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1.
O. O. Olorunsogo F. O. Agbolade S. O. Owojuyigbe J. A. Adebisi A. O. Adebayo W. G. Okunade 《Bioscience reports》1990,10(3):281-291
Calpain, a calcium-dependent, neutral cysteine-protease was purified from the erythrocyte cytosol of subjects having essential hypertension (HTN), sickle cell anaemia, (SCA), or kwashiorkor (KWA). Identical electrophoretic mobility on SDS-polyacrylamide gradient gel, sensitivity to micromolar amounts of Ca2+, absolute requirement for a reducing environment and a high susceptibility to inhibition by leupeptin and thiol-group modifying reagents confirm that calpain preparations from these erythrocytes are equivalent to calpain I. Whereas the extent of calpain activation of erythrocyte membrane Ca2+-pumping ATPase of normal subjects was almost equal to that due to calmodulin, calpain activation of the HTN and SCA pump was greater than activation by calmodulin. Like in normal membranes, exogenous calmodulin protected the Ca2+-pumping ATPase of these erythrocytes against calpainization; the degree of protection by calmodulin is least in SCA and HTN. Electrophoretic separation of erythrocyte membranes and the purified Ca2+-pumping ATPase of HTN, SCA and KWA subjects does not indicate the presence of fragments resulting from the proteolytic action of calpain.Abbreviations PMSF
phenylmethylsulfonylfluoride
- TLCK
N--tosyl-L-lysine chloromethyl ketone
- EGTA
ethyleneglycol-bis (-aminoethylether) N,N1-tetraacetic acid
- ATP
adenosine 51-triphosphate
- Hepes
4-(2 hydroxyethyl)-1-piperazine ethanesulphonic acid
- Tris-HC1
Tris (hydroxymethyl) aminomethane-hydrochloride
- SDS
sodium dodecyl sulphate 相似文献
2.
Kevin K. W. Wang Basil D. Roufogalis Antonio Villalobo 《The Journal of membrane biology》1989,112(3):233-245
Summary Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca2+ transport function of the Ca2+-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca2+ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca2+/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca2+-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca2+ translocation function of the Ca2+-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain. 相似文献
3.
Summary Observations described here provide the first demonstration that calpain (Ca2+-dependent cysteine protease) can degrade proteins of skeletal muscle plasma membranes. Frog muscle plasma membrane vesicles were incubated with calpain preparations and alterations of protein composition were revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Calpain II (activated by millimolar concentrations of Ca2+) was isolated from frog skeletal muscle, but the activity of calpain I (activated by micromolar concentrations of Ca2+) was lost during attempts at fractionation. Calpain I obtained from skeletal muscle and erythrocytes of rats was tested instead, and exerted effects similar to those of frog muscle calpain on the membrane proteins. All of the calpain preparations caused striking losses of a major membrane protein of molecular mass of approximately 97 kDa, designated band c, and diminution of a thinner band of approximately 200 kDa. There were concomitant increases in 83-and 77-kDa polypeptides. These effects were absolutely dependent on the presence of free Ca2+, and were completely blocked by calpastatin, a specific inhibitor of calpain action. Frog muscle calpain differed only in being relatively more active at 0°C than were the calpains from rat tissues. Experimental observations suggest that calpain acts at the cytoplasmic surface of the plasma membrane. 相似文献
4.
S. Shevchenko W. Feng M. Varsanyi V. Shoshan-Barmatz 《The Journal of membrane biology》1998,161(1):33-43
A 94 kDa large subunit thiol-protease, as identified by anti-calpain antibodies, has been isolated from skeletal muscle junctional
sarcoplasmic reticulum (SR). This protease cleaves specifically the skeletal muscle ryanodine receptor (RyR)/Ca2+ release channel at one site resulting in the 375 kDa and 150 kDa fragments. The 94 kDa thiol-protease degrades neither other
SR proteins nor the ryanodine receptor of cardiac nor brain membranes. The partially purified 94 kDa protease, like the SR
associated protease, had an optimal pH of about 7.0, was absolutely dependent on the presence of thiol reducing reagents,
and was completely inhibited by HgCl2, leupeptin and the specific calpain I inhibitor. However, while the SR membrane-associated protease requires Ca2+ at a submicromolar concentration, the isolated thiol-protease has lost the Ca2+ requirement.
