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2010-01-01
Catestatin, an endogenous peptide derived from bovine chromogranin A, and its active domain cateslytin display powerful antimicrobial activities. We have tested the activities of catestatin and other related peptides on the growth of Plasmodium falciparum in vitro. Catestatin inhibits growth of the chloroquine-sensitive strain of P. falciparum 3D7, exhibiting 88% inhibition at 20 μM. A similar partial inhibition of parasite growth was observed for the chloroquine-resistant strain, 7G8 (64%,) and the multidrug-resistant strain, W2 (62%). In the presence of parasite-specific lactate dehydrogenase, a specific protein–protein interaction between catestatin and plasmepsin II precursor was demonstrated. In addition, catestatin partially inhibited the parasite-specific proteases plasmepsin in...
Full Text Available.BackgroundAntimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides.Methodology/Principal FindingsPMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity.Conclusions/SignificanceFor the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems.
http://services.ands.org.au/home/orca/rda/view.php?key=71ffe97b-efec-4814-a657-4240fef7cae2
Neuronal nitric oxide synthase (nNOS) inhibition tests were carried out on peptides in addition to other bioactivity experiments, in particular, antibiotic activity. Inhibition of nNOS was measured by monitoring the conversion of [3H]arginine to [3H]citrulline. Information recorded: individual identifier for sample, peptide sequence, molecular weight, stock concentration (mg/ml), source (native peptide, synthetic derivative), solvent and concentration.
http://hdl.handle.net/2440/43291
Src signalling and transduction are directly involved in cell growth, cell cycle, malignant transformation and cell migration, providing therapeutic opportunities through inhibition of Src. Here we report virtual screening for novel compounds that inhibit the Src-SH3 protein–protein interaction with a proline-rich peptide ligand. Computational docking of the ZINC compound database was performed using GOLD. Top-scoring compounds were assayed using a fluorescence polarization-based assay. A benzoquinoline derivative showed micromolar inhibition of binding between Src-SH3 and the proline-rich peptide. Several analogues were subsequently assayed showing the requirement of a linker between the benzoquinoline and phenyl rings, and electron donating substituents on the phenyl ring.Noor Atatreh, Cvetan Stojkoski, Phillippa Smith, Grant W. Booker, Caroline Dive, A. David Frenkel, Sally Freeman, and Richard A. Brycehttp://www.elsevier.com/wps/find/journaldescription.cws_home/972/description#description Publisher: Pergamon-Elsevier Science Ltd Contributor: School of Molecular and Biomedical Science Other identifier: Bioorganic & Medicinal Chemistry Letters, 2007; 18 (3):1217-1222; 0960-894X; 0020075037; 10.1016/j.bmcl.2007.11.115 Language: en
http://hdl.handle.net/10072/29371
The first solution state structural analysis (NMR) of the C-terminal sequence of human GL that binds to glycogen phosphorylase a (GPa), PEWPSYLGYEKLGPYY-NH2 (1), showed it to be in a random coil conformation. This was supported by molecular dynamics simulation (modelled in solution) using NAMD 2.6. The conformational ambiguity of the peptide makes the structural arrangement of the peptide (and internal residues) strongly dependent on the environment. Thirteen tetra-peptide fragments of the C-terminal sequence, YEKLG-NH2, and the corresponding tri- and di-peptide sequences were used in a fragment screen against GPa. Compound 2 (H-GPYY-NH2) did not give an IC50 value, whereas PEWPSYLGYEKLGPYY-NH2 (1) displayed an IC50 of 34 µM against GPa. Truncated peptides derived from 1, (EKL-NH2, EKLG-NH2, and AcEKNH2) inhibited GPa (21%, 32%, 63%, respectively at 22 mM). These studies suggest key residues within the peptide chain have additional molecular interactions with GPa. The interaction of intra-sequence residues in combination with the terminal residues of PEWPSYLGYEKLGPYY with GPa may form the basis for the design of new inhibitors of GPa. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. Publisher: http://dx.doi.org/10.1002/psc.1140; John Wiley & Sons Ltd; United Kingdom Relation: Journal of Peptide Science; 442; 450; N; 15 Other identifier: 1075-2617 Language: en_AU Rights: Copyright 2009 European Peptide Society and John Wiley & Sons, Ltd. Self-archiving of the author-manuscript version is not yet supported by the European Peptide Society and John Wiley & Sons, Ltd. Please refer to the journal link for access to the definitive, published version or contact the authors for more information.; Y
http://hdl.handle.net/10072/14074
A T cell antigen receptor (TCR) transmembrane sequence derived peptide (CP) has been shown to inhibit T cell activation both in vitro and in vivo at the membrane level of the receptor signal transduction. To examine the effect of sugar or lipid conjugations on CP function, we linked CP to 1-aminoglucosesuccinate (GS), N-myristate (MYR), mono-di-tripalmitate (LP1, LP2, or LP3), and a lipoamino acid (LA) and examined the effects of these compounds on T cell activation in vitro and by using a rat model of adjuvant-induced arthritis, in vivo. In vitro, antigen presentation results demonstrated that lipid conjugation enhanced CP's ability to lower IL-2 production from 56.99%+/-15.69 S.D. observed with CP, to 12.08%+/-3.34 S.D. observed with LA. The sugar conjugate GS resulted in only a mild loss of in vitro activity compared to CP (82.95%+/-14.96 S.D.). In vivo, lipid conjugation retarded the progression of adjuvant-induced arthritis by approximately 50%, whereas the sugar conjugated CP, GS, almost completely inhibited the progression of arthritis. This study demonstrates that hydrophobic peptide activity is markedly enhanced in vitro and in vivo by conjugation to lipids or sugars. This may have practical applications in drug delivery and bioavailability of hydrophobic peptides. Publisher: Elsevier BV; Netherlands Contributor: E L F Holzbauer Relation: 8; Biochimica et Biophysica Acta (BBA) - Molecular Cell Research; 879; 888; N; 1763 Other identifier: 0167-4889 Language: en_AU Rights: Y
http://espace.library.uq.edu.au/view/UQ:80307
A T cell antigen receptor (TCR) transmembrane sequence derived peptide (CP) has been shown to inhibit T cell activation both in vitro and in vivo at the membrane level of the receptor signal transduction. To examine the effect of sugar or lipid conjugations on CP function, we linked CP to 1-aminoglucosesuccinate (GS), N-myristate (MYR), mono-di-tripalmitate (LP1, LP2, or LP3), and a lipoamino acid (LA) and examined the effects of these compounds on T cell activation in vitro and by using a rat model of adjuvant-induced arthritis, in vivo. In vitro, antigen presentation results demonstrated that lipid conjugation enhanced CP's ability to lower IL-2 production from 56.99% +/- 15.69 S.D. observed with CP, to 12.08% +/- 3.34 S.D. observed with LA. The sugar conjugate GS resulted in only a mild loss of in vitro activity compared to CP (82.95% +/- 14.96 S.D.). In vivo, lipid conjugation retarded the progression of adjuvant-induced arthritis by approximately 50%, whereas the sugar. conjugated CP, GS, almost completely inhibited the progression of arthritis. This study demonstrates that hydrophobic peptide activity is markedly enhanced in vitro and in vivo by conjugation to lipids or sugars. This may have practical applications in drug delivery and bioavailability of hydrophobic peptides. (c) 2006 Elsevier B.V. All rights reserved. Publisher: Elsevier Science BV Coverage: 2006-08-01T00:00:00Z
http://espace.library.uq.edu.au/view/UQ:73251
The sliding clamp of the Escherichia coli replisome is now understood to interact with many proteins involved in DNA synthesis and repair. A universal interaction motif is proposed to be one mechanism by which those proteins bind the E. coli sliding clamp, a homodimer of the beta subunit, at a single site on the dimer. The numerous beta(2)-binding proteins have various versions of the consensus interaction motif, including a related hexameric sequence. To determine if the variants of the motif could contribute to the competition of the beta-binding proteins for the beta(2) site, synthetic peptides derived from the putative beta(2)-binding motifs were assessed for their abilities to inhibit protein-beta(2) interactions, to bind directly to beta(2), and to inhibit DNA synthesis in vitro. A hierarchy emerged, which was consistent with sequence similarity to the pentameric consensus motif, QL(S/D)LF, and peptides containing proposed hexameric motifs were shown to have activities comparable to those containing the consensus sequence. The hierarchy of peptide binding may be indicative of a competitive hierarchy for the binding of proteins to beta(2) in various stages or circumstances of DNA replication and repair. Publisher: American Chemical Society Coverage: 2004-01-01T00:00:00Z
http://espace.library.uq.edu.au/view/UQ:180818
The first solution state structural analysis (NMR) of the C-terminal sequence of human GL that binds to glycogen phosphorylase a (GPa), PEWPSYLGYEKLGPYY-NH2 (1), showed it to be in a random coil conformation. This was supported by molecular dynamics simulation (modelled in solution) using NAMD 2.6. The conformational ambiguity of the peptide makes the structural arrangement of the peptide (and internal residues) strongly dependent on the environment. Thirteen tetra-peptide fragments of the C-terminal sequence, YEKLG-NH2, and the corresponding tri- and di-peptide sequences were used in a fragment screen against GPa. Compound 2 (H-GPYY-NH2) did not give an IC50 value, whereas PEWPSYLGYEKLGPYY-NH2 (1) displayed an IC50 of 34 µM against GPa. Truncated peptides derived from 1, (EKL-NH2, EKLG-NH2, and AcEKNH2) inhibited GPa (21%, 32%, 63%, respectively at 22 mM). These studies suggest key residues within the peptide chain have additional molecular interactions with GPa. The interaction of intra-sequence residues in combination with the terminal residues of PEWPSYLGYEKLGPYY with GPa may form the basis for the design of new inhibitors of GPa. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. Publisher: John Wiley & Sons Ltd. Contributor: J R Jones, Luis Moroder (Editor-in-Chief); Luis Moroder (Editor-in-Chief) Coverage: 2009-06-01T00:00:00Z
