Different anesthetic sensitivities of skeletal and cardiac isoforms of the Ca-ATPase.

We have previously shown that low levels of the volatile anesthetic halothane activate the Ca-ATPase in skeletal sarcoplasmic reticulum (SR), but inhibit the Ca-ATPase in cardiac SR. In this study, we ask whether the differential inhibition is due to (a) the presence of the regulatory protein phospholamban in cardiac SR, (b) different lipid environments in skeletal and cardiac SR, or (c) the different Ca-ATPase isoforms present in the two tissues. By expressing skeletal (SERCA 1) and cardiac (SERCA 2a) isoforms of the Ca-ATPase in Sf21 insect cell organelles, we found that differential anesthetic effects in skeletal and cardiac SR are due to differential sensitivities of the SERCA 1 and SERCA 2a isoforms to anesthetics. Low levels of halothane inhibit the SERCA 2a isoform of the Ca-ATPase, and have little effect on the SERCA 1 isoform. The biochemical mechanism of halothane inhibition involves stabilization of E2 conformations of the Ca-ATPase, suggesting direct anesthetic interaction with the ATPase. This study establishes a biochemical model for the mechanism of action of an anesthetic on a membrane protein, and should lead to the identification of anesthetic binding sites on the SERCA 1 and SERCA 2a isoforms of the Ca-ATPase.     

The isolation of aggregates of spectrin from bovine erythrocyte membranes.

Aggregated states of spectrin from bovine erythrocyte membranes can be detected in sedimentation velocity experiments. These aggregates have been isolated by means of gel filtration on columns of 4% agarose. They appear to be stable over a wide range of pH and ionic strength, although they are dissociated by sodium dodecyl sulphate. Sedimentation equilibrium measurements yielded values of 960 000 and 480 000 for the molecular weights of the major aggregates, corresponding to a tetramer and dimer, respectively. The presence of different aggregated states in spectrin preparations may explain the wide variation in the reported physical properties of spectrin.     

Thapsigargin inhibits the sarcoplasmic or endoplasmic reticulum Ca-ATPase family of calcium pumps.

The role of ATP-dependent calcium uptake into intracellular storage compartments is an essential feature of hormonally induced calcium signaling. Thapsigargin, a non-phorboid tumor promoter, increasingly is being used to manipulate calcium stores because it induces a hormone-like elevation of cytosolic calcium. It has been suggested that thapsigargin acts through inhibition of the endoplasmic reticulum calcium pump. We have directly tested the specificity of thapsigargin on all of the known intracellular-type calcium pumps (referred to as the sarcoplasmic or endoplasmic reticulum Ca-ATPase family (SERCA]. Full-length cDNA clones encoding SERCA1, SERCA2a, SERCA2b, and SERCA3 enzymes were expressed in COS cells, and both calcium uptake and calcium-dependent ATPase activity were assayed in microsomes isolated from them. Thapsigargin inhibited all of the SERCA isozymes with equal potency. Furthermore, similar doses of thapsigargin abolished the calcium uptake and ATPase activity of sarcoplasmic reticulum isolated from fast twitch and cardiac muscle but had no influence on either the plasma membrane Ca-ATPase or Na,K-ATPase. The interaction of thapsigargin with the SERCA isoforms is rapid, stoichiometric, and essentially irreversible. These properties demonstrate that thapsigargin interacts with a recognition site found in, and only in, all members of the endoplasmic and sarcoplasmic reticulum calcium pump family.     

An immunosuppressive peptide from the horsefly inhibits inflammation by repressing macrophage maturation and phagocytosis

Ectoparasites repress host immune responses while they obtain nutrition from their hosts. Understanding the immunosuppressive mechanisms between ectoparasites and their hosts will provide new strategies to develop potential immunosuppressive drugs against immune disorder diseases. Previously, we have discovered that a small peptide, immunoregulin HA, from the horsefly (Hybomitra atriperoides) may play an immunosuppressive role in rat splenocytes. However, the targeting cells and detailed mechanisms of immunoregulin HA in immunosuppressive reactions are not well defined. Here, we show that immunoregulin HA reduces the secretion of proinflammatory cytokines upon lipopolysaccharide (LPS) stimulation. Interestingly, we discover that the major cytokines repressed by immunoregulin HA are secreted by macrophages, rather than by T cells. Furthermore, immunoregulin HA inhibits macrophage maturation and phagocytosis. Mechanically, the activations of c-JUN N-terminal kinase and extracellular signal-regulated kinase upon LPS stimulation are decreased by immunoregulin HA. Consistently, immunoregulin HA treatment exhibits an anti-inflammatory activity in a mouse model of adjuvant-induced paw inflammation. Taken together, our data reveal that immunoregulin HA conducts the anti-inflammatory activity by blocking macrophage functions.     

