Showing papers in "FEBS Journal in 1999"
TL;DR: Although the precise role of A-SAA in host defense during inflammation has not been defined, many potential clinically important functions have been proposed for individual SAA family members, including involvement in lipid metabolism/transport, induction of extracellular-matrix-degrading enzymes, and chemotactic recruitment of inflammatory cells to sites of inflammation.
Abstract: The serum amyloid A (SAA) family comprises a number of differentially expressed apolipoproteins, acute-phase SAAs (A-SAAs) and constitutive SAAs (C-SAAs). A-SAAs are major acute-phase reactants, the in vivo concentrations of which increase by as much as 1000-fold during inflammation. A-SAA mRNAs or proteins have been identified in all vertebrates investigated to date and are highly conserved. In contrast, C-SAAs are induced minimally, if at all, during the acute-phase response and have only been found in human and mouse. Although the liver is the primary site of synthesis of both A-SAA and C-SAA, extrahepatic production has been reported for most family members in most of the mammalian species studied. In vitro, the dramatic induction of A-SAA mRNA in response to pro-inflammatory stimuli is due largely to the synergistic effects of cytokine signaling pathways, principally those of the interleukin-1 and interleukin-6 type cytokines. This induction can be enhanced by glucocorticoids. Studies of the A-SAA promoters in several mammalian species have identified a range of transcription factors that are variously involved in defining both cytokine responsiveness and cell specificity. These include NF-κB, C/EBP, YY1, AP-2, SAF and Sp1. A-SAA is also post-transcriptionally regulated. Although the precise role of A-SAA in host defense during inflammation has not been defined, many potential clinically important functions have been proposed for individual SAA family members. These include involvement in lipid metabolism/transport, induction of extracellular-matrix-degrading enzymes, and chemotactic recruitment of inflammatory cells to sites of inflammation. A-SAA is potentially involved in the pathogenesis of several chronic inflammatory diseases: it is the precursor of the amyloid A protein deposited in amyloid A amyloidosis, and it has also been implicated in the pathogenesis of atheroscelerosis and rheumatoid arthritis.
1,038 citations
TL;DR: This work tries to identify the mechanistic aspects of mitochondrial involvement in cell death as can be derived from current understanding of mitochondrial physiology, with special emphasis on the permeability transition and its consequences (like onset of swelling, cytochrome c release and respiratory inhibition).
Abstract: Mitochondria are involved in cell death for reasons that go beyond ATP supply. A recent advance has been the discovery that mitochondria contain and release proteins that are involved in the apoptotic cascade, like cytochrome c and apoptosis inducing factor. The involvement of mitochondria in cell death, and its being cause or consequence, remain issues that are extremely complex to address in situ. The response of mitochondria may critically depend on the type of stimulus, on its intensity, and on the specific mitochondrial function that has been primarily perturbed. On the other hand, the outcome also depends on the integration of mitochondrial responses that cannot be dissected easily. Here, we try to identify the mechanistic aspects of mitochondrial involvement in cell death as can be derived from our current understanding of mitochondrial physiology, with special emphasis on the permeability transition and its consequences (like onset of swelling, cytochrome c release and respiratory inhibition); and to critically evaluate methods that are widely used to monitor mitochondrial function in situ.
743 citations
TL;DR: A classification containing 10 different groups of toxins is proposed in this review, based on functional and structural features of the known toxins, and the limited success obtained in the search for the site through which these peptides bind to the channels.
Abstract: Na+-channel specific scorpion toxins are peptides of 60-76 amino acid residues in length, tightly bound by four disulfide bridges. The complete amino acid sequence of 85 distinct peptides are presently known. For some toxins, the three-dimensional structure has been solved by X-ray diffraction and NMR spectroscopy. A constant structural motif has been found in all of them, consisting of one or two short segments of alpha-helix plus a triple-stranded beta-sheet, connected by variable regions forming loops (turns). Physiological experiments have shown that these toxins are modifiers of the gating mechanism of the Na+-channel function, affecting either the inactivation (alpha-toxins) or the activation (beta-toxins) kinetics of the channels. Many functional variations of these peptides have been demonstrated, which include not only the classical alpha- and beta-types, but also the species specificity of their action. There are peptides that bind or affect the function of Na+-channels from different species (mammals, insects or crustaceans) or are toxic to more than one group of animals. Based on functional and structural features of the known toxins, a classification containing 10 different groups of toxins is proposed in this review. Attempts have been made to correlate the presence of certain amino acid residues or 'active sites' of these peptides with Na+-channel functions. Segments containing positively charged residues in special locations, such as the five-residue turn, the turn between the second and the third beta-strands, the C-terminal residues and a segment of the N-terminal region from residues 2-11, seems to be implicated in the activity of these toxins. However, the uncertainty, and the limited success obtained in the search for the site through which these peptides bind to the channels, are mainly due to the lack of an easy method for expression of cloned genes to produce a well-folded, active peptide. Many scorpion toxin coding genes have been obtained from cDNA libraries and from polymerase chain reactions using fragments of scorpion DNAs, as templates. The presence of an intron at the DNA level, situated in the middle of the signal peptide, has been demonstrated.
635 citations
TL;DR: It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of .OH from O2.- and H2O2 (iron-catalyzed Haber-Weiss reaction).