http://espace.library.uq.edu.au/view/UQ:164226
ACE inhibitory peptides are biologically active peptides that play a role in blood pressure regulation. When derived from food proteins during food processing or gastrointestinal digestion, these peptides could function as efficient agents in treating and preventing hypertension. However, in order to exert an antihypertensive effect by inhibition of the ACE enzyme, they have to reach the bloodstream intact. The aim of this research was to assess if the known ACE inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg, derived from a tryptic digest of β-lactoglobulin, could be absorbed through a Caco-2 Bbe cell monolayer in an Ussing chamber and reach the serosal side undegraded. Samples of the mucosal compartment showed high ACE inhibitory activity. No or only little ACE inhibitory activity was detected in the serosal compartment. However, when the serosal sample was concentrated three-fold, a substantial ACE inhibitory activity was registered. Concomitantly, HPLC and MS clearly showed the presence of Ala-Leu-Pro-Met-His-Ile-Arg in the mucosal compartment, whereas in the serosal compartment only MS was able to detect the heptapeptide. In conclusion, under the observed experimental conditions, the ACE inhibitory peptide Ala-Leu-Pro-Met-His- Ile-Arg was transported intact through the Caco-2 Bbe monolayer, but in concentrations too low to exert an ACE inhibitory activity. Publisher: John Wiley & Sons Coverage: 2002-03-01T00:00:00Z
http://espace.library.uq.edu.au/view/UQ:163725
Angiotensin-converting enzyme (ACE) plays a major role in the regulation of blood pressure. A diagnostic assay to measure angiotensin-converting enzyme (ACE) activity was transformed into an enzyme inhibition assay and optimised, which led to a more sensitive and less expensive assay. By this spectrophotometric method, ACE inhibition is measured using the substrate furanacryloyl-Phe-Gly-Gly and as ACE source rabbit lung acetone extract. The optimised as well as the original ACE inhibition assay were used to verify the ACE inhibitory activity of captopril. The ACE inhibition assay was further validated by enalapril, its active derivative enalaprilat and the ACE-inhibitory peptide Ala-Leu-Pro-Met-His-Ile-Arg, corresponding to a tryptic fragment of bovine β-lactoglobulin. Sigmoid curves could be fit adequately to the data points representing ACE inhibition in function of inhibitor concentration. IC50 values for these compounds corresponded well with literature data. Furthermore, pea and whey protein hydrolysates obtained by digestion with trypsin showed ACE inhibitory activity in the ACE inhibition assay. Hence, this optimised assay is suitable to screen for ACE inhibitory peptides derived from food proteins with a possible antihypertensive effect in vivo. Publisher: Elsevier Science B.V. Coverage: 2002-03-04T00:00:00Z
http://espace.library.uq.edu.au/view/UQ:143855
kappa-Conotoxin-PVIIA (kappa-PVIIA) belongs to a family of peptides derived from a hunting marine snail that tar gets to a wide variety of ion channels and receptors. kappa-PVIIA is a small, structurally constrained, 27-residue peptide that inhibits voltage-gated K channels. Three disulfide bonds shape a characteristic four-loop folding. The spatial localization of positively charged residues in K-PVIIA exhibits strong structural mimicry to that of charybdotoxin, a scorpion toxin that occludes the pore of K channels. Me studied the mechanism by which this peptide inhibits Shaker, K channels expressed in Xenopus oocytes with the N-type inactivation removed. Chronically applied to whole oocytes or outside-out patches, kappa-PVIIA inhibition appears as a voltage-dependent relaxation in response to the depolarizing pulse used to activate the channels. At any applied voltage, the relaxation rare depended linearly on the toxin concentration, indicating a bimolecular stoichiometry. Time constants and voltage dependence of the current relaxation produced by chronic applications agreed with that of rapid applications to open channels. Effective valence of the voltage dependence, z delta, is similar to 0.55 and resides primarily in the rare of dissociation from the channel, while the association rate is voltage independent with a magnitude of 10(7)-10(8) M-1 s(-1), consistent with diffusion-limited binding. Compatible with a purely competitive interaction for a site in the external vestibule, tetraethylammonium, a well-known Ii-pore blocker, reduced kappa-PVIIA's association rate only. Removal of internal K+ reduced, but did not eliminate, the effective valence of the toxin dissociation rate to a value <0.3. This trans-pore effect suggests that: (a) as in the alpha-KTx, a positively changed side chain, possibly a Lys, interacts electrostatically with ions residing inside the Shaker pore, and (b) a part of the toxin occupies an externally accessible K+ binding site, decreasing the degree of pore occupancy by permeant ions, We conclude that, although evolutionarily distant to scorpion toxins, kappa-PVIIA shares with them a remarkably similar mechanism of inhibition of K channels. Publisher: Rockfeller Univ. Press Coverage: 1999-07-01T00:00:00Z
Full Text Available.BackgroundTesticular germ cell tumors (TGCTs) are classified as seminonas or non-seminomas of which a major subset is embryonal carcinoma (EC) that can differentiate into diverse tissues. The pluripotent nature of human ECs resembles that of embryonic stem (ES) cells. Many Wnt signalling species are regulated during differentiation of TGCT-derived EC cells. This study comprehensively investigated expression profiles of Wnt signalling components regulated during induced differentiation of EC cells and explored the role of key components in maintaining pluripotency.MethodsHuman embryonal carcinoma cells were stably infected with a lentiviral construct carrying a canonical Wnt responsive reporter to assess Wnt signalling activity following induced differentiation. Cells were differentiated with all-trans retinoic acid (RA) or by targeted repression of pluripotency factor, POU5F1. A Wnt pathway real-time-PCR array was used to evaluate changes in gene expression as cells differentiated. Highlighted Wnt pathway genes were then specifically repressed using siRNA or stable shRNA and transfected EC cells were assessed for proliferation, differentiation status and levels of core pluripotency genes.ResultsCanonical Wnt signalling activity was low basally in undifferentiated EC cells, but substantially increased with induced differentiation. Wnt pathway gene expression levels were compared during induced differentiation and many components were altered including ligands (WNT2B), receptors (FZD5, FZD6, FZD10), secreted inhibitors (SFRP4, SFRP1), and other effectors of Wnt signalling (FRAT2, DAAM1, PITX2, Porcupine). Independent repression of FZD5, FZD7 and WNT5A using transient as well as stable methods of RNA interference (RNAi) inhibited cell growth of pluripotent NT2/D1 human EC cells, but did not appreciably induce differentiation or repress key pluripotency genes. Silencing of FZD7 gave the greatest growth suppression in all human EC cell lines tested including NT2/D1, NT2/D1-R1, Tera-1 and 833K cells.ConclusionDuring induced differentiation of human EC cells, the Wnt signalling pathway is reprogrammed and canonical Wnt signalling induced. Specific species regulating non-canonical Wnt signalling conferred growth inhibition when targeted for repression in these EC cells. Notably, FZD7 repression significantly inhibited growth of human EC cells and is a promising therapeutic target for TGCTs.
BackgroundTesticular germ cell tumors (TGCTs) are classified as seminonas or non-seminomas of which a major subset is embryonal carcinoma (EC) that can differentiate into diverse...Full Text Available
1991-02-01
The effects of U-61,431F, ciprostene, a stable prostacyclin analogue, were examined on the proliferation of cultured quiescent bovine aortic endothelial cells (EC) and smooth muscle cells (SMC). After stimulation with 5% fetal calf serum, U-61,431F suppressed both the DNA synthesis and proliferation of SMC dose-dependently at the concentration of 3-100 microM, but had no effect on either of them in EC at a concentration of up to 30 microM. The inhibitory effect on DNA synthesis was greater in SMC than in EC at 3-50 microM. When SMC were stimulated with platelet-derived growth factor (PDGF) for 2 hrs followed by a 22-hr incubation with insulin, U-61,431F (1-50 microM) administered at the time of PDGF stimulation did not inhibit DNA synthesis. SMC initiated and terminated DNA synthesis at about 15-18 h and 24 h after stimulation with serum, respectively. Inhibition of DNA synthesis in serum-stimulated SMC as a function of the addition time of U-61,431F reduced at 3-12 h after the stimulation. U-61,431F raised the cyclic AMP (cAMP) content in SMC. Moreover, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, and a more specific cAMP phosphodiesterase inhibitor, Ro 20-1724, augmented the inhibition of DNA synthesis in SMC concomitant with further elevation of cAMP level. These results suggest that U-61,431F inhibits DNA synthesis of SMC acting in the progression stage rather than in the competence stage, with little antiproliferative effect on EC. cAMP may play an important role in its antiproliferative action in SMC.