Disassembly from both ends of thick filaments in rabbit skeletal muscle fibers. An optical diffraction study.

We show in this paper that the change of the internal structure of a sarcomere in a rabbit glycerinated psoas muscle fiber can be examined by analyzing the intensity change of the first- and the second-order optical diffraction lines. A unit-cell (sarcomere)-structure model has been applied to the estimation of the length of thick filaments in a muscle fiber while they undergo dissociation. The optical factors, except for the unit-cell-structure factor, hardly changed during the dissociation of the filaments. Our results show that thick filaments dissociate from both ends on increasing the KCl concentration in the presence of 10 mM pyrophosphate and 5 mM MgCl2. Micromolar concentrations of Ca2+ suppressed to some extent the dissociation of thick filaments. The disassembly of thick filaments occurred at higher KCl concentrations in the absence of pyrophosphate. There was a correlation between the stability of the thick filament structure and cross-bridge formation, which was induced either by the addition of micromolar concentrations of Ca2+ in the presence of Mg-pyrophosphate or by removal of Mg-pyrophosphate.     

Characterization and partial sequence of di-iodosulphophenyl isothiocyanate-binding peptide from human erythrocyte anion-transport protein.

We investigated the presumed anion-binding domain of the anion-transport protein from human erythrocyte membranes, using 2,6-di-iodo-4-sulphophenyl isothiocyanate, an inhibitor of anion transport. The 125I-labelled reagent binds covalently to the protein with a half-maximal inhibitory concentration of 86 microM. Treatment of unsealed erythrocyte 'ghosts' with chymotrypsin yielded a membrane-bound fragment (mol.wt. 14 500 +/- 1000) that contained all the protein-bound radioactivity. The binding of the inhibitor to this peptide gave a pattern very similar to that obtained for the effect of the compound on phosphate transport into erythrocytes. The peptide is therefore presumed to be intimately involved in the mediation of anion exchange. Cleavage of the 14 500-mol.wt. transmembrane fragment with CNBr resulted in the production of two peptides with apparent molecular weights of 8800 and 4700. The 4700-mol.wt. peptide is the N-terminal portion of the 14 500-mol.wt. peptide. The attachment site for 2,6-di-iodo-4-sulphophenyl isothiocyanate is situated near the C-terminal of the 8800-mol.wt. peptide. This locates the inhibitor-binding site near the chymotrypsin cleavage point at the extracellular surface of the membrane. A partial sequence (residues 1--38) of the 8800-mol.wt. peptide was obtained.     

Wortmannin inhibits both insulin- and contraction-stimulated glucose uptake and transport in rat skeletal muscle

Wojtaszewski, Jørgen F. P., Bo F. Hansen, BirgitteUrsø, and Erik A. Richter. Wortmannin inhibits both insulin-and contraction-stimulated glucose uptake and transport in rat skeletal muscle. J. Appl. Physiol. 81(4):1501-1509, 1996.The role of phosphatidylinositol (PI) 3-kinasefor insulin- and contraction-stimulated muscle glucose transport wasinvestigated in rat skeletal muscle perfused with a cell-freeperfusate. The insulin receptor substrate-1-associated PI 3-kinaseactivity was increased sixfold upon insulin stimulation but wasunaffected by contractions. In addition, the insulin-stimulated PI3-kinase activity and muscle glucose uptake and transport in individualmuscles were dose-dependently inhibited by wortmannin with one-halfmaximal inhibition values of ~10 nM and total inhibition at 1 µM.This concentration of wortmannin also decreased thecontraction-stimulated glucose transport and uptake by ~30-70%without confounding effects on contractility or on muscle ATP andphosphocreatine concentrations. At higher concentrations(3 and 10 µM), wortmannin completely blocked thecontraction-stimulated glucose uptake but also decreased thecontractility. In conclusion, inhibition of PI 3-kinase with wortmanninin skeletal muscle coincides with inhibition of insulin-stimulated glucose uptake and transport. Furthermore, in contrast to recent findings in incubated muscle, wortmannin also inhibitedcontraction-stimulated glucose uptake and transport. The inhibitoryeffect of wortmannin on contraction-stimulated glucose uptake may beindependent of PI 3-kinase activity or due to inhibition of asubfraction of PI 3-kinase with low sensitivity to wortmannin.