Abstract: Peroxidases catalyze the dehydrogenation by hydrogen peroxide (H2O2) of various phenolic and endiolic substrates in a peroxidatic reaction cycle. In addition, these enzymes exhibit an oxidase activity mediating the reduction of O2 to superoxide (O2.-) and H2O2 by substrates such as NADH or dihydroxyfumarate. Here we show that horseradish peroxidase can also catalyze a third type of reaction that results in the production of hydroxyl radicals (.OH) from H2O2 in the presence of O2.-. We provide evidence that to mediate this reaction, the ferric form of horseradish peroxidase must be converted by O2.- into the perferryl form (Compound III), in which the haem iron can assume the ferrous state. It is concluded that the ferric/perferryl peroxidase couple constitutes an effective biochemical catalyst for the production of .OH from O2.- and H2O2 (iron-catalyzed Haber-Weiss reaction). This reaction can be measured either by the hydroxylation of benzoate or the degradation of deoxyribose. O2.- and H2O2 can be produced by the oxidase reaction of horseradish peroxidase in the presence of NADH. The .OH-producing activity of horseradish peroxidase can be inhibited by inactivators of haem iron or by various O2.- and .OH scavengers. On an equimolar Fe basis, horseradish peroxidase is 1-2 orders of magnitude more active than Fe-EDTA, an inorganic catalyst of the Haber-Weiss reaction. Particularly high .OH-producing activity was found in the alkaline horseradish peroxidase isoforms and in a ligninase-type fungal peroxidase, whereas lactoperoxidase and soybean peroxidase were less active, and myeloperoxidase was inactive. Operating in the .OH-producing mode, peroxidases may be responsible for numerous destructive and toxic effects of activated oxygen reported previously.
441 citations
TL;DR: The targeted derivative of vitamin E protected mitochondrial function from oxidative damage far more effectively than vitamin E itself and suggests a new family of mitochondrially targeted antioxidants, redox-active and pharmacologically active molecules designed to prevent damage or manipulate mitochondrial function.
Abstract: Mitochondrial oxidative damage contributes significantly to a range of human disorders, including neurodegenerative diseases, ischaemia-reperfusion injury and ageing-associated dysfunction. To prevent this damage we have delivered a molecule containing the active antioxidant moiety of vitamin E to mitochondria. This was carried out by covalently coupling the antioxidant moiety to a lipophilic triphenylphosphonium cation. This mitochondrially targeted antioxidant, 2-[2-(triphenylphosphonio)ethyl]-3,4-dihydro-2, 5,7,8-tetramethyl-2H-1-benzopyran-6-ol bromide (TPPB), accumulated several-hundred fold within the mitochondrial matrix, driven by the organelle's large membrane potential. When cells were incubated with micromolar concentrations of TPPB, they accumulated millimolar concentrations within their mitochondria. The amount of TPPB taken up by mitochondria was approximately 80-fold greater than endogenous levels of vitamin E. Consequently the targeted derivative of vitamin E protected mitochondrial function from oxidative damage far more effectively than vitamin E itself. The mitochondrially targeted antioxidant TPPB has potential as an antioxidant therapy for disorders involving mitochondrial oxidative damage. It also suggests a new family of mitochondrially targeted antioxidants, redox-active and pharmacologically active molecules designed to prevent damage or manipulate mitochondrial function.
437 citations
TL;DR: Redundant systems of acyltransferases, the interplay of organelles, regulation of the pathway on the compartmental level, and the contribution of alternative pathways to PtdOH biosynthesis appear to be required for the balanced formation of this important lipid intermediate.
Abstract: Phosphatidic acid (PtdOH) is a key intermediate in glycerolipid biosynthesis. Two different pathways are known for de novo formation of this compound, namely (a) the Gro3P (glycerol 3-phosphate) pathway, and (b) the GrnP (dihydroxyacetone phosphate) pathway. Whereas the former route of PtdOH synthesis is present in bacteria and all types of eukaryotes, the GrnP pathway is restricted to yeast and mammalian cells. In this review article, we describe the enzymes catalyzing de novo formation of PtdOH, their properties and their occurrence in different cell types and organelles. Much attention has recently been paid to the subcellular localization of enzymes involved in the biosynthesis of PtdOH. In all eukaryotic cells, microsomes (ER) harbour the complete set of enzymes catalyzing these pathways and are thus the usual organelle for PtdOH formation. In contrast, the contribution of mitochondria to PtdOH synthesis is restricted to certain enzymes and depends on the cell type. In addition, chloroplasts of plants, lipid particles of the yeast, and peroxisomes of mammalian cells are significantly involved in PtdOH biosynthesis. Redundant systems of acyltransferases, the interplay of organelles, regulation of the pathway on the compartmental level, and finally the contribution of alternative pathways (phosphorylation of diacylglycerol and cleavage of phospholipids by phospholipases) to PtdOH biosynthesis appear to be required for the balanced formation of this important lipid intermediate. Dysfunction of enzymes involved in PtdOH synthesis can result in severe defects of various cellular processes. In this context, the possible physiological role(s) of PtdOH and its related metabolites, lysophosphatidic acid and diacylglycerol, will be discussed.
368 citations
TL;DR: The purpose of this review is to describe the domain structure of plant transcription factors, and to relate this information to processes that control the synthesis and action of these proteins.
Abstract: A typical plant transcription factor contains, with few exceptions, a DNA-binding region, an oligomerization site, a transcription-regulation domain, and a nuclear localization signal Most transcription factors exhibit only one type of DNA-binding and oligomerization domain, occasionally in multiple copies, but some contain two distinct types DNA-binding regions are normally adjacent to or overlap with oligomerization sites, and their combined tertiary structure determines critical aspects of transcription factor activity Pairs of nuclear localization signals exist in several transcription factors, and basic amino acid residues play essential roles in their function, a property also true for DNA-binding domains Multigene families encode transcription factors, with members either dispersed in the genome or clustered on the same chromosome Distribution and sequence analyses suggest that transcription factor families evolved via gene duplication, exon capture, translocation, and mutation The expression of transcription factor genes in plants is regulated at transcriptional and post-transcriptional levels, while the activity of their protein products is modulated post-translationally The purpose of this review is to describe the domain structure of plant transcription factors, and to relate this information to processes that control the synthesis and action of these proteins
363 citations
TL;DR: Insights into the progression of the exocytotic vesicle from docking to fusion and how these processes are precisely regulated by proteins and second messengers may provide the basis for new therapeutic principles.