The 94 kDa thiol-protease had no effect on ryanodine binding but modified the channel activity of RyR reconstituted into planar
lipid bilayer: in a time-dependent manner, the channel activity decreases and within several minutes the channel is converted
into a subconducting state. The protease-modified channel activity is still Ca2+-dependent and ryanodine sensitive.
This 94 kDa thiol-protease cross react with anti-calpain antibodies thus, may represent the novel large subunit of the skeletal
muscle specific calpain p94.
Received: 10 December 1996/Revised: 11 August 1997 相似文献
5.
Further characterization of calpain-mediated proteolysis of the human erythrocyte plasma membrane Ca2+-ATPase 总被引:1,自引:0,他引:1
The membrane-bound form and a solubilized and purified form of the Ca2+-ATPase from human erythrocyte have been proteolyzed under controlled conditions by highly purified Ca2+-dependent neutral cysteine-protease, calpain I, in the absence and in the presence of the calmodulin-calcium complex. In the absence of calmodulin the 136-kDa enzyme was transformed into a group of fragments of 125-124 kDa, followed by the slower formation of a second group of fragments of 82-80 kDa. These heterogeneous fragments were capable of forming an acylphosphate intermediate. The 125- and 82-kDa minor components of each heterogeneous group of fragments (125-124 and 82-80 kDa) were capable of binding calmodulin, whereas the 124- and the 80-kDa major components did not. In the presence of calmodulin, however, the native enzyme was transformed into a 127-kDa fragment followed by the slower formation of an 85-kDa fragment. Both fragments (127 and 85 kDa) formed an acylphosphate intermediate and were capable of binding calmodulin. The presence of calmodulin during calpain action effectively protected the Ca2+-ATPase from proteolytic activation (K.K.W. Wang, A. Villalobo, and B.D. Roufogalis (1988) Arch. Biochem. Biophys. 260, 696-704) and prevented the formation of the calmodulin-insensitive 124- and 80-kDa fragments. Smaller fragments not capable of forming the acylphosphate intermediate were also produced, in particular a 39-37 kDa doublet band retaining the capacity to bind calmodulin. In contrast to the membrane-bound form, the purified form of the Ca2+-ATPase was proteolyzed by calpain at a slower rate. 相似文献
6.
Mira Malcov Dalit Ben-Yosef Tova Glaser Ruth Shalgi 《Molecular reproduction and development》1997,48(1):119-126
Resumption of meiosis at fertilization is mediated by increased levels of calcium which activate several calcium-dependent enzymes. Calpain, a neutral calcium-activated thiol protease, is present in the cytoplasm of many cells. Its activation is associated with limited autolysis and relocalization in the cell. Calpain is thought to participate in the regulation of mitosis and resumption of meiosis in Xenopus oocytes. In this study we followed the activation and localization of calpain during maturation and fertilization in rat eggs using a polyclonal antibody raised against chicken muscle calpain. A band of 80 kDa was detected in GV oocytes and its level increased in unfertilized MII eggs. At the early stages of fertilization, we observed a transient decrease in the level of calpain which was regained at the pronuclear stage. Adding Ca2+ to lysate of MII eggs resulted in an additional band, representing the degraded fragment of the activated protein. In eggs activated by ionomycin, calpain level decreased, followed by an increase in a dynamic similar to that observed in fertilized eggs. Egg activation also led to changes in calpain localization. A homogenous distribution was observed in GV and in MII eggs, while in activated eggs it was localized predominantly overlying the metaphase plate. In the current study we demonstrate the presence of calpain in the rat egg. During maturation, calpain level increases; however, during egg activation, in response to [Ca2+]i changes, calpain undergoes autolysis, translocation, and fluctuation in its level. We therefore suggest a correlation between calpain activation and fertilization. Mol. Reprod. Dev. 48:119–126, 1997. © 1997 Wiley-Liss, Inc. 相似文献
7.