1991-01-01
The effects of U-61,431F, ciprostene, a stable prostacyclin analogue, were examined on the proliferation of cultured quiescent bovine aortic endothelial cells (EC) and smooth muscle cells (SMC). After stimulation with 5% fetal calf serum, U-61,431F suppressed both the DNA synthesis and proliferation of SMC dose-dependently at the concentration of 3-100 microM, but had no effect on either of them in EC at a concentration of up to 30 microM. The inhibitory effect on DNA synthesis was greater in SMC than in EC at 3-50 microM. When SMC were stimulated with platelet-derived growth factor (PDGF) for 2 hrs followed by a 22-hr incubation with insulin, U-61,431F (1-50 microM) administered at the time of PDGF stimulation did not inhibit DNA synthesis. SMC initiated and terminated DNA synthesis at about 15-18 h and 24 h after stimulation with serum, respectively. Inhibition of DNA ... >>
BackgroundAntimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs...Full Text Available
Trichostatin A inhibits beta-casein expression in mammary epithelial cells
2002-01-01
Many aspects of cellular behavior are affected by information derived from association of the extracellular matrix (ECM) and with cell membrane receptors. When cultured in the presence of laminin-containing ECM and prolactin (Prl), normal mammary epithelial cells express the milk protein beta-casein. Previously, we defined the minimal ECM- and Prl-responsive enhancer element BCE-1 from the upstream region of the beta-casein gene. We also found that BCE-1 was only active when stably integrated into chromatin, and that trichostatin A (TSA), a reagent that leads to alterations in chromatin structure, was able to activate the integrated enhancer element. We now show that endogenous b-casein gene, which is controlled by a genetic assembly that is highly similar to that of BCE-1 and which is also activated by incubation in ECM and Prl, is instead inhibited by TSA. We provide ... >>
1993-09-01
These studies describe progress made in the definition of the transport capabilities and reaction mechanism of the V-PPase through biochemical analyses of native membrane vesicles isolated from etiolated hypocotyls of Vigna radiate and by reconstitution of the purified enzyme into artificial liposomes; delineation of the ligand requirements of the V-PPase; and the delineation of sequence motifs implicated in substrate-binding through the development of strategies for selective cleavage of the M{sub r} 66,000 polypeptide and the mapping of covalently modified peptide fragments.
The origins of phagocytosis and eukaryogenesis
Full Text Available.BackgroundPhagocytosis, that is, engulfment of large particles by eukaryotic cells, is found in diverse organisms and is often thought to be central to the very origin of the eukaryotic cell, in particular, for the acquisition of bacterial endosymbionts including the ancestor of the mitochondrion.ResultsComparisons of the sets of proteins implicated in phagocytosis in different eukaryotes reveal extreme diversity, with very few highly conserved components that typically do not possess readily identifiable prokaryotic homologs. Nevertheless, phylogenetic analysis of those proteins for which such homologs do exist yields clues to the possible origin of phagocytosis. The central finding is that a subset of archaea encode actins that are not only monophyletic with eukaryotic actins but also share unique structural features with actin-related proteins (Arp) 2 and 3. All phagocytic processes are strictly dependent on remodeling of the actin cytoskeleton and the formation of branched filaments for which Arp2/3 are responsible. The presence of common structural features in Arp2/3 and the archaeal actins suggests that the common ancestors of the archaeal and eukaryotic actins were capable of forming branched filaments, like modern Arp2/3. The Rho family GTPases that are ubiquitous regulators of phagocytosis in eukaryotes appear to be of bacterial origin, so assuming that the host of the mitochondrial endosymbiont was an archaeon, the genes for these GTPases come via horizontal gene transfer from the endosymbiont or in an earlier event.ConclusionThe present findings suggest a hypothetical scenario of eukaryogenesis under which the archaeal ancestor of eukaryotes had no cell wall (like modern Thermoplasma) but had an actin-based cytoskeleton including branched actin filaments that allowed this organism to produce actin-supported membrane protrusions. These protrusions would facilitate accidental, occasional engulfment of bacteria, one of which eventually became the mitochondrion. The acquisition of the endosymbiont triggered eukaryogenesis, in particular, the emergence of the endomembrane system that eventually led to the evolution of modern-type phagocytosis, independently in several eukaryotic lineages.ReviewersThis article was reviewed by Simonetta Gribaldo, Gaspar Jekely, and Pierre Pontarotti. For the full reviews, please go to the Reviewers' Reports section.
The origins of phagocytosis and eukaryogenesis
BackgroundPhagocytosis, that is, engulfment of large particles by eukaryotic cells, is found in diverse organisms and is often thought to be central to the very origin of the eukaryotic...Full Text Available
2010-01-01
Streptomyces coelicolor mutants lacking the zinc-responsive Zur repressor are conditionally defective in sporulation, presumably due to the overexpression of one or more Zur target...Full Text Available
2010-01-01
Full Text Available.Streptomyces coelicolor mutants lacking the zinc-responsive Zur repressor are conditionally defective in sporulation, presumably due to the overexpression of one or more Zur target genes. Gene disruption analyses revealed that deregulation of previously known Zur targets was not responsible for the sporulation phenotype. We used microarrays to identify further Zur targets and discovered that Zur controls a cluster of genes predicted to direct synthesis of an uncharacterized siderophore-related non-ribosomally encoded peptide designated coelibactin. Disruption of a key coelibactin biosynthetic gene suppressed the Zur sporulation phenotype, suggesting that deregulation of coelibactin synthesis inhibits sporulation.
2009-07-01
Phytochrome A (phyA) is the primary photoreceptor for sensing extremely low amounts of light and for mediating various far-red light-induced responses in higher plants. Translocation from the cytosol...Full Text Available
2009-07-01
Full Text Available.Phytochrome A (phyA) is the primary photoreceptor for sensing extremely low amounts of light and for mediating various far-red light-induced responses in higher plants. Translocation from the cytosol to the nucleus is an essential step in phyA signal transduction. EID1 (for EMPFINDLICHER IM DUNKELROTEN LICHT1) is an F-box protein that functions as a negative regulator in far-red light signaling downstream of the phyA in Arabidopsis (Arabidopsis thaliana). To identify factors involved in EID1-dependent light signal transduction, pools of ethylmethylsulfonate-treated eid1-3 seeds were screened for seedlings that suppress the hypersensitive phenotype of the mutant. The phenotype of the suppressor mutant presented here is caused by a missense mutation in the PHYA gene that leads to an amino acid transition in its histidine kinase-related domain. The novel phyA-402 allele alters the spectral sensitivity and the persistence of far-red light-induced high-irradiance responses. The strong eid1-3 suppressor phenotype of phyA-402 contrasts with the moderate phenotype observed when phyA-402 is introgressed into the wild-type background, which indicates that the mutation mainly alters functions in an EID1-dependent signaling cascade. The mutation specifically inhibits nuclear accumulation of the photoreceptor molecule upon red light irradiation, even though it still interacts with FHY1 (for far-red long hypocotyl 1) and FHL (for FHY1-like protein), two factors that are essential for nuclear accumulation of phyA. Degradation of the mutated phyA is unaltered even under light conditions that inhibit its nuclear accumulation, indicating that phyA degradation may occur mostly in the cytoplasm.
2009-08-03
Full Text Available.Down syndrome critical region gene 1 (DSCR-1) short variant (DSCR-1s) is an inhibitor of calcineurin/NFAT signaling encoded by exons 4–7 of DSCR1. We previously reported that VEGF induces DSCR-1s expression in endothelial cells, which in turn negatively feeds back to attenuate endothelial cell activation. Here, in order to characterize the role of the promoter that drives DSCR-1s expression in mediating inducible expression in vivo and to determine the functional relevance of DSCR-1s in inflammation, we targeted a DNA construct containing 1.7 kb of the human DSCR1s promoter coupled to the lacZ reporter to the hypoxanthine guanine phosphoribosyl transferase (Hprt) locus of mice. We determined that lacZ was uniformly expressed in the endothelium of transgenic embryos but was markedly downregulated postnatally. Systemic administration of VEGF or LPS in adult mice resulted in cyclosporine A–sensitive reactivation of the DSCR1s promoter and endogenous gene expression in a subset of organs, including the heart and brain. The DSCR1s promoter was similarly induced in the endothelium of tumor xenografts. In a mouse model of endotoxemia, DSCR-1s–deficient mice demonstrated increased sepsis mortality, whereas adenovirus-mediated DSCR-1s overexpression protected against LPS-induced lethality. Collectively, these data suggest that the DSCR1s promoter directs vascular bed–specific expression in activated endothelium and that DSCR-1s serves to dampen the host response to infection.
2009-08-03
Down syndrome critical region gene 1 (DSCR-1) short variant (DSCR-1s) is an inhibitor of calcineurin/NFAT signaling encoded by exons 4–7 of DSCR1. We previously reported that...Full Text Available
Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth
2009-08-01
The development of short interfering RNA (siRNA), has provided great hope for therapeutic targeting of specific genes responsible for patholological disorders. However, the poor cellular uptake and...Full Text Available
Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth
2009-08-01
Full Text Available.The development of short interfering RNA (siRNA), has provided great hope for therapeutic targeting of specific genes responsible for patholological disorders. However, the poor cellular uptake and bioavailability of siRNA remain a major obstacle to their clinical development and most strategies that propose to improve siRNA delivery remain limited for in vivo applications. In this study, we report a novel peptide-based approach, MPG-8 an improved variant of the amphipathic peptide carrier MPG, that forms nanoparticles with siRNA and promotes their efficient delivery into primary cell lines and in vivo upon intra-tumoral injection. Moreover, we show that functionalization of this carrier with cholesterol significantly improves tissue distribution and stability of siRNA in vivo, thereby enhancing the efficiency of this technology for systemic administration following intravenous injection without triggering any non-specific inflammatory response. We have validated the therapeutic potential of this strategy for cancer treatment by targeting cyclin B1 in mouse tumour models, and demonstrate that tumour growth is compromised. The robustness of the biological response achieved through this approach, infers that MPG 8-based technology holds a strong promise for therapeutic administration of siRNA.