Abstract: Secretion of the peptide hormone insulin from pancreatic beta cells constitutes an important step in the regulation of body homeostasis. Insulin is stored in large dense core vesicles and released by exocytosis, a multistage process involving transport of vesicles to the plasma membrane, their docking, priming and finally their fusion with the plasma membrane. Some of the protein components necessary for this process have been identified in beta cells. The export of potent and potentially harmful substances has to be tightly controlled. The secretory response in pancreatic beta cells requires the concerted action of nutrients together with enteric hormones and neurotransmitters acting on G-protein coupled receptors. It is well established that glucose and other metabolizable nutrients depolarize the beta-cell membrane and the ensuing Ca2+ influx through voltage-dependent channels constitutes a main stimulus for insulin exocytosis. Theoretical considerations and recent observations suggest in addition an organizing role for the Ca2+ channel similar to neurotransmission. A second regulatory control on exocytosis is exerted by monomeric and heterotrimeric G-proteins. The monomeric GTPase Rab3A controls insulin secretion through cycling between a guanosine triphosphate liganded vesicle-bound form and a guanosine diphosphate liganded, cytosolic form. The effect of neurohormones is transduced by the heterotrimeric GTPases. Whereas pertussis-toxin sensitive alpha-subunits exert direct inhibition at the level of exocytosis, the Gbeta gamma-subunits are required for stimulation. It is possible that these GTPases exert immediate regulation, while protein kinases and phosphatases may modulate long-term adaptation at the exocytotic machinery itself. The molecular nature of their activators and effectors still await identification. Insights into the progression of the exocytotic vesicle from docking to fusion and how these processes are precisely regulated by proteins and second messengers may provide the basis for new therapeutic principles.
362 citations
TL;DR: It is postulate that the homology of AChE to the cell adhesion proteins, gliotactin, glutactin and the neurexins, which have more established functions in nervous system development, is the basis of its morphogenic functions.
Abstract: Apart from its catalytic function in hydrolyzing acetylcholine, acetylcholinesterase (AChE) affects cell proliferation, differentiation and responses to various insults, including stress. These responses are at least in part specific to the three C-terminal variants of AChE which are produced by alternative splicing of the single ACHE gene. 'Synaptic' AChE-S constitutes the principal multimeric enzyme in brain and muscle; soluble, monomeric 'readthrough' AChE-R appears in embryonic and tumor cells and is induced under psychological, chemical and physical stress; and glypiated dimers of erythrocytic AChE-E associate with red blood cell membranes. We postulate that the homology of AChE to the cell adhesion proteins, gliotactin, glutactin and the neurexins, which have more established functions in nervous system development, is the basis of its morphogenic functions. Competition between AChE variants and their homologs on interactions with the corresponding protein partners would inevitably modify cellular signaling. This can explain why AChE-S exerts process extension from cultured amphibian, avian and mammalian glia and neurons in a manner that is C-terminus-dependent, refractory to several active site inhibitors and, in certain cases, redundant to the function of AChE-like proteins. Structural functions of AChE variants can explain their proliferative and developmental roles in blood, bone, retinal and neuronal cells. Moreover, the association of AChE excess with amyloid plaques in the degenerating human brain and with progressive cognitive and neuromotor deficiencies observed in AChE-transgenic animal models most likely reflects the combined contributions of catalytic and structural roles.
359 citations
TL;DR: It is found that ionomycin and lipopolysaccharide cause a 24-fold and 2.5-fold stimulation of 2-AG levels in J774 cells, respectively, thus providing unprecedented evidence that this cannabimimetic metabolite can be synthesized by macrophages.
Abstract: The stimulus-induced biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) in intact mouse J774 macrophages and the inactivation of 2-AG by the same cells or by rat circulating macrophages was studied. By using gas chromatography-mass spectrometry, we found that ionomycin (5 µm) and lipopolysaccharide (LPS, 200 µg·mL−1) cause a 24-fold and 2.5-fold stimulation of 2-AG levels in J774 cells, respectively, thus providing unprecedented evidence that this cannabimimetic metabolite can be synthesized by macrophages. In J774 cells, LPS also induced a 7.8-fold increase of the levels of the other endocannabinoid, anandamide, and, in rat circulating macrophages, an almost twofold increase of 2-AG levels. Extracellular [3H]2-AG was cleared from the medium of intact J774 macrophages (t1/2 = 19–28 min) and esterified to phospholipids, diacylglycerols and triglycerides or hydrolyzed to [3H]arachidonic acid and glycerol. These catabolic processes were attenuated differentially by various enzyme inhibitors. Rat circulating macrophages were shown to contain enzymatic activities for the hydrolysis of 2-AG, including: (a) fatty acid amide hydrolase (FAAH), the enzyme responsible for anandamide breakdown and previously shown to catalyse also 2-AG hydrolysis, and (b) a 2-AG hydrolase activity different from FAAH and down-regulated by LPS. High levels of FAAH mRNA were found in circulating macrophages but not platelets, which, however, contain a 2-AG hydrolase. Both platelets and macrophages were shown to express the mRNA for the CB1 cannabinoid receptor. A macrophage 2-AG hydrolase with apparent Km = 110 µm and Vmax = 7.9 nmol·min−1·(mg protein)−1 was partially characterized in J774 cells and found to exhibit an optimal pH of 6–7 and little or no sensitivity to typical FAAH inhibitors. These findings demonstrate for the first time that macrophages participate in the homeostasis of the hypotensive and immunomodulatory endocannabinoid 2-AG through metabolic mechanisms that are subject to regulation.
305 citations
TL;DR: Export of phosphatidylethanolamine from mitochondria to the endoplasmic reticulum was shown to be energy-independent in the reconstituted yeast system, in contrast with previous observations with permeabilized yeast cells.