V. Shoshan-Barmatz S. Weil H. Meyer M. Varsanyi L. M. G. Heilmeyer 《The Journal of membrane biology》1994,142(3):281-288
The association of an endogenous, Ca2+-dependent cysteine-protease with the junctional sarcoplasmic reticulum (SR) is demonstrated. The activity of this protease is strongly stimulated by dithiothreitol (DTT), cysteine and β-mercaptoethanol, and is inhibited by iodoacetamide, mercuric chloride and leupeptin, but not by PMSF. The activity of this thiol-protease is dependent on Ca2+ with half-maximal activity obtained at 0.1 μm and maximal activity at 10 μm. Mg2+ is also an activator of this enzyme (CI50=22 μm). These observations, together with the neutral pH optima and inhibition by the calpain I inhibitor, suggest that this enzyme is of calpain I type. This protease specifically cleaves the ryanodine receptor monomer (510 kD) at one site to produce two fragments with apparent molecular masses of 375 and 150 kD. The proteolytic fragments remain associated as shown by purification of the cleaved ryanodine receptor. The calpain binding site is identified as a PEST (proline, glutamic acid, serine, threonine-rich) region in the amino acid sequence GTPGGTPQPGVE, at positions 1356–1367 of the RyR and the cleavage site, the calmodulin binding site, at residues 1383–1400. The RyR cleavage by the Ca2+-dependent thiol-protease is prevented in the presence of ATP (1–5 mm) and by high NaCl concentrations. This cleavage of the RyR has no effect on ryanodine binding activity but stimulates Ca2+ efflux. A possible involvement of this specific cleavage of the RyR/Ca2+ release channel in the control of calpain activity is discussed. 相似文献
8.
Neil R. Brandt Anthony H. Caswell Shu-Rong Wen Jane A. Talvenheimo 《The Journal of membrane biology》1990,113(3):237-251
Summary Isolated triadic proteins were employed to investigate the molecular architecture of the triad junction in skeletal muscle. Immunoaffinity-purified junctional foot protein (JFP), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), aldolase and partially purified dihydropyridine (DHP) receptor were employed to probe protein-protein interactions using affinity chromatography, protein overlay and crosslinking techniques. The JFP, an integral protein of the sarcoplasmic reticulum (SR) preferentially binds to GAPDH and aldolase, peripheral proteins of the transverse (T)-tubule. No direct binding of JFP to the DHP receptor was detected. The interactions of JFP with GAPDH and aldolase appear to be specific since other glycolytic enzymes associated with membranes do not bind to the JFP. The DHP receptor, an integral protein of the T-tubule, also binds GAPDH and aldolase. A ternary complex between the JFP and the DHP receptor can be formed in the presence of GAPDH. In addition, the DHP receptor binds to a previously undetectedM
r 95 K protein which is distinct from the SR Ca2+ pump and phosphorylaseb. TheM
r 95 K protein is an integral protein of the junctional domain of the SR terminal cisternae. It is also present in the newly identified strong triads (accompanying paper). From these findings, we propose a new model for the triad junction. 相似文献
9.
The effects of Ca2+ and calmodulin on endogenously catalyzed ADP-ribosylation were investigated in adipocyte plasma membranes. Four specific proteins of 70, 65, 61 and 52 kDa were labeled with [32P]ADP-ribose and ADP-ribosylation of the proteins was highly dependent upon the conditions employed. ADP-ribosylation of the 70 kDa protein was observed only in membranes supplemented with Ca2+. Maximal incorporation of [32P] into the protein was achieved with free Ca2+ concentrations of 90 μM. Calcium-stimulated ADP-ribosylation of the 70 kDa protein was inhibited by calmodulin. Half-maximal inhibition was observed in membranes incubated with 1.2 μM calmodulin. The effect of calmodulin was characterized by an inhibition of the incorporation of [32P]ADP-ribose as opposed to a stimulation of its removal. ADP-ribosylation of the 61 kDa protein was not altered by added Ca2+ and/or calmodulin whereas ADP-ribosylation of the 65 kDa protein was partially (50%) inhibited by free Ca2+ concentrations between 10−6 – 10−5 M. These results provide evidence that the adipocyte plasma membrane contains ADP-ribosyltransferase activities and demonstrate that ADP-ribosylation of a 70 kDa protein is regulated by Ca2+ and calmodulin. 相似文献
10.
