| 8959854 |
Comparison of Sandimmun with a new cyclosporin derivative (IMM 125) in renal transplant patients with stable renal function. IMM 125 Multicentre Study Group |
10.1007/978-3-662-00818-8_78. |
Transpl Int |
Comparison of Sandimmun with a new cyclosporin derivative (IMM 125) in renal transplant patients with stable renal function. IMM 125 Multicentre Study Group
Abstract
- A double-blind switch-over study was carried out on 70 renal transplant patients to assess the value of a new cyclosporin derivative, IMM 125. Preclinical in vitro and in vivo studies indicated that IMM 125 was as equally immunosuppressive as Sandimmun, but that its therapeutic index should be superior. The duration of the treatment was 24 weeks. The assumption that the dosage of IMM 125 could be 2.5 times lower than Sandimmun proved to be false; three patients suffered acute rejection episodes, probably as a consequence of the low dosage, and dosage adjustments had to be made for all patients receiving IMM 125 after only a few weeks. Although IMM 125 is an effective immunosuppressive agent, it does not appear to offer advantages over Sandimmun with regard to renal function. In addition, IMM 125 causes some disturbances in liver function.
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| 8960363 |
Molecular mechanisms underlying he interaction of motuporin and microcystins with type-1 and type-2A protein phosphatases |
10.1139/o96-061. |
Biochem Cell Biol |
Molecular mechanisms underlying he interaction of motuporin and microcystins with type-1 and type-2A protein phosphatases
Abstract
- Heptapeptide microcystin and pentapeptide motuporin (nodularin-V) are equipotent inhibitors of type-1 and type-2A protein phosphatase catalytic subunits (PP-1c and PP-2Ac). Herein we describe elucidation of the molecular mechanisms involved in the interaction of these structurally similar hepatotoxins with PP-1c/PP-2Ac and identification of an important functional difference between their mode of interaction with these enzymes. Microcystin-LR, microcystin-LA, and microcystin-LL were found to interact with PP-2Ac and PP-1c by a two-step mechanism involving rapid binding and inactivation of the protein phosphatase (PPase) catalytic subunit, followed by a slower covalent interaction (within hours). Covalent adducts comprising PPase-toxin complexes were separated from free PPase by C-18 reverse-phase liquid chromatography, thus allowing the time course of covalent adduct formation to be quantitated. In contrast to microcystins, motuporin (nodularin-V) and nodularin-R were unable to form covalent complexes with either PP-1c or PP-2Ac even after 96 h incubation. Specific reduction of microcystin-LA to dihydromicrocystin-LA abolished the ability of the toxin to form a covalent adduct with PP-2Ac. Specific methyl esterification of the single Glu residue in microcystin-LR rendered this toxin inactive as a PPase inhibitor and abolished subsequent formation of a covalent adduct. Our data indicate that inactivation of PP-2Ac/PP-1c by microcystins precedes covalent modification of the PPases via a Michael addition reaction between a nucleophilic phosphatase residue and Mdha in the heptapeptide toxin. In contrast, following rapid inactivation of PP-2Ac/PP-1c by motuporin, the equivalent N-methyldehydrobutyrine residue in this toxin is unreactive and does not form a covalent bond with the PPases. These results are consistent with structural data for (i) the NMR solution structures of microcystin-LR and motuporin, which indicate a striking difference in the relative positions of their corresponding dehydroamino acids in the toxin peptide backbone, and (ii) X-ray crystallographic data on an inactive complex between PP-1c and microcystin-LR, which show a covalent bond between Cys-273 and the bound toxin.