Abstract: Membrane association between mitochondria and the endoplasmic reticulum of the yeast Saccharomyces cerevisiae is probably a prerequisite for phospholipid translocation between these two organelles. This association was visualized by fluorescence microscopy and computer-aided three-dimensional reconstruction of electron micrographs from serial ultrathin sections of yeast cells. A mitochondria-associated membrane (MAM), which is a subfraction of the endoplasmic reticulum, was isolated and re-associated with mitochondria in vitro. In the reconstituted system, phosphatidylserine synthesized in MAM was imported into mitochondria independently of cytosolic factors, bivalent cations, ATP, and ongoing synthesis of phosphatidylserine. Proteolysis of mitochondrial surface proteins by treatment with proteinase K reduced the capacity to import phosphatidylserine. Phosphatidylethanolamine formed in mitochondria by decarboxylation of phosphatidylserine is exported to the endoplasmic reticulum where part of it is converted into phosphatidylcholine. In contrast with previous observations with permeabilized yeast cells [Achleitner, G., Zweytick, D., Trotter, P., Voelker, D. & Daum, G. (1995) J. Biol. Chem.270, 29836–29842], export of phosphatidylethanolamine from mitochondria to the endoplasmic reticulum was shown to be energy-independent in the reconstituted yeast system.
TL;DR: The main emphasis is placed on recent data characterizing the alpha-neurotoxin interactions with nicotinic acetylcholine receptors.
Abstract: The review is mainly devoted to snake venom alpha-neurotoxins which target different muscle-type and neuronal nicotinic acetylcholine receptors. The primary and spatial structures of other snake venom proteins as well as mammalian proteins of the Ly-6 family, which structurally resemble the 'three-finger' snake proteins, are also briefly discussed. The main emphasis is placed on recent data characterizing the alpha-neurotoxin interactions with nicotinic acetylcholine receptors.
TL;DR: The experimental data suggest that the proteins described here could be involved in contact chemoreception in Orthoptera, in disagreement with the hypothesis that this class of proteins is involved in the CO2-sensing cascade.
Abstract: Soluble low-molecular-mass protein isoforms were purified from chemosensory organs (antennae, tarsi and labrum) of the desert locust Schistocerca gregaria. Five genes encoding proteins of this group were amplified by PCR from cDNAs of tarsi and sequenced. Their expression products are polypeptide chains of 109 amino acids showing 40–50% sequence identity with putative olfactory proteins from Drosophila melanogaster and Cactoblastis cactorum. Direct structural investigation on isoforms purified from chemosensory organs revealed the presence in the expression products of two of the genes cloned. Two additional protein isoforms were detected and their molecular structure exhaustively characterized. MS analysis of all isoforms demonstrated that the four cysteine residues conserved in the polypeptide chain were involved in disulfide bridges (Cys29–Cys38 and Cys57–Cys60) and indicated the absence of any additional post-translational modifications. Immunocytochemistry experiments, performed with rabbit antiserum raised against the protein isoform mixture, showed selective labelling of the outer lymph in contact sensilla of tarsi, maxillary palps and antennae. Other types of sensilla were not labelled, nor were the cuticle and dendrites of the sensory cells. No binding of radioactively labelled glucose or bicarbonate was detected, in disagreement with the hypothesis that this class of proteins is involved in the CO2-sensing cascade. Our experimental data suggest that the proteins described here could be involved in contact chemoreception in Orthoptera.
TL;DR: The cloning of a mouse GCN2 cDNA (MGCN2) is described, which represents the first mammalian GCn2 homolog and provides evidence that GCN 2 is the unique eIF2alpha kinase present in all eukaryotes from yeast to mammals and underscores the role of MGCN 2 kinase in translational control and its potential physiological significance.
Abstract: In eukaryotic cells, protein synthesis is regulated in response to various environmental stresses by phosphorylating the α subunit of the eukaryotic initiation factor 2 (eIF2α). Three different eIF2α kinases have been identified in mammalian cells, the heme-regulated inhibitor (HRI), the interferon-inducible RNA-dependent kinase (PKR) and the endoplasmic reticulum-resident kinase (PERK). A fourth eIF2α kinase, termed GCN2, was previously characterized from Saccharomyces cerevisiae, Drosophila melanogaster and Neurospora crassa. Here we describe the cloning of a mouse GCN2 cDNA (MGCN2), which represents the first mammalian GCN2 homolog. MGCN2 has a conserved motif, N-terminal to the kinase subdomain V, and a large insert of 139 amino acids located between subdomains IV and V that are characteristic of the known eIF2α kinases. Furthermore, MGCN2 contains a class II aminoacyl-tRNA synthetase domain and a degenerate kinase segment, downstream and upstream of the eIF2α kinase domain, respectively, and both are singular features of GCN2 protein kinases. MGCN2 mRNA is expressed as a single message of ≈ 5.5 kb in a wide range of different tissues, with the highest levels in the liver and the brain. Specific polyclonal anti-(MGCN2) immunoprecipitated an eIF2α kinase activity and recognized a 190 kDa phosphoprotein in Western blots from either mouse liver or MGCN2-transfected 293 cell extracts. Interestingly, serum starvation increased eIF2α phosphorylation in MGCN2-transfected human 293T cells. This finding provides evidence that GCN2 is the unique eIF2α kinase present in all eukaryotes from yeast to mammals and underscores the role of MGCN2 kinase in translational control and its potential physiological significance.
TL;DR: Findings clearly demonstrate that glycosaminoglycans act at the earliest stage of fibril formation, namely amyloid-beta nucleation, and are not simply involved in the lateral aggregation of preformed fibrils or nonspecific adhesion to plaques.