Bozóky Z Róna G Klement É Medzihradszky KF Merényi G Vértessy BG Friedrich P 《PloS one》2011,6(5):e19546
Background
Calpain proteases drive intracellular signal transduction via specific proteolysis of multiple substrates upon Ca2+-induced activation. Recently, dUTPase, an enzyme essential to maintain genomic integrity, was identified as a physiological calpain substrate in Drosophila cells. Here we investigate the potential structural/functional significance of calpain-activated proteolysis of human dUTPase.Methodology/Principal Findings
Limited proteolysis of human dUTPase by mammalian m-calpain was investigated in the presence and absence of cognate ligands of either calpain or dUTPase. Significant proteolysis was observed only in the presence of Ca(II) ions, inducing calpain action. The presence or absence of the dUTP-analogue α,β-imido-dUTP did not show any effect on Ca2+-calpain-induced cleavage of human dUTPase. The catalytic rate constant of dUTPase was unaffected by calpain cleavage. Gel electrophoretic analysis showed that Ca2+-calpain-induced cleavage of human dUTPase resulted in several distinctly observable dUTPase fragments. Mass spectrometric identification of the calpain-cleaved fragments identified three calpain cleavage sites (between residues 4SE5; 7TP8; and 31LS32). The cleavage between the 31LS32 peptide bond specifically removes the flexible N-terminal nuclear localization signal, indispensable for cognate localization.Conclusions/Significance
Results argue for a mechanism where Ca2+-calpain may regulate nuclear availability and degradation of dUTPase. 相似文献11.
Two forms of Ca2+-pump were identified in bovine brain synaptic membranes as aspartylphosphate intermediates and were characterized. The 140 kDa and 97 kDa phosphoproteins were digested by calpain, producing two phosphorylated fragments, of M.W. 124 and 80 kDa respectively, not inhibited by thapsigargin, and displayed a trypsin digestion pattern with the formation of one phosphorylatable fragment of about 80 kDa. These results suggest that both pumps belong to the Plasma Membrane-type of Ca2+ ATPases, differing from the Sarco- or Endoplasmic Reticulum kind. A plasma membrane Ca2+-ATPase proteinaceous inhibitor with molecular weight between 6,000 and 10,000 Da was resolved from synaptic terminal cytosol, where it is enriched by fourfold with respect to frontal cortex brain cytosol. Such enrichment is already evident in the correspondent crude fractions. The presence of calcium pump and its proteinaceous inhibitor inside the synaptic terminals from bovine brain is discussed in terms of free calcium level regulation in neuron synaptoplasm. 相似文献
12.
Many short-lived proteins which are devoid of proteolytic activity contain PEST sequences which are segments along the polypeptide chain that are rich in proline (P), glutamate (E), serine (S) and threonine (T). These designated PEST sequences are believed to be putative intramolecular signals for rapid proteolytic degradation. Calmodulin is a ubiquitous, 17kDa, acidic Ca2+-binding protein which plays an important role in the regulation of many physiological processes through its interaction with a wide range of calmodulin-binding proteins. Several calmodulin-binding proteins are known to contain PEST sequences and are susceptible to proteolysis by endogenous neutral proteases such as calpain I and calpain II. In this report, we discuss the functions of PEST sequences in calmodulin-binding proteins and assess the correlation between calmodulin-binding proteins and PEST sequences. 相似文献
13.
《Comparative biochemistry and physiology. B, Comparative biochemistry》1992,101(4):951-954
- 1.1. About 0.3–0.4% of total water-soluble protein extracted from sea urchin embryos at the two-cell and early-gastrula stages was Ca2+-dependently bound to immobilized calmodulin.
- 2.2. SDS-PAGE of calmodulin-binding proteins revealed at least 20 polypeptides ranging from 200 to 15.5 kDa, and 70–80% of the protein belonged to a dozen major polypeptides. Polypeptides of 70, 55, 50, 45 and 18 kDa seemed to be the same as those that were detected earlier (Iwasa and Mohri, J. Biochem.94, 575–587, 1983).
- 3.3. The polypeptide spectrum of calmodulin target proteins changed significantly, e.g. the major polypeptides of 70 and 41 kDa increased, and the 200 and 43 kDa polypeptides decreased sharply during development from the two-cell to the early-gastrula stage.