|
| 8962717 |
Analysis of the glycosylation patterns of the extracellular domain of the epidermal growth factor receptor expressed in Chinese hamster ovary fibroblasts |
10.3109/08977199609034572. |
Growth Factors |
Analysis of the glycosylation patterns of the extracellular domain of the epidermal growth factor receptor expressed in Chinese hamster ovary fibroblasts
Abstract
- The extracellular domain (621 N-terminal amino acids) of the p170 epidermal growth factor (EGF) receptor has eleven consensus N-linked glycosylation sites. When expressed in Chinese hamster ovary cells this was glycosylated with a combination of high mannose and complex chains. The latter chains were shown by chromatographic separation and mass spectrometric analysis of tryptic digests to be clustered in the EGF-binding domain. Treatment with the endoglycosidase, peptide-N-glycosidase F (PNGase F), reduced the molecular weight from 110 kDa to 75 kDa. Released oligosaccharides were characterised at high sensitivity by high pH anion exchange chromatography with pulsed amperometric detection and gas-liquid chromatography/mass spectrometry. The data were consistent with the complex chains being trisialylated tetra-antennary oligosaccharides fucosylated on the reducing terminal GlcNAc. The large hydrodynamic mass of these oligosaccharides could influence ligand binding, an effect which is likely to vary with the difference in consensus glycosylation sites of proteins related to p170 i.e. p185erbB2/neu, p180erbB3 and p180erbB4.
|
| 8980754 |
Antibacterial activity in bovine lactoferrin-derived peptides |
10.1128/AAC.41.1.54. |
Antimicrob Agents Chemother |
Antibacterial activity in bovine lactoferrin-derived peptides
Abstract
- Several peptides sharing high sequence homology with lactoferricin B (Lf-cin B) were generated from bovine lactoferrin (Lf) with recombinant chymosin. Two peptides were copurified, one identical to Lf-cin B and another differing from Lf-cin B by the inclusion of a C-terminal alanine (lactoferricin). Two other peptides were copurified from chymosin-hydrolyzed Lf, one differing from Lf-cin B by the inclusion of C-terminal alanyl-leucine and the other being a heterodimer linked by a disulfide bond. These peptides were isolated in a single step from chymosin-hydrolyzed Lf by membrane ion-exchange chromatography and were purified by reverse-phase high-pressure liquid chromatography (HPLC). They were characterized by N-terminal Edman sequencing, mass spectrometry, and antibacterial activity determination. Pure lactoferricin, prepared from pepsin-hydrolyzed Lf, was purified by standard chromatography techniques. This peptide was analyzed against a number of gram-positive and gram-negative bacteria before and after reduction of its disulfide bond or cleavage after its single methionine residue and was found to inhibit the growth of all the test bacteria at a concentration of 8 microM or less. Subfragments of lactoferricin were isolated from reduced and cleaved peptide by reverse-phase HPLC. Subfragment 1 (residues 1 to 10) was active against most of the test microorganisms at concentrations of 10 to 50 microM. Subfragment 2 (residues 11 to 26) was active against only a few microorganisms at concentrations up to 100 microM. These antibacterial studies indicate that the activity of lactoferricin is mainly, but not wholly, due to its N-terminal region.
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| 8984160 |
Oscillapeptin G, a tyrosinase inhibitor from toxic Oscillatoria agardhii |
10.1021/np9600210. |
J Nat Prod |
Oscillapeptin G, a tyrosinase inhibitor from toxic Oscillatoria agardhii
Abstract
- Oscillapeptin G, a tyrosinase inhibitor, was isolated from the freshwater toxic cyanobacterium Oscillatoria agardhii. The structure was determined to be 1 by chemical degradation and 2D NMR analyses.