Abstract: Proteoglycans and their constituent glycosaminoglycans are associated with all amyloid deposits and may be involved in the amyloidogenic pathway. In Alzheimer's disease, plaques are composed of the amyloid-beta peptide and are associated with at least four different proteoglycans. Using CD spectroscopy, fluorescence spectroscopy and electron microscopy, we examined glycosaminoglycan interaction with the amyloid-beta peptides 1-40 (Abeta40) and 1-42 (Abeta42) to determine the effects on peptide conformation and fibril formation. Monomeric amyloid-beta peptides in trifluoroethanol, when diluted in aqueous buffer, undergo a slow random to amyloidogenic beta sheet transition. In the presence of heparin, heparan sulfate, keratan sulfate or chondroitin sulfates, this transition was accelerated with Abeta42 rapidly adopting a beta-sheet conformation. This was accompanied by the appearance of well-defined amyloid fibrils indicating an enhanced nucleation of Abeta42. Incubation of preformed Abeta42 fibrils with glycosaminoglycans resulted in extensive lateral aggregation and precipitation of the fibrils. The glycosaminoglycans differed in their relative activities with the chondroitin sulfates producing the most pronounced effects. The less amyloidogenic Abeta40 isoform did not show an immediate structural transition that was dependent upon the shielding effect by the phosphate counter ion. Removal or substitution of phosphate resulted in similar glycosaminoglycan-induced conformational and aggregation changes. These findings clearly demonstrate that glycosaminoglycans act at the earliest stage of fibril formation, namely amyloid-beta nucleation, and are not simply involved in the lateral aggregation of preformed fibrils or nonspecific adhesion to plaques. The identification of a structure-activity relationship between amyloid-beta and the different glycosaminoglycans, as well as the condition dependence for glycosaminoglycan binding, are important for the successful development and evaluation of glycosaminoglycan-specific therapeutic interventions.
TL;DR: Extracts of the granular haemocytes of Carcinus maenas were subjected to ion-exchange chromatography and reverse-phase (RP)-HPLC to investigate the presence of an antibacterial protein, which was found to be cationic and hydrophobic and active only against marine Gram-positive bacteria.
Abstract: Extracts of the granular haemocytes of Carcinus maenas were subjected to ion-exchange chromatography and reverse-phase (RP)-HPLC to investigate the presence of an antibacterial protein of approximately 11 kDa. This protein was isolated, characterized and subjected to partial amino acid sequence analysis. It was found by mass spectrometry to have a molecular mass of 11 534 Da, to be cationic and hydrophobic and active only against marine Gram-positive bacteria. In addition its activity is stable after heating to 100 degrees C and is retained at concentrations as low as 10 microgram.mL-1. It has an unusual amino acid sequence, unlike any known antibacterial peptide described in the literature but bears a consensus disulphide domain signature, indicating that it might be a member of the four-disulphide core proteins. Partial cDNA sequence data has been obtained.
TL;DR: The isolation of two isoforms of a novel cysteine-rich peptide from haemocytes and plasma of the mussel, Mytilus galloprovincialis, suggests that myticins are synthesized as preproproteins and then processed by various proteolytic events before storage of the active peptide in the haemocyte.
Abstract: We report here the isolation of two isoforms of a novel cysteine-rich peptide from haemocytes (isoform A of 4.438 Da and B of 4.562 Da) and plasma (isoform A) of the mussel, Mytilus galloprovincialis. The two molecules display antibacterial activity against gram-positive bacteria, whereas only isoform B is active against the fungus Fusarium oxysporum and a gram-negative bacteria Escherichia coli D31. Complete peptide sequences were determined by a combination of Edman degradation, mass spectrometry and cDNA cloning using a haemocyte cDNA library. The mature molecules, named myticins, comprise 40 residues with four intramolecular disulfide bridges and a cysteine array in the primary structure different to that of the previously characterized cysteine-rich antimicrobial peptides. Sequence analysis of the cloned cDNAs revealed that myticin precursors consist of 96 amino acids with a putative signal peptide of 20 amino acids, the antimicrobial peptide sequence and a 36-residue C-terminal extension. This structure suggests that myticins are synthesized as preproproteins and then processed by various proteolytic events before storage of the active peptide in the haemocytes. Myticin precursors are expressed mainly in the haemocytes as revealed by Northern blot analysis.
TL;DR: It is demonstrated that the human ST2 gene has two alternative promoters followed by distinct noncoding first exons, which are located more than 8 kb apart and are spliced to the common exon 2 containing the translation initiation site.
Abstract: The ST2 gene encodes receptor-like molecules that are very similar to the type I interleukin-1 receptor. Two distinct types of the ST2 gene products, ST2 (a soluble secreted form) and ST2L (a transmembrane form) are produced by alternative splicing. Here we demonstrate that the human ST2 gene has two alternative promoters followed by distinct noncoding first exons, which are located more than 8 kb apart and are spliced to the common exon 2 containing the translation initiation site. Within 1001 bp upstream of the transcription initiation site of the cloned distal promoter, there are four GATA-1. The main promoter used for the expression of the ST2 gene in UT-7, a human leukaemic cell line, is distinct from that in TM12, a human fibroblastic cell line. Although UT-7 cells use both distal and proximal promoters, the distal promoter is used dominantly for expression of both ST2 and ST2L mRNA. On the other hand, almost all transcription in TM12 cells starts from the proximal promoter. These results contrast with those of former studies on the rat system, in which ST2 and ST2L mRNA were generated by use of the proximal and distal promoters, respectively. Furthermore, UT-7 cells use multiple transcription initiation sites in both the proximal and distal promoters, whereas the transcription of the ST2 gene in TM12 cells starts at a unique site. Intriguingly, these results suggest that ST2 and ST2L proteins have distinct functions in different cells within different biological systems, such as those of growth control, differentiation and immunological responses.
TL;DR: Data suggest that the activation of p38 and JNK by 1O2 occurs only when the electronically excited molecule is generated intracellularly.