     

Human cathelicidin peptide LL37 inhibits both attachment capability and biofilm formation of Staphylococcus epidermidis

Aims: The aim of this work was to investigate the possible effect of human cathelicidin antimicrobial peptide LL37 on biofilm formation of Staphylococcus epidermidis, a major causative agent of indwelling device‐related infections. Methods and Results: We performed initial attachment assay and biofilm formation solid surface assay in microtitre plates, as well as growth experiment in liquid medium using laboratory strain Staph. epidermidis ATCC35984. We found that already a low concentration of the peptide LL37 (1 mg l^?1) significantly decreased both the attachment of bacteria to the surface and also the biofilm mass. No growth inhibition was observed even at 16 mg l^?1 concentration of LL37, indicating a direct effect of the peptide on biofilm production. Conclusions: As biofilm protects bacteria during infections in humans and allows their survival in a hostile environment, inhibition of biofilm formation by LL37 may have a key role to prevent bacterial colonization on indwelling devices. Significance and Impact of the Study: Our findings suggest that this host defence factor can be a potential candidate in prevention and treatment strategies of Staph. epidermidis infections in humans.     

Identification of an anticoagulant peptide that inhibits both fXIa and fVIIa/tissue factor from the blood-feeding nematode Ancylostoma caninum

Factor VIIa-tissue factor complex (fVIIa/TF) and factor XIa (fXIa) play important roles in the initiation and amplification of coagulation, respectively. They may be good targets for the development of novel anticoagulants to treat and prevent thromboembolic disease. In this study, we cloned, expressed and identified a novel anticoagulant peptide, AcaNAP10, from the blood-feeding nematode Ancylostoma caninum. AcaNAP10 showed potent anticoagulant activity and doubled the activated partial thromboplastin and prothrombin times at estimated concentrations of 92.9 nM and 28.8 nM, respectively. AcaNAP10 demonstrated distinct mechanisms of action compared with known anticoagulants. It inhibited fXIa and fVIIa/TF with IC₅₀ values of 25.76 ± 1.06 nM and 123.9 ± 1.71 nM, respectively. This is the first report on an anticoagulant that can inhibit both fXIa and fVIIa/TF. This anticoagulant peptide may be an alternative molecule for the development of novel anticoagulants.     

Purification and characterization of acylphosphatase erythrocyte isoenzyme from turkey muscle

An acylphosphatase has been purified from turkey muscle in a rapid and high-yield way. The enzyme has been characterized for structural, kinetic, and immunological parameters, as well as with regard to its stability to thermal, urea, and phenylglyoxal inactivation. The enzyme is quite different from the turkey muscular isoenzyme, and shows structural and kinetic properties that are very similar to those previously reported for the erythrocyte isoenzyme from human erythrocytes and from chicken muscle. From the data reported it appears that this enzyme corresponds to the acylphosphatase erythrocyte isoenzyme. Unlike the erythrocyte isoenzymes studied so far, this enzyme is able to cross-react with antibodies that are raised against the muscular isoenzyme.     