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| 8995282 |
Functional association between the insulin receptor and the transmembrane protein-tyrosine phosphatase LAR in intact cells |
None |
J Biol Chem |
Functional association between the insulin receptor and the transmembrane protein-tyrosine phosphatase LAR in intact cells
Abstract
- The receptor-type protein-tyrosine phosphatase LAR (for leukocyte common antigen-related) has been implicated as a physiological regulator of the insulin receptor. To demonstrate a functional interaction between LAR and the insulin receptor, we incubated CHO cells overexpressing the human insulin receptor with an antibody to the extracellular domain of LAR and found a 47% decrease in insulin receptor autophosphorylation and kinase activity. A physical association between LAR and the insulin receptor was then shown by immunoprecipitation of LAR from cell lysates and immunoblotting with antibody to the insulin receptor, or vice versa. Up to 11.8% of the LAR protein in the lysates of CHO cells overexpressing both the insulin receptor and LAR co-immunoprecipitated with the insulin receptor. The LAR/insulin receptor association was related to the level of LAR or insulin receptor overexpression and was increased 6.5-fold by chemical cross-linking and 3.9-fold by treatment with insulin, suggesting that receptor activation influences the affinity of LAR for the insulin receptor. In insulin-stimulated rat liver, LAR was temporally enriched in endosomes with the insulin receptor, and incubation of endosomes with neutralizing LAR antibodies decreased insulin receptor dephosphorylation in situ by 28% (p = 0.01 versus control). These data provide more direct evidence of a role for LAR in the physiological regulation of insulin action at the receptor level.
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| 8998456 |
Identification in the rat brain of cyclo-prolylglycine, a novel endogenous peptide with nootropic activity |
None |
Dokl Akad Nauk |
Identification in the rat brain of cyclo-prolylglycine, a novel endogenous peptide with nootropic activity
Abstract
|
| 8999839 |
Protein-tyrosine phosphatase 1B complexes with the insulin receptor in vivo and is tyrosine-phosphorylated in the presence of insulin |
10.1074/jbc.272.3.1639. |
J Biol Chem |
Protein-tyrosine phosphatase 1B complexes with the insulin receptor in vivo and is tyrosine-phosphorylated in the presence of insulin
Abstract
- In response to insulin, protein-tyrosine phosphatase 1B (PTPase 1B) dephosphorylates 95- and 160-180-kDa tyrosine phosphorylated (PY) proteins (Kenner, K. A., Anyanwu, E., Olefsky, J. M., and Kusari, J. (1996) J. Biol. Chem. 271, 19810-19816). To characterize these proteins, lysates from control and insulin-treated cells expressing catalytically inactive PTPase 1B (CS) were immunoadsorbed and subsequently immunoblotted using various combinations of phosphotyrosine, PTPase 1B, and insulin receptor (IR) antibodies. Anti-PTPase 1B antibodies coprecipitated a 95-kDa PY protein from insulin-stimulated cells, subsequently identified as the IR beta-subunit. Similarly, anti-IR antibodies coprecipitated the 50-kDa PY-PTPase 1B protein from insulin-treated cells. To identify PTPase 1B tyrosine (Tyr) residues that are phosphorylated in response to insulin, three candidate sites (Tyr66, Tyr152, and Tyr153) were replaced with phenylalanine. Replacing Tyr66 or Tyr152 and Tyr153 significantly reduced insulin-stimulated PTPase 1B phosphotyrosine content, as well as its association with the IR. Studies using mutant IRs demonstrated that IR autophosphorylation is necessary for the PTPase 1B-IR interaction. These results suggest that PTPase 1B complexes with the autophosphorylated insulin receptor in intact cells, either directly or within a complex involving additional proteins. The interaction requires multiple tyrosine phosphorylation sites within both the receptor and PTPase 1B.
|
| 9021170 |
Effects of arenastatin A and its synthetic analogs on microtubule assembly |
10.1016/s0009-2797(96)03743-x. |
Chem Biol Interact |
Effects of arenastatin A and its synthetic analogs on microtubule assembly
Abstract
- Inhibition of microtubule assembly by arenastatin A (1) and five synthetic analogs (3-7) was examined. Arenastatin A and the triamide 6 showed potent and moderately strong inhibitory activities, respectively (IC50; 2.3 microM for 1, 7.8 microM for 6) and also depolymerized preformed microtubules. The other analogs tested showed no activity.