Abstract: Ultraviolet A (UVA; 320–400 nm) radiation in human skin fibroblasts induces a pattern of mitogen-activated protein kinase (MAPK) activation consisting of a rapid and transient induction of p38 and c-Jun-N-terminal kinase (JNK) activity but not extracellular signal-regulated kinases (ERK). UVA activation of p38 can be inhibited by the singlet oxygen (1O2) quenchers azide and imidazole, but not by the hydroxyl radical scavengers mannitol or dimethylsulfoxide, pointing to the involvement of 1O2. The same effect has been shown for JNK. Like UVA, 1O2 generated intracellularly upon photoexcitation of Rose Bengal activates p38 and JNK but not ERK. p38 and JNK activation was also elicited by chemiexcitation for the intracellular generation of 1O2 by the lipophilic 1,4-endoperoxide of N,N′-di(2,3-dihydroxypropyl)-1,4-naphthalene dipropionamide. In contrast, extracellular generation of 1O2, by irradiation of Rose Bengal immobilized on agarose beads or by chemiexcitation employing the hydrophilic 1,4-endoperoxide of disodium 3,3′-(1,4-naphthylidene) dipropionate, was ineffective in activating p38 or JNK. These data suggest that the activation of p38 and JNK by 1O2 occurs only when the electronically excited molecule is generated intracellularly.
TL;DR: The high mRNA stability under selenium restriction, the speed of biosynthesis upon Selenium repletion and the marginal effect of selenum on the SECIS efficiency indicate that of the GPx isotypes, GI-GPx ranks highest in the hierarchy of seLenoproteins and point to a vital role of GI- GPx in the gastrointestinal tract.
Abstract: The recently described gastrointestinal glutathione peroxidase (GI-GPx) is the fourth member of the family of the selenoenzymes glutathione peroxidases (GPx). In contrast to the more uniform distribution of, for example, the classical glutathione peroxidase (cGPx), it is expressed exclusively in the gastrointestinal tract and has, therefore, been suggested to function as a primary barrier against alimentary hydroperoxides. In order to get an idea of its relative importance we investigated its position in the hierarchy of selenoprotein expression. The selenium-dependent expression of GI-GPx was analyzed in comparison with that of other GPx types at the level of mRNA and protein in HepG2 and CaCo-2 cells. Furthermore, the selenocysteine insertion sequence (SECIS) efficiencies of GI-GPx, phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cGPx in response to selenium were determined by a reporter-gene assay in human hepatoma cells and baby hamster kidney cells. GI-GPx mRNA levels increased during selenium deficiency, whereas cGPx mRNA levels decreased and PHGPx mRNA levels remained almost unaffected. In cells grown in selenium-poor media, all GPx-types were low in both activity and immunochemical reactivity. Upon selenium repletion immunoreactive GI-GPx protein reached a plateau after 10 h, whereas cGPx started to be expressed at 24 h and did not reach its maximum level before 3 days. SECIS efficiencies decreased in the order PHGPx > cGPx > GI-GPx. The augmentation of SECIS efficiencies by selenium was highest for cGPx and intermediate for PHGPx, whereas it was marginal for GI-GPx. The high mRNA stability under selenium restriction, the speed of biosynthesis upon selenium repletion and the marginal effect of selenium on the SECIS efficiency indicate that of the GPx isotypes, GI-GPx ranks highest in the hierarchy of selenoproteins and point to a vital role of GI-GPx in the gastrointestinal tract.
TL;DR: Findings indicate that PDE10A functions in these tissues, and its presence in the neurons of the striatum and the olfactory tubercle regions of the brain is indicated.
Abstract: PDE10A, a phosphodiesterase (PDE) exhibiting properties of a cAMP PDE and a cAMP-inhibited cGMP PDE, was cloned and investigated in detail in rats. PDE10A transcripts were abundant in the brain and testis. In situ hybridization analysis using a PDE10A riboprobe demonstrated the presence of PDE10A transcripts in the neurons of the striatum and the olfactory tubercle regions of the brain. Rat PDE10A cDNAs were isolated from a brain cDNA library and nucleotide sequence analysis revealed several N-terminal variants. The deduced amino-acid sequence of one of the major variant forms contained 794 amino acids, and it was 96% identical to that of the human PDE10A2. The other major form has a distinct N-terminal sequence that is not found in humans. PDE10A was partially purified from rat striatum and testis, and characterized with respect to Km, inhibitor sensitivity and immunoreactivity to an anti-PDE10A serum. These findings indicate that PDE10A functions in these tissues.
TL;DR: An extended analysis of the organization of green plant photosystem II and its associated light-harvesting antenna using electron microscopy and image analysis and it is suggested that CP26 and CP24 are not required for the binding of trimeric LHCII at any of the three binding positions.
Abstract: We present an extended analysis of the organization of green plant photosystem II and its associated lightharvesting antenna using electron microscopy and image analysis. The analysis is based on a large dataset of 16 600 projections of negatively stained PSII‐LHCII supercomplexes and megacomplexes prepared by means of three different pretreatments. In addition to our previous work on this system [Boekema, E.J., van Roon, H., Calkoen, F., Bassi, R. and Dekker, J.P. (1999) Biochemistry 38, 2233‐2239], the following results were obtained. The rotational orientation of trimeric LHCII at the S, M and L binding positions was determined. It was found that compared to the S trimer, the M and L trimers are rotationally shifted by about 2208 and 2508, respectively. The number of projections with empty CP29, CP26 and CP24 binding sites was found to be about 0, 18 and 4%, respectively. We suggest that CP26 and CP24 are not required for the binding of trimeric LHCII at any of the three binding positions. A new type of megacomplex was observed with a characteristic windmill-like shape. This type III megacomplex consists of two C2S2 supercomplexes connected at their CP26 tips. Structural variation in the region of the central dimeric photosystem II complex was found to occur at one specific position near the periphery of the complex. We attribute this variation to the partial absence of an extrinsic polypeptide or one or more small intrinsic membrane proteins.
TL;DR: It is shown that the classical mitogenic pathway involving Ras-Erk, PKC-ErK or Rac-JNK, among others, is activated by growth factors or endogenously by oncogenes, and constitutively activates uPA and uPAR expression.