2009-04-01
Immune sensing of a microbe occurs via multiple receptors. How signals from different receptors are coordinated to yield a specific immune response is poorly understood. We demonstrate that...Full Text Available
2009-04-01
Full Text Available.Immune sensing of a microbe occurs via multiple receptors. How signals from different receptors are coordinated to yield a specific immune response is poorly understood. We demonstrate that the different pathogen recognition receptors, TLR2 and dectin-1, recognizing the same microbial stimulus, stimulate distinct innate and adaptive responses. TLR2 signaling induced splenic dendritic cells (DCs) to express the retinoic acid (RA) metabolizing enzyme Raldh2 and IL-10, and to metabolize vitamin A and stimulate Foxp3+ T regulatory cells (Treg cells). RA acted on DCs to induce Socs3 expression, which suppressed activation of p38 MAPK and pro-inflammatory cytokines. Consistent with this, TLR2 signaling induced Treg cells, and suppressed IL-23 and TH-17/ TH-1 mediated autoimmune responses in vivo. In contrast, dectin-1 signaling mostly induced IL-23 and pro-inflammatory cytokines, and augmented TH-17/ TH-1 mediated autoimmune responses in vivo. These data define a new mechanism for the systemic induction of RA and immune suppression against autoimmunity.
Synthetic Nano-Low Density Lipoprotein as Targeted Drug DeliveryVehicle for Glioblastoma Multiforme
2006-06-14
This paper discribes a synthetic low density lipoprotein(LDL) made by complexing a 29 amino acid that consists of a lipid bindingdomain and the LDL receptor binding domain with a lipid microemulsion.The nano-LDL particles were intermdiate in size between LDL and HDL andbound to LDL receptors on GBM brain tumor cells. Synthetic nano-LDLuptake by GBM cells was LDL receptor specific and dependent on cellreceptor number. It is suggested that these synthetic particles can serveas a delivery vehicle for hydophobic anti-tumor drugs by targeting theLDL receptor.
2010-05-01
Non-antibody scaffold proteins are used for a range of applications, especially the assessment of protein–protein interactions within human cells. The search for a versatile, robust and biologically...Full Text Available
2010-05-01
Full Text Available.Non-antibody scaffold proteins are used for a range of applications, especially the assessment of protein–protein interactions within human cells. The search for a versatile, robust and biologically neutral scaffold previously led us to design STM (stefin A triple mutant), a scaffold derived from the intracellular protease inhibitor stefin A. Here, we describe five new STM-based scaffold proteins that contain modifications designed to further improve the versatility of our scaffold. In a step-by-step approach, we introduced restriction sites in the STM open reading frame that generated new peptide insertion sites in loop 1, loop 2 and the N-terminus of the scaffold protein. A second restriction site in ‘loop 2’ allows substitution of the native loop 2 sequence with alternative oligopeptides. None of the amino acid changes interfered significantly with the folding of the STM variants as assessed by circular dichroism spectroscopy. Of the five scaffold variants tested, one (stefin A quadruple mutant, SQM) was chosen as a versatile, stable scaffold. The insertion of epitope tags at varying positions showed that inserts into loop 1, attempted here for the first time, were generally well tolerated. However, N-terminal insertions of epitope tags in SQM had a detrimental effect on protein expression.
1992-08-01
Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by [sup 31]P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 [Angstrom] of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an [alpha]-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.
1992-08-01
Catalytic immunoglobin fragments were studied Nuclear Magnetic Resonance spectroscopy to identify amino acid residues responsible for the catalytic activity. Small, hybrid sequence peptides were analyzed for helix propagation following covalent initiation and for activity related to the protein from which the helical sequence was derived. Hydrolysis of p-nitrophenyl carbonates and esters by specific immunoglobins is thought to involve charge complementarity. The pK of the transition state analog P-nitrophenyl phosphate bound to the immunoglobin fragment was determined by {sup 31}P-NMR to verify the juxtaposition of a positively charged amino acid to the binding/catalytic site. Optical studies of immunoglobin mediated photoreversal of cis, syn cyclobutane thymine dimers implicated tryptophan as the photosensitizing chromophore. Research shows the chemical environment of a single tryptophan residue is altered upon binding of the thymine dimer. This tryptophan residue was localized to within 20 {Angstrom} of the binding site through the use of a nitroxide paramagnetic species covalently attached to the thymine dimer. A hybrid sequence peptide was synthesized based on the bee venom peptide apamin in which the helical residues of apamin were replaced with those from the recognition helix of the bacteriophage 434 repressor protein. Oxidation of the disufide bonds occured uniformly in the proper 1-11, 3-15 orientation, stabilizing the 434 sequence in an {alpha}-helix. The glycine residue stopped helix propagation. Helix propagation in 2,2,2-trifluoroethanol mixtures was investigated in a second hybrid sequence peptide using the apamin-derived disulfide scaffold and the S-peptide sequence. The helix-stop signal previously observed was not observed in the NMR NOESY spectrum. Helical connectivities were seen throughout the S-peptide sequence. The apamin/S-peptide hybrid binded to the S-protein (residues 21-166 of ribonuclease A) and reconstituted enzymatic activity.
Structural determination of larger proteins using stable isotope labeling and NMR spectroscopy
1996-04-01
The project sought to employ stable isotope labeling and NMR spectroscopy to study protein structures and provide insight into important biochemical problems. A methylotrophic bacterial expression system has been developed for uniform deuterium and carbon-13 labeling of proteins for structural studies. These organisms grow using methanol as the sole source of carbon and energy. Because isotopically labeled methanol is relatively inexpensive, the methylotrophs are ideal for expressing proteins labeled uniformly with deuterium and/or carbon-13. This expression system has been employed to prepare deuterated troponin C. NMR spectroscopy measurements have been made on the inhibitory peptide from troponin I (residues 96--115), both as the free peptide and the peptide complexed with deuterated troponin C. Proton-NMR spectroscopy resonance-signal assignments have been made for the free peptide.
Stimulatory effects of neuronally released norepinephrine on renin release in vitro
1988-01-01
Extracellular high potassium inhibits renin release in vitro by increasing calcium concentrations in the juxtaglomerular cells. The authors found that the decreased response of renin release from rat kidney cortical slices in high potassium solution changed to a strikingly increased one in the presence of nifedipine at doses over 10-6 M. They then examined the stimulatory effect of extracellular high potassium in the presence of nifedipine on renin release. The enhancement of release was significantly suppressed either by propranolol or by metoprolol but not by prazosin. High potassium plus nifedipine-induced increase in renin release was markedly attenuated by renal denervation. The enhancing effect was not observed when the slices were incubated in calcium-free medium. Divalent cations such as Cd2+, Co2+, and Mn2+ ... >>
1991-02-01
We have previously demonstrated the presence of binding sites for atrial natriuretic peptide (ANP) in human platelets. These sites have pharmacological characteristics similar to those of rat vascular smooth muscle. They are subject to regulation by circulating levels of ANP in plasma, varying inversely with the latter after high sodium intake, in arterial hypertension and congestive heart failure. We have now solubilized these platelet receptors with the nonionic detergent Triton X-100 (0.6%). The preparations were incubated with (125I)ANP in the presence of increasing concentrations of ANP-(99-126), ANP-(101-126), ANP-(103-126), and ANP-(103-123). The order of potency of these peptides to displace (125I)ANP was similar for the solubilized and particulate receptor. Bound (125I)ANP was covalently cross-linked to the receptor with 5 mM disuccinimidyl suberate. Autoradiography of the sodium dodecyl sulfate-polyacrylamide gel showed that (125I)ANP specifically interacts with a 125-kDa membrane component, some of which may be reduced by 2% mercaptoethanol or 10 mmol/L dithiothreitol to a 70-kDa species. A small proportion of a 70-kDa peptide is also found under nonreducing conditions. The concentration of ANP-(99-126) that inhibits binding of (125I)ANP by 50% to both the 125-kDa and the 70-kDa species was 0.1 nM, while that for ANP-(103-123) was 3 nM. The internally ring-deleted analog Des(Gln116,Ser117,Gly118,Leu119,Gly120)ANP -(102-121) or C-ANP displaced with equal potency ANP binding to the high and low mol wt (Mr) bands, as also found in cultured rat vascular smooth muscle cells, but not in the mesemteric arteries these cells are derived from. In the latter, C-ANP displaced only binding from the lower Mr band. These results show that the ANP receptor in human platelets is heterogeneous.
1991-01-01
We have previously demonstrated the presence of binding sites for atrial natriuretic peptide (ANP) in human platelets. These sites have pharmacological characteristics similar to those of rat vascular smooth muscle. They are subject to regulation by circulating levels of ANP in plasma, varying inversely with the latter after high sodium intake, in arterial hypertension and congestive heart failure. We have now solubilized these platelet receptors with the nonionic detergent Triton X-100 (0.6%). The preparations were incubated with [125I]ANP in the presence of increasing concentrations of ANP-(99-126), ANP-(101-126), ANP-(103-126), and ANP-(103-123). The order of potency of these peptides to displace [125I]ANP was similar for the solubilized and particulate receptor. Bound [125I]ANP was covalently cross-linked to the receptor with 5 mM disuccinimidyl ... >>
2005-01-01
S100A6 (calcyclin), a member of the S100 family of EF-hand Ca2+ binding proteins, has been implicated in the regulation of cell growth and proliferation. We have previously shown that S100B, another member of the S100 family, is induced postinfarction and limits the hypertrophic response of surviving cardiac myocytes. We presently report that S100A6 expression is also increased in the periinfarct zone of rat heart postinfarction and in cultured neonatal rat myocytes by treatment with several trophic agents, including platelet-derived growth factor (PDGF), the alpha1-adrenergic agonist phenylephrine (PE), and angiotensin II (AII). Cotransfection of S100A6 in cultured neonatal rat cardiac myocytes inhibits induction of the cardiac fetal gene promoters skeletal alpha-actin (skACT) and beta-myosin ... >>
2009-05-01
Full Text Available.Adoptively transferred T cells possess anticancer activities partially mediated by T-cell FasL engagement of Fas tumor targets. However, antigen-induced T-cell activation and clonal expansion, which stimulates FasL activity, is often inefficient in tumors. As a gene therapy approach to overcome this obstacle, we have created oncoretroviral vectors to overexpress FasL or non-cleavable FasL (ncFasL) on murine T cells of a diverse T-cell receptor repertoire. Expression of c-FLIP was also engineered to prevent apoptosis of transduced cells. Retroviral transduction of murine T lymphocytes has historically been problematic, and we describe optimized T-cell transduction protocols involving CD3/CD28 co-stimulation of T cells, transduction on ice using concentrated oncoretrovirus, and culture with IL-15. Genetically modified T cells home to established prostate cancer tumors in vivo. Co-stimulated T cells expressing FasL, ncFasL and ncFasL/c-FLIP each mediated cytotoxicity in vitro against RM-1 and LNCaP prostate cancer cells. To evaluate the compatibility of this approach with current prostate cancer therapies, we exposed RM-1, LNCaP, and TRAMP-C1 cells to radiation, mitoxantrone, or docetaxel. Fas and H-2b expression were upregulated by these methods. We have developed a novel FasL-based immuno-gene therapy for prostate cancer that warrants further investigation given the apparent constitutive and inducible Fas pathway expression in this malignancy.