|
| 9023920 |
Isolation and characterization of a bacteriocin (Butyrivibriocin AR10) from the ruminal anaerobe Butyrivibrio fibrisolvens AR10: evidence in support of the widespread occurrence of bacteriocin-like activity among ruminal isolates of B. fibrisolvens |
10.1128/aem.63.2.394-402.1997. |
Appl Environ Microbiol |
Isolation and characterization of a bacteriocin (Butyrivibriocin AR10) from the ruminal anaerobe Butyrivibrio fibrisolvens AR10: evidence in support of the widespread occurrence of bacteriocin-like activity among ruminal isolates of B. fibrisolvens
Abstract
- Forty-nine isolates of Butyrivibrio fibrisolvens and a single isolate of Butyrivibrio crossotus were screened for the production of inhibitors by a deferred plating procedure. Twenty-five isolates produced factors which, to various degrees, inhibited the growth of the other Butyrivibrio isolates. of the inhibitory activity was due to bacteriophages. The inhibitory products from 18 of the producing strains were sensitive to protease digestion. Differences in the ranges of activity among the Butyrivibrio isolates and protease sensitivity profiles suggest that a number of different inhibitory compounds are produced. These findings suggest that the production of bacteriocin-like inhibitors may be a widespread characteristic throughout the genus Butyrivibrio. The bacteriocin-like activity from one isolate, B. fibrisolvens AR10, was purified and confirmed to reside in a single peptide. Crude bacteriocin extracts were prepared by ammonium sulfate and methanol precipitation of spent culture supernatants, followed by dialysis and high-speed centrifugation. The active component was isolated from the semicrude extract by reverse-phase chromatography. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity, having an estimated molecular mass of approximately 4,000 Da. The N terminus of the peptide was blocked. A cyanogen bromide cleavage fragment of the native peptide yielded a sequence of 20 amino acids (M)GIQLAPAXYQDIVNXVAAG. No homology with previously reported bacteriocins was found. Butyrivibriocin AR10 represents the first bacteriocin isolated from a ruminal anaerobe.
|
| 9027581 |
Cyclosporin A and its non-immunosuppressive derivative exhibit a differential effect on cell-mediated mineralization in culture |
None |
J Cell Biochem |
Cyclosporin A and its non-immunosuppressive derivative exhibit a differential effect on cell-mediated mineralization in culture
Abstract
- Chronic immunosuppressive treatment with cyclosporin A (CsA) is associated with decreased bone density. However, in culture, CsA inhibits osteoclast differentiation and bone resorption. This raises the question as to whether CsA also affects osteoblast function. Immunophilin, one of the CsA-binding cyclophilins that is implicated in the immunosuppressive action of CsA via calcineurin, is a peptidyl prolyl cis-trans isomerase (PPl). CsA also binds a mitochondrial membrane PPl which is implicated in controlling permeability transition pores. Therefore, in the present study we tested the effect of CsA on cell mediated mineralization in parallel with mitochondrial rhodamine retention as an indicator of mitochondrial membrane potential Rat marrow stromal cells were grown in dexamethasone (DEX) medium to stimulate mineralization in culture, and CsA was added to various cultures using different treatment schedules. Low dose (0.1 microM) CsA inhibited mineralization, compared to controls, when present in the cultures during days 3-11 of DEX stimulation. Contrarily, high dose CsA (1.0 microM) resisted the inhibitory effect of the low dose. SDZ 220-384 (SDZ), a non-immunosuppressive derivative of CsA which is known, like CsA, to bind to mitochondrial cyclophilin but does not inhibit calcineurin, was also tested. Both high and low doses of SDZ decreased mineralization when present in the cultures from day 3 or from day 0. The similar effect of the low CsA dose and SDZ on mineralization is in accord with their ability to block permeability transition pores. The differential effect, on day 21 mineralization, between high CsA dose and SDZ took place in parallel to their opposing effects on mitochondrial membrane potential. On days 4-8, mitochondrial rhodamine retention was higher under CsA than under SDZ. Under these conditions there was no significant difference between the effects of these drugs on cell proliferation measured on day 11; there was a minor decrease in specific alkaline phosphatase activity by SDZ, too small to explain the extent of mineralization inhibition by SDZ. These results suggest that permeability transition pores might be involved in controlling mineralization. Unlike SDZ, CsA exhibits an additional effect on the mitochondrial membrane potential and on mineralization when applied at a high dose on day 3. Therefore identifying the additional activity of high dose CsA, which is missing in SDZ, may be beneficial. Such activity is expected to resist changes in rhodamine retention and decreased mineralization induced by SDZ, and yet enable preservation of immunosuppressive activity of CsA.