Structures of the Ca-ATPase complexes with ATP, AMPPCP and AMPPNP. An FTIR study

We studied binding of ATP and of the ATP analogs adenosine 5′-(β,γ-methylene)triphosphate (AMPCP) and β,γ-imidoadenosine 5′-triphosphate (AMPPNP) to the Ca²⁺-ATPase of the sarcoplasmic reticulum membrane (SERCA1a) with time-resolved infrared spectroscopy. In our experiments, ATP reacted with ATPase which had AMPPCP or AMPPNP bound. These experiments monitored exchange of ATP analog by ATP and phosphorylation to the first phosphoenzyme intermediate Ca₂E1P. These reactions were triggered by the release of ATP from caged ATP. Only small differences in infrared absorption were observed between the ATP complex and the complexes with AMPPCP and AMPPNP indicating that overall the interactions between nucleotide and ATPase are similar and that all complexes adopt a closed conformation. The spectral differences between ATP and AMPPCP complex were more pronounced at high Ca²⁺ concentration (10 mM). They are likely due to a different position of the γ-phosphate which affects the β-sheet in the P domain.     

黑腹果蝇抗真菌肽Drosomycin同系物的免疫原性和抗真菌活性

通过黑腹果蝇 Drosophila melanogaster抗真菌肽Drosomycin（Drs）及其同系物Drs-lC和Drs-lE的抗体制备及Western blotting 结果,分析了Drs同系物的免疫原性与其抗真菌活性的关系。研究采用了2种技术路线,分别将Drs、Drs-lC和Drs-lE 基因构建成与细胞生长因子基因 afgf 融合的重组表达质粒 pET-afgf-Drs、pET-afgf-C和pET-afgf-E,以及通过基因同向串连获得重组表达质粒 pRSET-2Drs、4Drs、6Drs 和 pRSET-2E、4E、6E,并将这些重组表达质粒转化到BL21（DE3）plysS受体菌进行诱导表达。分离纯化后的融合蛋白afgf-Drs、afgf-C和afgf-E 以及串连蛋白 4 Drs、4 Drs-lE分别免疫小白鼠获得相应的抗血清。Western blotting免疫原性检测结果表明,Drs及其同系物与各自的抗血清具有强的免疫反应,同时相互间也有交叉免疫反应,提示它们具有相似的主要抗原决定簇,这些抗原决定簇可能与抗真菌活性无关。同系物之间抗真菌活性的差异可能来源于某些细微结构上的差异。     

Synthesis and characterization of a chimeric peptide derived from fasciculin that inhibits acetylcholinesterase.

Fasciculins are peptides isolated from mamba (Dendroaspis) venoms which exert their toxic action by inhibiting acetylcholinesterase (AChE). They contain a characteristic triple stranded antiparallel beta-sheet formed by residues 22-27, 34-39 and 48-53. A chimeric peptide named Fas-C, encompassing most of these sequences was synthesized using SPPS/Boc-chemistry and characterized chemically, structurally and functionally. Fas-C has two disulfide bridges, formed sequentially using dual cysteine protection. SDS-PAGE patterns, HPLC profiles and MS proved the peptide identity. Circular dichroism indicated the presence of 13.6% and 41.6% of beta-sheet and beta-turn, respectively, comparable to values observed in the native toxin. An inhibitory effect on eel AChE was displayed by the peptide (Ki71.6 +/- 18.3 microM), although not reaching the affinity level of the parent native toxin (Ki 0.3 nM). It is confirmed that the principal binding region of fasciculin to AChE resides within loop II.     

The nucleotide-binding site of the sarcoplasmic reticulum Ca-ATPase is conformationally altered in aged skeletal muscle.

Cellular conditions in senescent skeletal muscle have been shown to result in the loss of conformational stability of the sarcoplasmic reticulum (SR) Ca-ATPase. To identify underlying structural features of age-modified Ca-ATPase, we have utilized the fluorescence properties of protein-bound probes to assess both local and global structure. We find conformational changes that include an age-related decrease in the apparent binding affinity to high affinity calcium sites detected by fluorescence signals in both tryptophans within nearby membrane-spanning helices and fluorescein isothiocyanate (FITC) bound distally to Lys(515) within the nucleotide-binding site. In addition, a substantial (80%) age-related increase in the accessibility to soluble quenchers of fluorescence of FITC is observed without concomitant changes in bimolecular quenching constants (k(q)) for protein-bound IAEDANS, also within the nucleotide-binding domain, and tryptophans within the membrane. Using fluorescence resonance energy transfer to measure distances between IAEDANS and FITC across the nucleotide-binding domain, we find no significant age-related change in the mean donor-acceptor distance; however, significant increases are observed in the conformational heterogeneity of this domain, as assessed by the width at half-maximum (HW) of the distance distribution, increasing with age from 29.4 +/- 0.8 A to 42.5 +/- 1. 1 A. Circular dichroism indicates that the average secondary structure is unaltered with age. Thus, these data suggest tertiary structural alterations in specific regions around the nucleotide-binding site rather than global conformational changes.     