2009-05-01
Adoptively transferred T cells possess anticancer activities partially mediated by T-cell FasL engagement of Fas tumor targets. However, antigen-induced T-cell activation and clonal expansion,...Full Text Available
Response of tonoplast H sup + -pump and ATPase to cadmium
1991-05-01
It has been demonstrated that Cd{sup 2+} accumulates in vacuoles of tobacco leaves exposed to 20 {mu}M Cd{sup 2+}, after 4 days of growth in Hoaglands medium. The accumulation of Cd{sup 2+} is also associated with the accumulation of Cd{sup 2+}-peptide (phytocelatin) in the vacuole. The transport of Cd{sup 2+} and/or Cd{sup 2+}-peptide across the tonoplast membrane may be energized by the H{sup +} electrochemical gradient that exists across this membrane and which is generated by an H{sup +}-pumping ATPase. In vitro 2 {mu}M Cd{sup 2+} inhibits oat root tonoplast H{sup +}-pumping ATPase by 50% with a Ki of approximately 4.0 {mu}M. However, exposure to 2- {mu}M Cd{sup 2+} for 4 days during germination and growth of oat seedlings causes a 100% increase in tonoplast H{sup +}-pumping ATPase activity. This increase on exposure to Cd{sup 2+} during growth may represent part of the physiological mechanism whereby plants accumulate Cd{sup 2+} and/or Cd{sup 2+}-peptide within the vacuole. This could also represent an acclimation of H{sup +}-pumping activity to Cd{sup 2+}. Current studies are testing the effects of Cd{sup 2+}-peptide, organic acids and other ligands on H{sup +}-pumping activity and Cd{sup 2+} transport in root derived tonoplast vesicles of oat and tobacco.
Reaction mechanisms in the radiolysis of peptides, polypeptides and proteins
1985-01-01
The purpose of this review is to bring together and to correlate the wide variety of experimental studies that provide information on the reaction products and reaction mechanisms involved in the radiolysis of peptides, polypeptides and proteins (including chromosomal proteins) in both aqueous and solid-state systems. The comparative radiation chemistry of these systems is developed in terms of specific reactions of the peptide main-chain and the aliphatic, aromatic-unsaturated and sulfur-containing side-chains. Information obtained with the various experimental techniques of product analysis, competition kinetics, spin-trapping, pulse radiolysis and ESR spectroscopy is included. 147 refs.
2009-09-01
Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate...Full Text Available
2009-09-01
Full Text Available.Insulin signaling can be modulated by several isoforms of PKC in peripheral tissues. Here, we assessed whether one specific isoform, PKC-θ, was expressed in critical CNS regions that regulate energy balance and whether it mediated the deleterious effects of diets high in fat, specifically palmitic acid, on hypothalamic insulin activity in rats and mice. Using a combination of in situ hybridization and immunohistochemistry, we found that PKC-θ was expressed in discrete neuronal populations of the arcuate nucleus, specifically the neuropeptide Y/agouti-related protein neurons and the dorsal medial nucleus in the hypothalamus. CNS exposure to palmitic acid via direct infusion or by oral gavage increased the localization of PKC-θ to cell membranes in the hypothalamus, which was associated with impaired hypothalamic insulin and leptin signaling. This finding was specific for palmitic acid, as the monounsaturated fatty acid, oleic acid, neither increased membrane localization of PKC-θ nor induced insulin resistance. Finally, arcuate-specific knockdown of PKC-θ attenuated diet-induced obesity and improved insulin signaling. These results suggest that many of the deleterious effects of high-fat diets, specifically those enriched with palmitic acid, are CNS mediated via PKC-θ activation, resulting in reduced insulin activity.
Origin and evolution of the Notch signalling pathway: an overview from eukaryotic genomes
Full Text Available.BackgroundOf the 20 or so signal transduction pathways that orchestrate cell-cell interactions in metazoans, seven are involved during development. One of these is the Notch signalling pathway which regulates cellular identity, proliferation, differentiation and apoptosis via the developmental processes of lateral inhibition and boundary induction. In light of this essential role played in metazoan development, we surveyed a wide range of eukaryotic genomes to determine the origin and evolution of the components and auxiliary factors that compose and modulate this pathway.ResultsWe searched for 22 components of the Notch pathway in 35 different species that represent 8 major clades of eukaryotes, performed phylogenetic analyses and compared the domain compositions of the two fundamental molecules: the receptor Notch and its ligands Delta/Jagged. We confirm that a Notch pathway, with true receptors and ligands is specific to the Metazoa. This study also sheds light on the deep ancestry of a number of genes involved in this pathway, while other members are revealed to have a more recent origin. The origin of several components can be accounted for by the shuffling of pre-existing protein domains, or via lateral gene transfer. In addition, certain domains have appeared de novo more recently, and can be considered metazoan synapomorphies.ConclusionThe Notch signalling pathway emerged in Metazoa via a diversity of molecular mechanisms, incorporating both novel and ancient protein domains during eukaryote evolution. Thus, a functional Notch signalling pathway was probably present in Urmetazoa.
Origin and evolution of the Notch signalling pathway: an overview from eukaryotic genomes
BackgroundOf the 20 or so signal transduction pathways that orchestrate cell-cell interactions in metazoans, seven are involved during development. One of these is the Notch signalling...Full Text Available
Neutron diffraction studies of amphipathic helices in phospholipid bilayers
1994-12-31
The structural feature which is thought to facilitate the interaction of many peptides with phospholipid bilayers is the ability to fold into an amphipathic helix. In most cases the exact location and orientation of this helix with respect to the membrane is not known, and may vary with factors such as pH and phospholipid content of the bilayer. The growing interest in this area is stimulated by indications that similar interactions can contribute to the binding of certain hormones to their cell-surface receptors. We have been using the techniques of neutron diffraction from stacked phospholipid bilayers in an attempt to investigate this phenomenon with a number of membrane-active peptides. Here we report some of our findings with three of these: the bee venom melittin; the hormone calcitonin; and a synthetic peptide representing the ion channel fragment of influenza A M2 protein.
Ner protein of phage Mu: Assignments using {sup 13}C/{sup 15}N-labeled protein
1994-12-01
The Ner protein is a small (74-amino acid) DNA-binding protein that regulates a switch between the lysogenic and lytic stages of phage Mu. It inhibits expression of the C repressor gene and down-regulates its own expression. Two-dimensional NMR experiments on uniformly {sup 15}N-labeled protein provided most of the backbone and some of the sidechain proton assignments. The secondary structure determination using two-dimensional NOESY experiments showed that Ner consists of five {alpha}-helices. However, because most of the sidechain protons could not be assigned, the full structure was not determined. Using uniformly {sup 13}C/{sup 15}N-labeled Ner and a set of three-dimensional experiments, we were able to assign all of the backbone and 98% of the sidechain protons. In particular, the CBCANH and CBCA(CO)NH experiments were used to sequentially assign the C{alpha} and C{beta} resonances; the HCCH-CTOCSY and HCCH-COSY were used to assign sidechain carbon and proton resonances.
2008-10-28
ObjectiveDetermination whether common variation at the CHGA locus increases susceptibility to hypertension.BackgroundFull Text Available
2008-10-28
Full Text Available.ObjectiveDetermination whether common variation at the CHGA locus increases susceptibility to hypertension.BackgroundChromogranin A (CHGA) regulates catecholamine storage and release. Previously we systematically identified genetic variants across CHGA.MethodsDense genotyping across the CHGA locus in >1000 individuals with the most extreme BPs in the population, as well twin pairs with autonomic phenotypes. Characterizing function of a trait-associated 3'-UTR variant with transfected CHGA 3'-UTR/luciferase reporter plasmids.ResultsCHGA was overexpressed in patients with hypertension, especially hypertensive men, and CHGA predicted catecholamines. In individuals with extreme BPs, CHGA genetic variants predicted BP, especially in men, with a peak association occurred in the 3'-UTR at C+87T, accounting for up to ~12/~9 mmHg. The C+87T genotype predicted CHGA secretion in vivo, with the +87T allele (associated with lower BP) also diminishing plasma CHGA by ~10%. The C+87T 3'-UTR variant also predicted the BP response to environmental (cold) stress; the same allele (+87T) that diminished basal BP in the population also decreased the SBP response to stress by ~12 mmHg, and the response was smaller in women (by ~6 mmHg). In a chromaffin cell-transfected CHGA 3'-UTR/luciferase reporter plasmid, the +87T allele associated with lower BP also decreased reporter expression by ~30%. In cultured chromaffin cells, reducing endogenous Chga expression by si-RNA caused ~2/3 depletion of catecholamine storage vesicles.ConclusionsCommon variant C+87T in the CHGA 3'-UTR is a functional polymorphism causally associated with hypertension especially in men of the population, and propose steps ("intermediate phenotypes") whereby in sex-dependent fashion this genetic variant influences the ultimate disease trait. These observations suggest new molecular strategies to probe the pathophysiology, risk, and rational treatment of hypertension.