- 4.4. According to our estimates, the molar concentrations of the calmodulin and targets were close enough, and therefore the Ca2+ signal should depend on the spatial-temporal distribution of free calmodulin in the cells.
14.
Advanced glycation end-products (AGEs) are important mediators of diabetic complications via incompletely understood pathways. AGEs bind to intracellular ERM proteins (ezrin, radixin and moesin) that modulate cell shape, motility, adhesion and signal transduction. AGEs bind to the N-terminal domain of ezrin but not full-length ezrin. The AGE binding site may be made accessible either by proteolysis releasing an N-terminal fragment or ezrin activation by phosphorylation. Increased intracellular calcium is a primary event in cell activation by high glucose or AGEs. Calpain activity is increased concomitantly, and ezrin is a calpain substrate. The present study assessed whether glycated proteins affect ezrin cleavage and activation in renal tubule epithelial cells. After 7?days, AGE-BSA decreased ezrin levels in MDCK renal tubular cells to 66?±?4% of control. AGE-RNAse, ribosylated fetal bovine serum and methylglyoxal-BSA all had similar effects. The AGE-BSA-induced decrease in ezrin was abolished by calpastatin peptide, a specific calpain inhibitor, and 1,2-bis-aminophenoxyethane-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a calcium chelator. Ezrin breakdown products were increased in AGE-BSA-treated cells, with a main fragment of ~?43?kDa. In?vitro, calpain?1 cleaved recombinant human ezrin, generating breakdown fragments including an N-terminal fragment of ~?43?kDa. Studies with ezrin mutants showed that non-phosphorylated ezrin was more susceptible to calpain cleavage. AGE-BSA decreased phosphorylated ERM levels to 31?±?12% in MDCK cells. Thus, AGE-BSA promotes calpain-mediated proteolysis of ezrin in MDCK cells by both increasing calpain activity and reducing phosphorylation. Therapies targeting both glycated proteins and calpain may provide protection against diabetic complications. Structured digital abstract ? Calpain-1?cleaves?Ezrin?by?protease assay?(View Interaction:?1,?2). 相似文献
15.
1. The intrinsic fluorescence of epoxysuccinyl-inhibited calpain II undergoes a Ca2(+)-dependent decrease which contrasts with the increase observed for calmodulin. 2. Calpain II was inhibited by the calmodulin antagonist toluidinylnaphthalenesulfonate (TNS), and a Ca2(+)-dependent increase in TNS fluorescence intensity was observed for epoxysuccinyl-inhibited calpain II. 3. The calmodulin antagonists calmidazolium CDZ and felodipine both caused decreases in the intrinsic fluorescence of epoxysuccinyl-inhibited calpain II. 4. Increasing concentrations of Ca2+ caused an increase in the fluorescence intensity of the inhibited enzyme in the presence of (CDZ), and a decrease in the presence of felodipine. 5. It is concluded from these studies that Ca2+ and calmodulin antagonists induce conformational changes in calpain II, and that changes occur in regions other than the Ca2(+)-binding domains. 相似文献
16.
In vitro proteolysis of brain spectrin by calpain I inhibits association of spectrin with ankyrin-independent membrane binding site(s). 总被引:5,自引:0,他引:5
This report demonstrates that specific proteolysis of brain spectrin by a calcium-dependent protease, calpain I, abolishes association of brain spectrin with the ankyrin-independent binding site(s) in brain membranes. Calpain I cleaves the beta subunit of spectrin at the N-terminal end leaving a 218-kDa fragment and cleaves the alpha subunit in the midregion to produce 150- and 130-kDa fragments. Calpain-proteolyzed spectrin almost completely loses the capacity to displace binding of intact spectrin to membranes. Spectrin digested by calpain I under conditions that almost completely destroyed membrane-binding remained associated as a tetramer and retained about 60% of the ability to associate with actin filaments. Cleavage of spectrin occurred at sites distinct from the membrane-binding site which is located on the beta subunit since the isolated 218-kDa fragment of the beta subunit as well as a reconstituted complex of alpha and 218-kDa beta subunit fragment partially regained binding activity. Moreover, cleavage of the alpha subunit alone reduced the affinity of spectrin for membranes by 2-fold. A consequence of distinct sites for calpain I cleavage and membrane-binding is that calpain I can digest spectrin while spectrin is complexed with other proteins and therefore has the potential to mediate disassembly of a spectrin-actin network from membranes. 相似文献
17.