Abstract: We review the evidence in support of the notion that, upon experimental oncogenic transformation or in spontaneous human cancers, mitogenesis and expression of urokinase (uPA) and its receptor (uPAR) are activated through common signaling complexes and pathways. It is well documented that uPA, uPAR or metalloproteinases (MMPs) are overexpressed in tumor cells of mesenchymal or epithelial origin and these molecules are required for tumor invasion and metastasis. Furthermore, oncogenic stimuli, which may render the transformed cells tumorigenic and metastatic in vivo, activate, in a constitutive fashion, the extracellular-regulated kinases (Erk 1 and 2) classical mitogenic pathway and others such as the NH2-Jun-kinase (Jnk). Cells from human tumors or oncogene-transformed cells overexpress uPA and uPAR, and also show a sustained activation of the above-mentioned signaling modules. In this paper we show that the classical mitogenic pathway involving Ras-Erk, PKC-Erk or Rac-JNK, among others, is activated by growth factors or endogenously by oncogenes, and constitutively activates uPA and uPAR expression. All the data obtained from human tumors or experimental systems, incorporated into a general model, indicate that oncogenic stimuli lead to the constitutive activation of mitogenesis and uPA and its receptor expression, through the activation of the same classical and nonclassical signaling complexes and pathways that regulate cell proliferation. We also discuss contrasting points of view. For instance, what governs the differential regulation of mitogenesis and the signal that leads to protease overexpression in a way that allows normal cells during physiological events to respond to growth factors, and proliferate without overexpressing extracellular matrix (ECM) proteases? Or how can cells remodel their microenvironment without proliferating? What restrains benign tumors from overexpressing tumor-associated proteases when they certainly have the mitogenic signal fully activated? This may occur by the differential regulation of transcriptional programs and recent reports reviewed in this paper may provide an insight into how this occurs at the signaling and transcriptional levels.
TL;DR: The results reported here suggest that the anti-apoptotic activity of resveratrol depends on the direct inhibition of the main arachidonate-metabolizing enzymes.
Abstract: The natural polyphenolic compound resveratrol (trans-3,4', 5-trihydroxystilbene) is shown to prevent apoptosis (programmed cell death) induced in human erythroleukemia K562 cells by hydrogen peroxide and other unrelated stimuli. Resveratrol reversed the elevation of leukotriene B4 (from 6.40 +/- 0.65 to 2.92 +/- 0.30 pmol.mg protein-1) and prostaglandin E2 (from 11.46 +/- 1.15 to 8.02 +/- 0.80 nmol.mg protein-1), induced by H2O2 challenge in K562 cells. The reduction of leukotriene B4 and prostaglandin E2 correlated with the inhibition of the 5-lipoxygenase activity, and the cyclooxygenase and peroxidase activity of prostaglandin H synthase, respectively. Resveratrol also blocked lipoperoxidation induced by hydrogen peroxide in K562 cell membranes. Resveratrol was found to act as a competitive inhibitor of purified 5-lipoxygenase and 15-lipoxygenase and prostaglandin H synthase, with inhibition constants of 4.5 +/- 0.5 microM (5-lipoxygenase), 40 +/- 5.0 microM (15-lipoxygenase), 35 +/- 4.0 microM (cyclooxygenase activity of prostaglandin H synthase) and 30 +/- 3.0 microM (peroxidase activity of prostaglandin H synthase). Altogether, the results reported here suggest that the anti-apoptotic activity of resveratrol depends on the direct inhibition of the main arachidonate-metabolizing enzymes.
TL;DR: It is demonstrated in this study that expression in yeast is appropriate for the large-scale production of functional penaeidins, whose activities are almost indistinguishable from those of the native molecules.
Abstract: Penaeidins are 5.5- to 6.6-kDa antimicrobial peptides recently isolated from the plasma and haemocytes of the tropical shrimp Penaeus vannamei. These molecules differ from the other classes of antimicrobial peptides in that they are composed of a proline-rich N-terminus and of a C-terminus containing six cysteine residues engaged in three disulfide bridges. In order to gain information on their antimicrobial activity, two penaeidins (Pen-2 and Pen-3a) were expressed in Saccharomyces cerevisiae. The recombinant Pen-2 and -3a were characterized in terms of primary structure by Edman degradation, mass spectrometry and gas chromatography. A protocol was then established to purify the amount of penaeidins required for the determination of their activity spectrum. We demonstrate in this study that expression in yeast is appropriate for the large-scale production of functional penaeidins, whose activities are almost indistinguishable from those of the native molecules. Data on Pen-2 and -3a activity demonstrate that penaeidins have a broad spectrum of antifungal properties associated with a fungicidal activity, and that their antibacterial activities are essentially directed against Gram-positive bacteria, with a strain-specific inhibition mechanism. Despite a better efficiency of Pen-3a on most of the tested strains, similar activity spectra and inhibition mechanisms were observed for both Pen-2 and -3a. Finally, no synergistic effect could be observed between the two molecules.
TL;DR: Results clearly indicate that expression of the genes for these carbohydrate-metabolizing enzymes, which are needed to process nectar into honey, in the hypopharyngeal gland is associated with the age-dependent role change of the worker bees.
Abstract: Worker honeybees change their behaviour from the role of nurse to that of forager with age. We have isolated cDNA clones for two honeybee (Apis mellifera L.) genes, encoding alpha-amylase and glucose oxidase homologues, that are expressed in the hypopharyngeal gland of forager bees. The predicted amino acid sequence of the putative Apis amylase showed 60.5% identity with Drosophila melanogaster alpha-amylase, whereas that of Apis glucose oxidase showed 23.8% identity with Aspergillus niger glucose oxidase. To determine whether the isolated cDNAs actually encode these enzymes, we purified amylase and glucose oxidase from homogenized forager-bee hypopharyngeal glands. We sequenced the N-terminal regions of the purified enzymes and found that they matched the corresponding cDNAs. mRNAs for both enzymes were detected by Northern blotting in the hypopharyngeal gland of the forager bee but not in the nurse-bee gland. These results clearly indicate that expression of the genes for these carbohydrate-metabolizing enzymes, which are needed to process nectar into honey, in the hypopharyngeal gland is associated with the age-dependent role change of the worker.