Mechanism of phosphoryl transfer and protein-protein interaction in the PTS system-an NMR study
1994-12-01
HPr and Enzyme IIA{sup Glc} are two of the components of the bacterial PTS (phosphoenolpyruvate: sugar phosphotranferase system) and are involved in the phosphorylation and concomitant translocation of sugars across the membrane. These PTS protein complexes also regulate sugar transport. HPr, phosphorylated at a histidine N1 site by Enzyme I and phosphoenol pyruvate, transfers the phosphoryl group to a histidine N3 position in Enzyme IIA{sup Glc}. HPrs from Gram-positive bacteria undergo regulatory phosphorylation at Ser{sup 46}, whereby phosphorylation of the histidine residue is inhibited. Conversely, histidine phosphorylation inhibits phosphorylation at Ser{sup 46}. HPrs from Gram-negative bacteria possess a serine residue at position 46, but do not undergo regulatory phosphorylation. HPr forms an open-faced sandwich structure with a four-strand S-sheet and 2 to 3 helices lying on top of the sheet. The active-site histidine and Ser{sup 46} occur in conformationally flexible regions. P-His-HPr from the Gram-positive bacterium Bacillus subtilus has been investigated by both homonuclear and heteronuclear two-dimensional and three-dimensional NMR experiments using an in-situ enzymatic regeneration system to maintain a constant level of P-His-HPr. The results show that localized conformational changes occur in the vicinity of the active-site histidine and also near Ser{sup 46}. HPr-Enzyme IIA{sup Glc} complexes from both Bacillus subtilis and Gram-negative Escherichia coli were also studied by a variety of {sup 15}N-edited two-dimensional NMR experiments, which were performed on uniformly {sup 15}N-labeled HPr complexed to unlabeled Enzyme IIA{sup Glc}. The complex is in fast exchange with a molecular weight of about 27 kDa. The focus of our work is to assess the changes undergone by HPr (the smaller of the two components), and so all the experiments were performed with excess Enzyme IIA present in the system.
Interaction of antimicrobial peptides with lipid membranes
2008-12-15
This study aims to investigate the difference in the interaction of antimicrobial peptides with two classes of zwitterionic peptides, phosphatidylethanolamines (PE) and phosphatidylcholines (PC). Further experiments were performed on model membranes prepared from specific bacterial lipids, lipopolysaccharides (LPS) isolated from Salmonella minnesota. The structure of the lipid-peptide aqueous dispersions was studied by small-and wide-angle X-ray diffraction during heating and cooling from 5 to 85 C. The lipids and peptides were mixed at lipid-to-peptide ratios 10-10000 (POPE and POPC) or 2-50 (LPS). All experiments were performed at synchrotron soft condensed matter beamline A2 in Hasylab at Desy in Hamburg, Germany. The phases were identified and the lattice parameters were calculated. Alamethicin and melittin interact in similar ways with the lipids. Pure POPC forms only lamellar phases. POPE forms lamellar phases at low temperatures that upon heating transform into a highly curved inverse hexagonal phase. Insertion of the peptide induced inverse bicontinuous cubic phases which are an ideal compromise between the curvature stress and the packing frustration. Melittin usually induced a mixture of two cubic phases, Im3m and Pn3m, with a ratio of lattice parameters close to 1.279, related to the underlying minimal surfaces. They formed during the lamellar to hexagonal phase transition and persisted during cooling till the onset of the gel phase. The phases formed at different lipid-to-peptide ratios had very similar lattice parameters. Epitaxial relationships existed between coexisting cubic phases and hexagonal or lamellar phases due to confinement of all phases to an onion vesicle, a vesicle with several layers consisting of different lipid phases. Alamethicin induced the same cubic phases, although their formation and lattice parameters were dependent on the peptide concentration. The cubic phases formed during heating from the lamellar phase and their onset temperature decreased with increasing peptide concentration. At low alamethicin concentrations, both Im3m and Pn3m formed and coexisted with the hexagonal phase. As the concentration of the peptide increased, the amount of hexagonal phase and Im3m decreased, until only Pn3m remained. Same epitaxial relationships were observed as for POPE with melittin. Lipopolysaccharides (LPS), strains R595 and R60 and their ''endotoxic principle'' lipid A, were studied. Longer sugar-chain LPS R60 and lipid A form cubic phases and LPS R595 lamellar phases at the employed water content around 95%. Melittin induced several lamellar phases and a hexagonal phase in all LPS varieties. (orig.)
There is an urgent need for new drugs against influenza type A and B viruses due to incomplete protection by vaccines and the emergence of resistance to current antivirals. The influenza virus polymerase...Full Text Available
Full Text Available.There is an urgent need for new drugs against influenza type A and B viruses due to incomplete protection by vaccines and the emergence of resistance to current antivirals. The influenza virus polymerase complex, consisting of the PB1, PB2 and PA subunits, represents a promising target for the development of new drugs. We have previously demonstrated the feasibility of targeting the protein-protein interaction domain between the PB1 and PA subunits of the polymerase complex of influenza A virus using a small peptide derived from the PA-binding domain of PB1. However, this influenza A virus-derived peptide did not affect influenza B virus polymerase activity. Here we report that the PA-binding domain of the polymerase subunit PB1 of influenza A and B viruses is highly conserved and that mutual amino acid exchange shows that they cannot be functionally exchanged with each other. Based on phylogenetic analysis and a novel biochemical ELISA-based screening approach, we were able to identify an influenza A-derived peptide with a single influenza B-specific amino acid substitution which efficiently binds to PA of both virus types. This dual-binding peptide blocked the viral polymerase activity and growth of both virus types. Our findings provide proof of principle that protein-protein interaction inhibitors can be generated against influenza A and B viruses. Furthermore, this dual-binding peptide, combined with our novel screening method, is a promising platform to identify new antiviral lead compounds.
Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells
1986-01-01
Aldosterone and insulin stimulate Na+ transport through mechanisms involving protein synthesis. Na+-K+-ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na+-K+-ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na+-K+-[32P]ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are ... >>
1994-12-01
To understand the details of macromolecular function, high-resolution structural and dynamic detail is essential. The polypeptide fold of the gramicidin channel has been effectively modeled for the past 20 years, yet the functional changes in conductance and channel lifetime associated with amino acid substitutions cannot be predicted. To accomplish this goal, high-resolution electrostatic modeling and the precise orientation of all dipoles are required. Furthermore, an enhanced knowledge of the complex molecular environment of this membrane-bound peptide is needed. An aqueous environment is relatively uniform and achiral. The membrane environment is very heterogenous and chiral. A knowledge of the interactions, specific and nonspecific, between peptide and lipid will aid in developing a better understanding of this environment. To accomplish this goal, it is necessary to study the peptide in an extended lipid bilayer, rather than in a vesicular or micellar form. These latter environments are likely to possess increased dynamics, increased water penetration, and distorted interactions between the polypeptide and membrane surface. To perform NMR studies on bilayer bound peptides, solid state NMR methods are required, and for specific site information, isotopic labels are incorporated using solid phase peptide synthesis.
Hepcidin modulation in human diseases: From research to clinic
2009-02-07
Full Text Available.By modulating hepcidin production, an organism controls intestinal iron absorption, iron uptake and mobilization from stores to meet body iron need. In recent years there has been important advancement in our knowledge of hepcidin regulation that also has implications for understanding the physiopathology of some human disorders. Since the discovery of hepcidin and the demonstration of its pivotal role in iron homeostasis, there has been a substantial interest in developing a reliable assay of the hormone in biological fluids. Measurement of hepcidin in biological fluids can improve our understanding of iron diseases and be a useful tool for diagnosis and clinical management of these disorders. We reviewed the literature and our own research on hepcidin to give an updated status of the situation in this rapidly evolving field.
Hepcidin modulation in human diseases: From research to clinic
2009-02-07
By modulating hepcidin production, an organism controls intestinal iron absorption, iron uptake and mobilization from stores to meet body iron need. In recent years there has been important advancement...Full Text Available
HES6 reverses nuclear reprogramming of insulin-producing cells following cell fusion
2007-01-01
To examine the mechanism by which growth-stimulated pancreatic beta-cells dedifferentiate, somatic cell fusions were performed between MIN6, a highly differentiated mouse insulinoma, and betalox5, a cell line derived from human beta-cells which progressively dedifferentiated in culture. MIN6/betalox5 somatic cells hybrids underwent silencing of insulin expression and a marked decline in PDX1, NeuroD, and MafA, indicating that betalox5 expresses a dominant transacting factor(s) that represses beta-cell differentiation. Expression of Hes1, which inhibits endocrine differentiation was higher in hybrid cells than in parental MIN6 cells. Hes6, a repressor of Hes1, was highly expressed in primary beta-cells as well as MIN6, but was repressed in hybrids. Hes6 overexpression ... >>
1994-12-31
Experimentally-determined structural models of fluid lipid bilayers are essential for verifying molecular dynamics simulations of bilayers and for understanding the structural consequences of peptide interactions. The extreme thermal motion of bilayers precludes the possibility of atomic-level structural models. Defining {open_quote}the structure{close_quote} of a bilayer as the time-averaged transbilayer distribution of the water and the principal lipid structural groups such as the carbonyls and double-bonds (quasimolecular fragments), one can represent the bilayer structure as a sum of Gaussian functions referred to collectively as the quasimolecular structure. One method of determining the structure is by neutron diffraction combined with exhaustive specific deuteration. This method is impractical because of the expense of the chemical syntheses and the limited amount of neutron beam time currently available. We have therefore developed the composition space refinement method for combining X-ray and minimal neutron diffraction data to arrive at remarkably detailed and accurate structures of fluid bilayers. The composition space representation of the bilayer describes the probability of occupancy per unit length across the width of the bilayer of each quasimolecular component and permits the joint refinement of X-ray and neutron lamellar diffraction data by means of a single quasimolecular structure that is fitted simultaneously to both data sets. Scaling of each component by the appropriate neutron or X-ray scattering length maps the composition-space profile to the appropriate scattering length space for comparison to experimental data. The difficulty with the method is that fluid bilayer structures are generally only marginally determined by the experimental data. This means that the space of possible solutions must be extensively explored in conjunction with a thorough analysis of errors.