An anticoagulant peptide from the human hookworm, Ancylostoma duodenale that inhibits coagulation factors Xa and XIa

A full-length cDNA encoding an anticoagulant peptide, named AduNAP4, was cloned and identified from the human hookworm Ancylostoma duodenale. AduNAP4 has 104 amino acids including a predicted 23-residue signal peptide and shows ?50% similarity with other known nematode anticoagulant protein/peptide (NAP). AduNAP4 is extremely efficient at prolonging the activated partial thromboplastin time, and is an inhibitor of both fXa (K_i = 7.34 ± 1.74 nM) and fXIa (K_i = 42.45 ± 3.25 nM). No fXIa inhibitor has previously been described from other blood-feeding animals. Our results suggest that hookworms have evolved a potent mechanism that interferes with coagulation by inhibition of fXIa to facilitate its blood-feeding lifestyle.     

Purification and characterization of two ryanodine-binding protein isoforms from sarcoplasmic reticulum of bullfrog skeletal muscle.

The two ryanodine-binding proteins (RyBPs) have been purified from sarcoplasmic reticulum of bullfrog skeletal muscle by Mono Q column chromatography following solubilization of SR by CHAPS and sucrose density gradient centrifugation. We conclude that the two RyBPs (alpha- and beta-RyBP) are isoforms on the basis (i) that each RyBP is distinguished by a specific polyclonal antibody and (ii) that distinct polypeptides are generated by limited tryptic digestion of the two RyBPs. Monomeric molecular weights for alpha- and beta-RyBP are estimated to be (690 +/- 10) and (570 +/- 10) kDa, respectively, as determined from mobilities on disc SDS-PAGE using the Weber-Osborn buffer system without 6 M urea, which gives an estimate of (590 +/- 10) kDa for RyBP of rabbit skeletal muscle. Similar determination in the presence of 6 M urea gave 630 kDa for alpha-RyBP and unchanged estimates for the other RyBPs. Both RyBPs show [3H]ryanodine-binding activities which are activated by Ca2+, AMPOPCP, and caffeine, and inhibited by ruthenium red, MgCl2, and procaine. beta-RyBP, however, has higher affinity for Ca2+. In the presence of Ca2+ and AMPOPCP, both RyBPs show single homogeneous binding sites for [3H]ryanodine with Kd = 2-5 nM. The values of Bmax for alpha- and beta-RyBP were 320-340 and 320-375 pmol/mg protein, respectively. These results are consistent with the conclusion that a homo-tetramer of each RyBP binds one ryanodine molecule, taking account of the estimated molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

Passive force generation and titin isoforms in mammalian skeletal muscle.

When relaxed striated muscle cells are stretched, a resting tension is produced which is thought to arise from stretching long, elastic filaments composed of titin (also called connectin). Here, I show that single skinned rabbit soleus muscle fibers produce resting tension that is several-fold lower than that found in rabbit psoas fibers. At sarcomere lengths where the slope of the resting tension-sarcomere length relation is low, electron microscopy of skinned fibers indicates that thick filaments move from the center to the side of the sarcomere during prolonged activation. As sarcomeres are stretched and the resting tension sarcomere length relation becomes steeper, this movement is decreased. The sarcomere length range over which thick filament movement decreases is higher in soleus than in psoas fibers, paralleling the different lengths at which the slope of the resting tension-sarcomere length relations increase. These results indicate that the large differences in resting tension between single psoas and soleus fibers are due to different tensions exerted by the elastic elements linking the end of each thick filament to the nearest Z-disc, i.e., the titin filaments. Quantitative gel electrophoresis of proteins from single muscle fibers excludes the possibility that resting tension is less in soleus than in psoas fibers simply because they have fewer titin filaments. A small difference in the electrophoretic mobility of titin between psoas and soleus fibers suggests the alternate possibility that mammalian muscle cells use at least two titin isoforms with differing elastic properties to produce variations in resting tension.