|
| 9030574 |
A molecular basis for different interactions of marine toxins with protein phosphatase-1. Molecular models for bound motuporin, microcystins, okadaic acid, and calyculin A |
10.1074/jbc.272.8.5087. |
J Biol Chem |
A molecular basis for different interactions of marine toxins with protein phosphatase-1. Molecular models for bound motuporin, microcystins, okadaic acid, and calyculin A
Abstract
- The hepatotoxic cyclic heptapeptide microcystins and cyclic pentapeptide nodularins are powerful liver tumor promoters and potent inhibitors of the catalytic subunits of protein phosphatase-1 and -2A (PP-1c and PP-2Ac). In marked contrast to microcystins, which interact covalently with PP-1 and PP-2A, the nodularins do not bind covalently to PP-1 and PP-2A and may additionally possess unique carcinogenic properties. The conformation of microcystin-LR has been determined in solution and bound to PP-1c. We show here that the free NMR solution structures of two distinct microcystin structural congeners (microcystin-LR and -LL) are remarkably similar to the bound crystal structure of microcystin-LR. We have exploited this finding by using Metropolis Monte Carlo modeling to dock the solution structures of microcystin-LL and the marine toxin motuporin (nodularin-V) onto the crystal structure of PP-1c. Both of these toxins occupy a position similar to that of microcystin-LR when bound to PP-1c. However, although there are relatively minor differences in the structural orientation of microcystin-LL compared with microcystin-LR, there is a striking difference in the position of the N-methyldehydrobutyrine residue in motuporin relative to the comparable N-methyldehydroalanine residue in microcystin-LR. We propose that this difference in orientation provides a molecular explanation for why nodularins are incapable of forming a covalent linkage with PP-1c. Furthermore, the predicted position of N-methyldehydrobutyrine in motuporin is at the surface of the PP-1c-toxin complex, which may thus facilitate chemical interaction with a further macromolecule(s) possibly relating to its carcinogenic properties. PP-1c and PP-2Ac are also targets for other marine toxins such as okadaic acid and calyculin A. It was therefore of interest to use Metropolis Monte Carlo modeling to dock the known free crystal structures of okadaic acid and calyculin A to the crystal structure of PP-1c. These experiments predict that both okadaic acid and calyculin A are strikingly similar to microcystins and motuporin in their tertiary structure and relative PP-1c binding position.
|
| 9039040 |
Caudal hindbrain neuromedin B-preferring receptors participate in the control of food intake |
10.1152/ajpregu.1997.272.1.R433. |
Am J Physiol |
Caudal hindbrain neuromedin B-preferring receptors participate in the control of food intake
Abstract
- Recent studies have identified two subtypes of bombesin (BN) receptors in the rat central nervous system: gastrin releasing-peptide (GRP) preferring and neuromedin B (NMB) preferring. To investigate a role for the NMB-preferring receptor subtype in feeding suppression elicited by fourth ventricular (4V) BN administration, we evaluated the ability of a selective NMB-preferring receptor antagonist, BIM-23127, to block suppression of glucose intake produced by 4V BN (10 pmol). Our results showed that 4V administration of BIM-23127 dose dependently antagonized the suppression of glucose intake produced by 4V BN. In addition, 4V administration of BIM-23127 alone increased glucose intake above that observed in the baseline condition. These results support a role for the NMB-preferring BN receptor subtype in the suppression of intake produced by 4V BN administration and suggest that endogenously released NMB participates in ingestive control.