Fibroblast growth factor-2 stimulates adipogenic differentiation of human adipose-derived stem cells
2007-01-01
Adipose-derived stem cells (ASCs) have demonstrated a capacity for differentiating into a variety of lineages, including bone, cartilage, or fat, depending on the inducing stimuli and specific growth and factors. It is acknowledged that fibroblast growth factor-2 (FGF-2) promotes chondrogenic and inhibits osteogenic differentiation of ASCs, but thorough investigations of its effects on adipogenic differentiation are lacking. In this study, we demonstrate at the cellular and molecular levels the effect of FGF-2 on adipogenic differentiation of ASCs, as induced by an adipogenic hormonal cocktail consisting of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, insulin, and indomethacin. FGF-2 significantly enhances the adipogenic differentiation of human ASCs. Furthermore, in cultures receiving FGF-2 before adipogenic induction, mRNA expression of ... >>
2007-01-01
F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein. Very recently, F2L was identified as an endogenous chemoattractant peptide acting specifically through formyl peptide receptor-like (FPRL)2. In the present study, we report that F2L stimulates chemotactic migration in human neutrophils. However, F2L inhibits formyl peptide receptor (FPR) and FPRL1 activities, resulting in the complete inhibition of intracellular calcium increases, and superoxide generation induced by N-formyl-Met-Leu-Phe, MMK-1, or Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) in human neutrophils. In terms of the inhibitory role of F2L on FPR- and FPRL-mediated signaling, we found that F2L competitively inhibits the binding of 125I-WKYMVm to its specific receptors, FPR and FPRL1. F2L is the first endogenous molecule that inhibits ... >>
1987-01-01
The binding of fatty acids by bovine serum albumin (BSA) is well documented. However, the interaction between the synthesis of prostaglandins (PGs) and the trapping of arachidonate released from cellular lipid stores is not as well understood. In this communication, we relate the trapping of fatty acids to the synthesis of PGs and the incorporation of [3H]acetate into platelet-activating factor (PAF). Our results show that, as determined by radioimmunoassay, BSA inhibits bradykinin (BK) (5 ng/ml) and ionophore A23187 (10 microM)-stimulated synthesis of PGs in human embryo lung fibroblasts (IMR-90) in a concentration-dependent manner. Experiments using prelabel with [3H]arachidonate followed by extraction and thin-layer chromatography show that, in the presence of 2 mg/ml BSA, IMR-90 release essentially only fatty acid following ... >>
1994-12-01
The conformations of two diazocine turn mimics, which were later incorporated into GPIIb/IIIa peptide antagonists, were investigated using nuclear magnetic resonance techniques. The two compounds, methyl (2,5-dioxo-3-(S)-(3-{omega}-tosylguanidino-propyl)-4-methyl-octahydro-1,4-dazocin-1-yl)acetate (1) and methyl (2,5-dioxo-3-(S)-(3-{omega}-tosyl-guanidino-propyl)-octahydro-1,5-diazocin-1-yl)acetate (2), differ only in their substituent at the diazocine position 4 nitrogen, yet this substitution results in a marked difference in the affinity of the resulting analogs for the GPIIb/IIIa receptor. It was of interest to determine if the difference observed in the antagonistic potency between these analogs was related to constitutional or, perhaps, conformational differences. The backbone conformations of these two molecules can be determined by measuring vicinal coupling constants along the trimethylene portion of the C8 ring backbone and by measuring interproton NOE intensities between the diazocine methine proton and the protons of the trimethylene group. For compound 1, {sup 3}J{sub HH} values measured from a P.E.COSY spectrum and interproton distances calculated from ROESY buildup curves indicated the presence of a single C8 ring backbone conformation where the trimethylene bridge adopted a staggered conformation and the H{alpha}1 and H{gamma}1 protons of the trimethylene group were 2.2 A from the methine proton. For compound 2, however, partial overlap of the central H{beta}1 and H{beta}2 protons made it impossible to measure {sup 3}J{sub HH} values from the P.E.COSY spectrum. We therefore used a {sup 13}C-filtered TOCSY experiment to measure the {sup 3}J{sub CH} values in both compounds 1 and 2. These heteronuclear vicinal coupling constants measured with {sup 13}C in natural abundance in conjunction with measured interproton NOE intensities indicate that these compounds share a common C8 ring backbone conformation.
2007-01-01
Bone marrow derived hematopoietic stem cells can function as endothelial progenitor cells. They are recruited to malignant tumors and differentiate into endothelial cells. This mechanism of neovascularization termed vasculogenesis is distinct from proliferation of pre-existing vessels. To better understand vasculogenesis we developed a cell culture model with expansion and subsequent endothelial differentiation of human CD133+ progenitor cells in vitro. alphavbeta3-integrins are expressed by endothelial cells and play a role in the attachment of endothelial cells to the extracellular matrix. We investigated the effect of Cilengitide, a peptide-like, high affinity inhibitor of alphavbeta3- and ... >>
1987-04-01
This study was undertaken to determine whether calcitonin inhibits Na/sup +/-(P/sub i/) cotransport across luminal brush-border membrane (BBM) of proximal tubules. Further, the authors determined the relative inhibitions of inorganic phosphate (P/sub i/) transport in BBM vesicles (BBMV) derived from superficial cortical tissue (BBMV-SC) and juxtamedullary tissue (BBMV-JM). The effects of maximally phosphaturic doses of calcitonin were compared with those of parathyroid hormone (PTH). Experiments were performed in acutely thyroparathyroidectomized (TPTX) rats fed either normal (NPD, 0.7%) P/sub i/ diets. After measurement of the fractional excretion of phosphate (FE/sub P/sub i//) by clearance, the BBMV-SC and BBMV-JM were prepared from kidneys of the same animals and uptakes of /sup 32/P/sub i/ were determined. Both calcitonin and PTH inhibited BBMV transport of P/sub i/ to a greater degree in BBMV-JM than in BBMV-SC, in rats fed NPD or LPD. Kinetic analysis shows that administration of calcitonin resulted in marked decrease of apparent V/sub max/ for P/sub i/ without any changes in apparent K/sub m/ for P/sub i/. They conclude that calcitonin administration decreases the capacity for Na/sup +/-P/sub i/ cotransport across BBM in proximal tubules of the acutely TPTX rat.
Calcitonin inhibits Na+ gradient-dependent phosphate uptake across renal brush-border membranes
1987-01-01
This study was undertaken to determine whether calcitonin inhibits Na+-(P/sub i/) cotransport across luminal brush-border membrane (BBM) of proximal tubules. Further, the authors determined the relative inhibitions of inorganic phosphate (P/sub i/) transport in BBM vesicles (BBMV) derived from superficial cortical tissue (BBMV-SC) and juxtamedullary tissue (BBMV-JM). The effects of maximally phosphaturic doses of calcitonin were compared with those of parathyroid hormone (PTH). Experiments were performed in acutely thyroparathyroidectomized (TPTX) rats fed either normal (NPD, 0.7%) P/sub i/ diets. After measurement of the fractional excretion of phosphate (FE/sub P/sub i//) by clearance, the BBMV-SC and BBMV-JM were prepared from kidneys of the same animals and uptakes of 32P/sub i/ were determined. Both calcitonin and PTH ... >>
Full Text Available.BackgroundImmunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment of tumors, which inhibits the functions of endogenous dendritic cells (DCs) that are necessary for the generation of anti-tumor CD8+ T cells. To overcome this problem, autologous DCs are generated ex vivo, loaded with tumor antigens, and activated in this non-suppressive environment before administration to patients. However, DC-based vaccines rarely induce tumor regression.Methodology/Principal FindingsWe examined the fate and function of these DCs following their injection using murine models, in order to better understand their interaction with the host immune system. Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells.Conclusions/SignificanceThis reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs.
BackgroundImmunotherapeutic strategies to stimulate anti-tumor immunity are promising approaches for cancer treatment. A major barrier to their success is the immunosuppressive microenvironment...Full Text Available
BRCA1/BRCA2-deficient cells are sensitive to mitomycin C and tirapazamine
2003-01-01
Full text: BRCA1- and BRCA2- deficient cells have defective repair of double-strand breaks (DSBs) via homologous recombination (HR). The toxicity profiles of these cells is of interest in the treatment of breast and ovarian cancer arising in mutation carriers. We focused on Mitomycin C (MMC), which produces DNA interstrand crosslinks, and Tirapazamine (TPZ), which is activated by hypoxia, and inhibits DNA replication and topoisomerase II. BRCA1-deficient HCC1937 cells, derived from a breast cancer in a BRCA1 carrier, were compared to cells corrected with wild-type (wt) BRCA1. BRCA2-deficient Capan-1 cells, derived from a pancreatic cancer in a BRCA2 carrier, were compared to a wt BRCA2-corrected clone. Expressing the BRC4 peptide in MCF-7 cells disrupted the association of BRCA2 with Rad51, thereby inactivating the function of BRCA2. Clonogenic cell survival was ... >>
Auxin-binding proteins without KDEL sequence in the moss Funaria hygrometrica
2009-11-01
Full Text Available.