Chung H Choi B Jang G Lee K Kim H Yoon S Im S Davis M Hines H 《Journal of applied genetics》2007,48(1):61-68
The ovine skeletal-muscle-specific calpain gene (p94), which is known also as the n-calpain or calpain 3 gene (CAPN3), was screened with primers. Selection of the PCR primers was based on the ovine cDNA sequence (GenBank accession No. AF087570). After sequence alignment between the ovine and human (AY902237) genes, exon and intron boundaries were determined. Polymorphisms were observed in the intron region for the CAPN31112 and CAPN31213 segments, and the sequences for these segments were submitted to the GenBank (AF309635 and AY102617, respectively). Body weight was recorded at birth, weaning and post-weaning. Calpain 3 genotypes of the CAPN31112 segment were associated with birth weight (P < 0.01), and a dominant gene effect was observed. Breeding group, birth type, and rearing type were significantly associated with weight traits. Allele frequencies were similar in purebred and crossbred animals. 相似文献
18.
Hui-Yan Lei Xiao-Long Zhou Zhi-Rong Ruan Wei-Cheng Sun Gilbert Eriani En-Duo Wang 《The Journal of biological chemistry》2015,290(43):26314-26327
Nine aminoacyl-tRNA synthetases (aaRSs) and three scaffold proteins form a super multiple aminoacyl-tRNA synthetase complex (MSC) in the human cytoplasm. Domains that have been added progressively to MSC components during evolution are linked by unstructured flexible peptides, producing an elongated and multiarmed MSC structure that is easily attacked by proteases in vivo. A yeast two-hybrid screen for proteins interacting with LeuRS, a representative MSC member, identified calpain 2, a calcium-activated neutral cysteine protease. Calpain 2 and calpain 1 could partially hydrolyze most MSC components to generate specific fragments that resembled those reported previously. The cleavage sites of calpain in ArgRS, GlnRS, and p43 were precisely mapped. After cleavage, their N-terminal regions were removed. Sixty-three amino acid residues were removed from the N terminus of ArgRS to form ArgRSΔN63; GlnRS formed GlnRSΔN198, and p43 formed p43ΔN106. GlnRSΔN198 had a much weaker affinity for its substrates, tRNAGln and glutamine. p43ΔN106 was the same as the previously reported p43-derived apoptosis-released factor. The formation of p43ΔN106 by calpain depended on Ca2+ and could be specifically inhibited by calpeptin and by RNAi of the regulatory subunit of calpain in vivo. These results showed, for the first time, that calpain plays an essential role in dissociating the MSC and might regulate the canonical and non-canonical functions of certain components of the MSC. 相似文献
19.
Iman Mehdizadeh Gohari Andrew M. Kropinski Scott J. Weese Valeria R. Parreira Ashley E. Whitehead Patrick Boerlin John F. Prescott 《PloS one》2016,11(2)
The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and unique plasmid-encoded locus. 相似文献
20.
Giguere CJ Covington MD Schnellmann RG 《Biochemical and biophysical research communications》2008,366(1):258-262
Calpains, Ca2+-activated cysteine proteases, have been implicated in the progression of multiple disease states. We recently identified calpain 10 as a mitochondrial calpain that is involved in Ca2+-induced mitochondrial dysfunction. The goals of this study were to characterize the expression and activity of renal mitochondrial calpain 10 in rabbit, mouse, and rat. Using shRNA technology and immunoblot analysis three previously postulated splice variants of calpain 10 were identified (50, 56, and 75 kDa). SLLVY-AMC zymography and immunoblot analysis was used to directly link calpeptin-sensitive calpain activity to calpain 10 splice variants. Rabbit, mouse, and rat kidney mitochondria contained 75 kDa (calpain 10a), 56 kDa (calpain 10c or 10d), and 50 kDa (calpain 10e) splice variants. Interestingly, zymography yielded distinct bands of calpain activity containing multiple calpain 10 splice variants in all species. These results provide evidence that several previously postulated splice variants of calpain 10 are localized to the mitochondria in kidneys of rabbits, rats, and mice. 相似文献