TL;DR: This work has characterized two H2O2-independent phenoloxidases with approximate molecular masses of 90 kDa and 110 kDa from cell walls of Populus euramericana xylem that are capable of oxidizing coniferyl alcohol.
Abstract: The nature of the enzyme(s) involved in the dehydrogenative polymerization of lignin monomers is still a matter of debate. Potential candidates include laccases which have recently received attention due to their capacity to oxidize lignin monomers and close spatial and temporal correlation with lignin deposition. We have characterized two H2O2-independent phenoloxidases with approximate molecular masses of 90 kDa and 110 kDa from cell walls of Populus euramericana xylem that are capable of oxidizing coniferyl alcohol. The 90-kDa protein was purified to apparent homogeneity and extensively characterized at the biochemical and structural levels. To our knowledge, this is the first report of a plant laccase purified to homogeneity from a lignifying tissue of an angiosperm. The cDNA clones corresponding to the 90-kDa and 110-kDa proteins, lac90 and lac110, were obtained by a PCR-based approach using specific oligonucleotides derived from peptide sequences. Sequence analysis indicated that lac90 and lac110 encoded two distinct laccases. In addition, heterologous screening using an Acer pseudoplatanus laccase cDNA enabled us to obtain three additional cDNAs (lac1, lac2, lac3) that did not correspond to lac90 and lac110. The five laccase cDNAs correspond to a highly divergent multigene family but Northern analysis with gene-specific probes indicated that all of the genes are exclusively and abundantly expressed in stems. These results highlight the polymorphism of plant laccases by an integrated biochemical and molecular approach, and provide the tools that will enable us to clearly determine the function of these enzymes in plants by molecular and genetic approaches.
TL;DR: A 1918-bp cDNA from a human adrenal cDNA library which encodes a novel thioredoxin reductase (TrxR2) of 521 amino acid residues with a calculated molecular mass of 56.2 kDa is isolated and it is shown that in vivo it is localized in mitochondria.
Abstract: We have isolated a 1918-bp cDNA from a human adrenal cDNA library which encodes a novel thioredoxin reductase (TrxR2) of 521 amino acid residues with a calculated molecular mass of 562 kDa It is highly homologous to the previously described cytosolic enzyme (TrxR1), including the conserved active site CVNVGC and the FAD-binding and NADPH-binding domains However, human TrxR2 differs from human TrxR1 by the presence of a 33-amino acid extension at the N-terminus which has properties characteristic of a mitochondrial translocation signal Northern-blot analysis identified one mRNA species of 22 kb with highest expression in prostate, testis and liver We expressed human TrxR2 as a fusion protein with green fluorescent protein and showed that in vivo it is localized in mitochondria Removal of the mitochondrial targeting sequence abolishes the mitochondrial translocation Finally, we determined the genomic organization of the human TrxR2 gene, which consists of 18 exons spanning about 67 kb, and its chromosomal localization at position 22q112
TL;DR: Akt-3 is a promising target for the discovery of novel chemotherapeutic agents which do not interfere with insulin signalling, but is not highly expressed in liver or skeletal muscle, suggesting that its principle function may not be in regulating insulin signalling.
Abstract: Akt (also known as PKB or RAC-PK) is an intracellular serine/threonine kinase involved in regulating cell survival. Although this makes it a promising target for the discovery of drugs to treat human cancer, a complicating factor may be the role played by Akt in insulin signalling. Two human isoforms, Akt-1 and Akt-2, have been described previously and a third isoform has been identified in rats (here termed Akt-3, but also called RAC-PK-g or PKB-g). We describe the identification of the corresponding human isoform of Akt-3. The gene encoding human Akt-3 was localized to chromosome 1q43‐44. The predicted protein sequence is 83% identical to human Akt-1 and 78% identical to human Akt-2, and contains a pleckstrin homology domain and a kinase domain. In contrast to the published rat Akt-3 isoform, human and mouse Akt-3 also possess a C-terminal ‘tail’ that contains a phosphorylation site (Ser472) thought to be involved in the activation of Akt kinases. In addition to phosphorylation of Ser472, phosphorylation of Thr305 also appears to contribute to the activation of Akt-3 because mutation of both these residues to aspartate increased the catalytic activity of Akt-3, whereas mutation to alanine inhibited activation. Akt-3 activity could be inhibited by the broad spectrum kinase inhibitor staurosporine and by the PKC inhibitor Ro 31-8220, but not by other PKC or PKA inhibitors tested. Although Akt-3 is expressed widely, it is not highly expressed in liver or skeletal muscle, suggesting that its principle function may not be in regulating insulin signalling. These observations suggest that Akt-3 is a promising target for the discovery of novel chemotherapeutic agents which do not interfere with insulin signalling.
TL;DR: The evolutionarily-conserved eukaryotic SMC proteins constitute an essential part of higher order complexes, which are involved in chromatin and DNA dynamics, and are discussed together with additional roles in gene dosage compensation and DNA recombination.
Abstract: The evolutionarily-conserved eukaryotic SMC (structural maintenance of chromosomes) proteins are ubiquitous chromosomal components in prokaryotes and eukaryotes. The eukaryotic SMC proteins form two kind of heterodimers: the SMC1/SMC3 and the SMC2/SMC4 types. These heterodimers constitute an essential part of higher order complexes, which are involved in chromatin and DNA dynamics. The two most prominent and best-characterized complexes are cohesin and condensin, necessary for sister chromatid cohesion and chromosome condensation. Here we discuss these functions together with additional roles in gene dosage compensation and DNA recombination.