|
| 9048550 |
NMR structure determination of a novel conotoxin, [Pro 7,13] alpha A-conotoxin PIVA |
10.1021/bi962301k. |
Biochemistry |
NMR structure determination of a novel conotoxin, [Pro 7,13] alpha A-conotoxin PIVA
Abstract
- A high-resolution solution conformation of a novel conotoxin, [Pro 7,13] alpha A-conotoxin PIVA, GCCGSYPNAACHPCSCKDROSYCGQ-NH2, has been determined by two-dimensional 1H NMR methods and distance geometry calculations. The total of 324 NOE-derived interproton distance restraints including 33 long-range NOE restraints as well as 11 phi and 7 chi 1 torsion angle restraints was used for computation of structures. Back-calculation from the experimental NOE spectrum has provided 49 new NOE restraints and yielded the final R-factors of Ra = 0.641 and Rb = 0.157. The final RMSD values are 0.90 and 1.16 A for the backbone and the heavy atoms, respectively. The C-terminal half of the molecule involving the residues 12-24 is extremely well-defined with a backbone RMSD value of 0.56 A, whereas the N-terminal 3-11 disulfide loop is relatively flexible, possessing a backbone RMSD value of 1.09 A. The [Pro 7,13] alpha A-conotoxin PIVA does not contain any significant secondary structure although the 21S-24G nearly completes one turn of a 3(10) helix. The overall protein fold is largely maintained by the three disulfide bridges of 2-16, 3-11, and 14-23. The presence of the three disulfide bridges imposes geometric constraints that force the molecule to form six continuous bends involving the following residues: 3C-5S, 7P-10A, 12H-14C, 15S-17K, 17K-19R, and 21S-25Q. The overall shape of the [Pro 7,13] alpha A-conotoxin PIVA can be described as an "iron". Residues 15S-19R form a loop that protrudes out of the "bottom plate" formed by the rest of the protein and constitute the handle of the iron. The N-terminal tip of the molecule is relatively immobile due to attractive electrostatic interactions between the gamma-hydroxyl group of 20 Hyp and the phenolic hydroxyl group of 22Y. The flexible 3-11 disulfide loop consists mostly of hydrophobic residues, while the best-defined 14-23 disulfide loop contains the highly charged hydrophilic 15S-19R "handle" domain exposed to the exterior of the protein. Binding to nicotinic acetylcholine receptor can be mediated through two different types of interactions: one involving the aromatic hydrophobic residues such as 6Y and 12H and the other involving the positively charged hydrophilic side chain of the 19R. The side chain of the 19R in the [Pro 7, 13] alpha A-conotoxin PIVA and that of the 9R of the alpha-conotoxin G1, and also the side chains of the 12H and 6Y in the former and those of 10H and 11Y in the latter can be aligned to point to the same direction when the corresponding backbone atoms are superimposed to an RMSD value of 2.5 A.
|
| 9057981 |
Interaction of arenastatin A with porcine brain tubulin |
10.1248/bpb.20.171. |
Biol Pharm Bull |
Interaction of arenastatin A with porcine brain tubulin
Abstract
- Arenastatin A, isolated from the Okinawan marine sponge Dysidea arenaria, is an antimitotic depsipeptide containing a 16-membered ring. Interaction of the compound with tubulin was investigated by the use of 3Harenastatin A and other microtubule disruptors. Scatchard analysis indicated the presence of one binding site for arenastatin A per tubulin heterodimer with a dissociation constant (Kd) of 1.8 x 10(-6) M. Rhizoxin was a competitive inhibitor of arenastatin A binding, and vinblastine also inhibited arenastatin A binding in a partially competitive manner. Arenastatin A had no inhibitory effect on colchicine binding to tubulin.
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