in R. ridibunda but no signal in R. lessonae. Experiments also showed differences between the fluorescence patterns of R. lessonae chromosome 13 in the Italian and Luxembourgian populations. These results sho w that the fluorescence replication banding using actinomycin D and DAPI is very effective in identifying individual frog chromosomes and detecting their structural changes. Received 7 June 1996; received after revision 23 July 1996; accepted 21 August 1996  相似文献   

16.
Synthetic peptides derived from the C-terminal end of the human complement serine protease C1s were analysed by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy. Circular dichroism indicates that peptides 656-673 and 653-673 are essentially unstructured in water and undergo a coil-to-helix transition in the presence of increasing concentrations of trifluoroethanol. Two-dimensional NMR analyses performed in water/trifluoroethanol solutions provide evidence for the occurrence of a regular α-helix extending from Trp659 to Ser668 (peptide 656-673), and from Tyr656 to Ser668 (peptide 653-673), the C-terminal segment of both peptides remaining unstructured under the conditions used. Based on these and other observations, we propose that the serine protease domain of C1s ends in a 13-residue α-helix (656Tyr-Ser668) followed by a five-residue C-terminal extension. The latter appears to be flexible and is probably locked within C1s through a salt bridge involving Glu672. Received 19 November 1997; accepted 24 November 1997  相似文献   

17.
Insulin action is initiated by binding to its cognate receptor, which then triggers multiple cellular responses by activating different signaling pathways. There is evidence that insulin receptor signaling may involve G protein activation in different target cells. We have studied the activation of G proteins in rat hepatoma (HTC) cells. We found that insulin stimulated binding of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-35S) to plasma membrane proteins of HTC cells, in a dose-dependent manner. This effect was completely blocked by pertussis toxin treatment of the membranes, suggesting the involvement of G proteins of the Gα i/Gα o family. The expression of these Gα proteins was checked by Western blotting. Next, we used blocking antibodies to sort out the specific Gα protein activated by insulin stimulation. Anti-Gα il,2 antibodies completely prevented insulin-stimulated GTP binding, whereas anti-Gα o,i3 did not modify this effect of insulin on GTP binding. Moreover, we found physical association of the insulin receptor with Gα i1,2 by copurification studies. These results further support the involvement of a pertussis toxin-sensitive G protein in insulin receptor signaling and provides some evidence of specific association and activation of Gα i1,2 protein by insulin. These findings suggest that Gα i1,2 proteins might be involved in insulin action. Received 23 September 1998; received after revision 23 November 1998; accepted 25 November 1998  相似文献   

18.
Avian egg whites are a rich source of protein inhibitors of proteinases belonging to all four mechanistic classes. Ovomucoid and ovoinhibitor are multidomain Kazal-type inhibitors with each domain containing an actual or putative reactive site for a serine proteinase. Cystatin is a cysteine proteinase inhibitor, while ovostatin inhibits proteinases of all four mechanistic classes. In this review we have summarized the general features, isolation, inhibitory mechanism and evolutionary aspects of these inhibitors. Received 6 March 1996; received after revision 17 June 1996; accepted 10 July 1996  相似文献   

19.
The assembly of the protein synthesis machinery occurs during translation initiation. In bacteria, this process involves the binding of messenger RNA(mRNA) start site and fMet-tRNAfMet to the ribosome, which results in the formation of the first codon-anticodon interaction and sets the reading frame for the decoding of the mRNA. This interaction takes place in the peptidyl site of the 30S ribosomal subunit and is controlled by the initiation factors IF1, IF2 and IF3 to form the 30S initiation complex. The binding of the 50S subunit and the ejection of the IFs mark the irreversible transition to the elongation phase. Visualization of these ligands on the ribosome has been achieved by cryo-electron microscopy and X-ray crystallography studies, which has helped to understand the mechanism of translation initiation at the molecular level. Conformational changes associated with different functional states provide a dynamic view of the initiation process and of its regulation. Received 16 July 2008; received after revision 31 August 2008; accepted 10 September 2008 A. Simonetti, S. Marzid: These authors contributed equally to this work.  相似文献   

20.
The neurotoxins produced by various species of Clostridia are the causative agents of botulism and tetanus. The ability of the toxins, specifically those of the botulinum neurotoxin family, to disrupt neurotransmission has been exploited for use in several medical indications and now represents the therapeutic option of choice in a number of cases. Clostridial neurotoxins have been discovered to have a multi-domain structure that is shared between the various proteins of the family, and it has also been determined that each domain contributes a specific role to the holotoxin. The extensive use of recombinant expression approaches, along with solution of multiple crystallographic structures of individual domains, has enabled researchers to explore structurefunction relationships of the toxin domains more closely. These advances have facilitated a greater understanding of the potential use of individual domains for a wide variety of purposes, including the development of new therapeutics. Received 21 October 2005; received after revision 10 November 2005; accepted 16 November 2005  相似文献   

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