1988-01-01
An insulin-independent teratoma-derived cell line, called 1246-3A, has been isolated from the adipogenic cell line 1246, which stringently requires insulin for proliferation. The 1246-3A cell line, which can proliferate in the absence of exogenous insulin, produces in its conditioned medium a growth factor similar to pancreatic insulin by its biological and immunological properties. This factor, called insulin-related factor (IRF), was purified and iodinated to study its binding to cell surface receptors. 125I-labeled IRF binding to intact 1246-3A cells is lower than to 1246 cells. Cell surface binding can be restored by culturing the 1246-3A cells in the presence of an anti-porcine insulin monoclonal antibody of by acid prewash of the cells prior to performing the binding. Scatchard analysis of binding indicates that IRF secreted by the 1246-3A cells ... >>
1985-01-01
Two site-specific antibodies that immunoprecipitate the human insulin receptor have been prepared by immunizing rabbits with chemically synthesized peptides derived from the cDNA-predicted amino acid sequence of the beta subunit of the proreceptor. Antibodies to the carboxyl terminus (AbP5) and to a domain around tyrosine-960 (AbP4) specifically recognize the beta subunit of the receptor on immunoblots. Both antibodies immunoprecipitated 125I-labeled insulin-receptor complexes and the autophosphorylated receptor. Although neither antibody inhibited insulin binding to the receptor, both insulin-dependent autophosphorylation and exogenous substrate phosphorylation were inhibited by AbP4. Inhibition by AbP4 was dependent upon the phosphorylation state of the receptor. It was not detected when the receptor was ... >>
Anti-inflammatory effect of a human prothrombin fragment-2-derived peptide, NSA9, in EOC2 microglia
2008-01-01
Pro-inflammatory mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2), and several cytokines (tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-6) are responsible for central nervous system (CNS) injuries that include ischemia, Alzheimer's disease, and neural death. Inhibition of these pro-inflammatory mediators would be an effective therapy to reduce the progression of neurodegenerative diseases. In this study, we examined the anti-inflammatory effects of a human prothrombin fragment-2-derived peptide, NSA9 (NSAVQLVEN), on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-activated brain microglia. NSA9 significantly inhibited the release of NO, PGE2, and pro-inflammatory cytokines in a dose-dependent manner. Furthermore, NSA9 reduced ... >>
2005-01-01
Previous studies demonstrate that one of the six plasminogen type 2 glycoforms, plasminogen 2epsilon, enhances invasiveness of the 1-LN human prostate tumor cell line in an in vitro model. Binding of plasminogen 2epsilon to CD26 on the cell surface induces a Ca2+ signaling cascade which stimulates the expression of matrix metalloproteinase-9, required by these cells to invade Matrigel registered . We now report that angiostatin, a fragment derived from plasminogen which prevents endothelial cell proliferation, is also a potent, direct inhibitor of 1-LN tumor cell invasiveness. We studied the effect of individual plasminogen 2 glycoform-derived angiostatins and found that only angiostatin 2epsilon binds to CD26 on the surface of 1-LN cells at a site also recognized by plasminogen 2epsilon. As a result, the ... >>
2009-03-01
Two factors contribute to Burkitt lymphoma (BL) pathogenesis, a chromosomal translocation leading to c-myc oncogene deregulation and infection with Epstein-Barr virus (EBV). Although the virus has B...Full Text Available
2009-03-01
Full Text Available.Two factors contribute to Burkitt lymphoma (BL) pathogenesis, a chromosomal translocation leading to c-myc oncogene deregulation and infection with Epstein-Barr virus (EBV). Although the virus has B cell growth–transforming ability, this may not relate to its role in BL since many of the transforming proteins are not expressed in the tumor. Mounting evidence supports an alternative role, whereby EBV counteracts the high apoptotic sensitivity inherent to the c-myc–driven growth program. In that regard, a subset of BLs carry virus mutants in a novel form of latent infection that provides unusually strong resistance to apoptosis. Uniquely, these virus mutants use Wp (a viral promoter normally activated early in B cell transformation) and express a broader-than-usual range of latent antigens. Here, using an inducible system to express the candidate antigens, we show that this marked apoptosis resistance is mediated not by one of the extended range of EBNAs seen in Wp-restricted latency but by Wp-driven expression of the viral bcl2 homologue, BHRF1, a protein usually associated with the virus lytic cycle. Interestingly, this Wp/BHRF1 connection is not confined to Wp-restricted BLs but appears integral to normal B cell transformation by EBV. We find that the BHRF1 gene expression recently reported in newly infected B cells is temporally linked to Wp activation and the presence of W/BHRF1-spliced transcripts. Furthermore, just as Wp activity is never completely eclipsed in in vitro–transformed lines, low-level BHRF1 transcripts remain detectable in these cells long-term. Most importantly, recognition by BHRF1-specific T cells confirms that such lines continue to express the protein independently of any lytic cycle entry. This work therefore provides the first evidence that BHRF1, the EBV bcl2 homologue, is constitutively expressed as a latent protein in growth-transformed cells in vitro and, in the context of Wp-restricted BL, may contribute to virus-associated lymphomagenesis in vivo.
A study of the yeast plasma membrane proton-translocating ATPase
1990-01-01
Four proteases have been used to assess the topology of the H+ ATPase from Saccharomyces cerevisiae reconstituted into phospholipid vesicles. Limited proteolysis by trypsin and alpha-chymotrypsin inactivates the enzyme and produces stable, membrane-bound fragments. Sequence analyses of these peptides have located the peptide bonds hydrolyzed. The labile bonds are on opposite sides of a central hydrophilic domain containing consensus sequences for the site of phosphorylation and fluorescein isothiocyanate binding of several related ATPases. Limited proteolysis of the ATPase by elastase cuts approximately 50 amino acids from the C-terminus, leaving the remaining membrane-bound fragments active. Proteolysis by carboxy-peptidase Y suggests that the C-terminus is on the inside of the vesicle. A model for the transmembrane arrangement of ... >>
1994-12-01
Advances in magnetic resonance and vibrational spectroscopy make it possible to derive detailed structural information about biomolecular structures in solution. These techniques are critically dependent on the availability of labeled compounds. For example, NMR techniques used today to derive peptide and protein structures require uniformity {sup 13}C-and {sup 15}N-labeled samples that are derived biosynthetically from (U-6-{sup 13}C) glucose. These experiments are possible now because, during the 1970s, the National Stable Isotope Resource developed algal methods for producing (U-6-{sup 13}C) glucose. If NMR techniques are to be used to study larger proteins, we will need sophisticated labelling patterns in amino acids that employ a combination of {sup 2}H, {sup 13}C, and {sup 15}N labeling. The availability of these specifically labeled amino acids requires a renewed investment in new methods for chemical synthesis of labeled amino acids. The development of new magnetic resonance or vibrational techniques to elucidate biomolecular structure will be seriously impeded if we do not see rapid progress in labeling technology. Investment in labeling chemistry is as important as investment in the development of advanced spectroscopic tools.
1986-03-01
Certain synthetic peptides derived from the Fc region of human IgG suppressed protein, RNA, and DNA synthesis during mixed lymphocyte reactions. Responder mononuclear cells were incubated with medium or agents that alter phosphorylation of cellular proteins before immunomodulatory Fc peptides and stimulator cells were added. Incubating cells with trifluoperazine which inhibits calcium binding to calmodulin and inhibits protein kinase C (PKC) increased inhibition of the MLR induced by Fc peptides. Conversely, incubating cells with dubutyryl cyclic AMP (DBcAMP), calmodulin, 1,2-diolein, or phorbol myristate acetate (PMA) abolished inhibition of the MLR induced by Fc peptides. Inhibition of the MLR by Fc ..gamma.. peptides was not affected when DBcAMP or PMA was added after peptide addition. The PKC activity of cell homogenates was decreased by 69% when Fc..gamma.. peptides were present during the MLR. The in vitro phosphorylation of histone Hl by partially purified PKC from lymphocytes was inhibited 74% in the presence of Fc..gamma.. peptides. These results indicate that suppression of the MLR induced by Fc..gamma.. peptides is dependent on inhibition of protein phosphorylation by kinases including protein kinase C. The inhibition of phosphorylation may be related to the ability of Fc..gamma.. peptides to reverse animal models of autoimmune disease.
Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator
1989-01-01
Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivates a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. Maitotoxin, by its action on calcium channels allows entry of extracellular calcium and inhibits PTH release. Our results suggest that calcium channels are involved in the release of PTH. Inhibition of PTH release by maitotoxin is not sensitive to pertussis toxin, suggesting that maitotoxin may act distal to the site interacting with a guanine nucleotide regulatory protein, or maitotoxin could interact with other ions or second messengers to inhibit PTH release.
Inhibition of parathyroid hormone release by maitotoxin, a calcium channel activator
1989-01-01
Maitotoxin, a toxin derived from a marine dinoflagellate, is a potent activator of voltage-sensitive calcium channels. To further test the hypothesis that inhibition of PTH secretion by calcium is mediated via a calcium channel we studied the effect of maitotoxin on dispersed bovine parathyroid cells. Maitotoxin inhibited PTH release in a dose-dependent fashion, and inhibition was maximal at 1 ng/ml. Chelation of extracellular calcium by EGTA blocked the inhibition of PTH by maitotoxin. Maitotoxin enhanced the effects of the dihydropyridine calcium channel agonist (+)202-791 and increased the rate of radiocalcium uptake in parathyroid cells. Pertussis toxin, which ADP-ribosylates and inactivates a guanine nucleotide regulatory protein that interacts with calcium channels in the parathyroid cell, did not affect the inhibition of PTH secretion by maitotoxin. ... >>
Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments
2007-01-01
HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1IIIB infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 ... >>
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