| 1629289 |
Studies on cyclic peptides related to gratisin by reversed-phase high-performance liquid chromatography |
10.1016/0378-4347(92)80098-b. |
J Chromatogr |
Studies on cyclic peptides related to gratisin by reversed-phase high-performance liquid chromatography
Abstract
- The chromatographic behaviour of several synthetic peptides related to the antibiotic peptide gratisin was investigated using reversed-phase high-performance liquid chromatography. The influence of the concentration of the peptides, column temperature and flow-rate on the chromatographic separation was examined. Of these peptides, some analogues with the D-X-D-Y-L-Pro or L-Pro-D-X-D-Y sequences and strong activities gave double peaks in the chromatogram. The isomeric conformers were separated from each of the double peaks and were in equilibrium with each other at low temperatures. It is proposed that the presence of the sequences of D-X-D-Y-L-Pro and L-Pro-D-X-D-Y in these peptides gives rise to the stabilization and various degrees of hydrophobicity of their conformers.
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| 1637876 |
Enzymatic biosynthesis of cyclosporin A and analogues |
10.1016/0300-9084(92)90092-s. |
Biochimie |
Enzymatic biosynthesis of cyclosporin A and analogues
Abstract
- The final assembly of the undecapeptide chain of cyclosporin A and its cyclization is accomplished in Beauveria nivea by cyclosporin synthetase. This multienzyme is the largest integrated enzyme structure so far reported. Its size has been estimated at approximately 1,400 kDa by two different methods: 1), by 3% SDS-PAGE using the related multienzymes ACV synthetase and gramicidin S synthetase 2 as references (420 and 556 kDa, respectively); and 2), by CsCl density gradient centrifugation experiments using fluorescence-labeled cyclosporin synthetase. Besides cyclosporin A and a number of cyclosporins known from fermentation studies cyclosporin synthetase is capable of synthesizing some new cyclosporins which are so far unobtainable by fermentation. So, for example the synthesis of N-methyl-(+)-2-amino-3-hydroxy-4,4-dimethyloctanoic acid1CyA, dihydro-CyA, L-norvaline2,5, N-methyl-L-norvaline11CyA, L-allo-isoleucine5, N-methyl-L-allo-isoleucine11CyA, D-2-aminobutyric acid8CyA, beta-chloro-D-alanine8CyA and some related compounds could be established. By using a related but different enzyme from Cylindrotrichum Bonorden, the peptolide L-threonine2, L-leucine5,10, D-2-hydroxyisovaleric acid8CyA could be synthesized in vitro. We were able to synthesize these cyclosporins in sufficient quantities to examine their structure by FAB mass spectroscopy and explore their immunosuppressivity. It was found that all new cyclosporins so far synthesized in the in vitro system are immunosuppressive.
|
| 1637988 |
Molecular conformation of ascidiacyclamide, a cytotoxic cyclic peptide from Ascidian: X-ray analyses of its free form and solvate crystals |
10.1002/bip.360320204. |
Biopolymers |
Molecular conformation of ascidiacyclamide, a cytotoxic cyclic peptide from Ascidian: X-ray analyses of its free form and solvate crystals
Abstract
- In order to investigate the conformational variation of ascidiacyclamide, a cytotoxic cyclic peptide from marine tunicate Ascidian, single crystals were prepared from ethanol and aqueous ethanol solutions as its free form (crystal I) and H2O/0.5 C2H5OH solvate (crystal II), respectively, and were determined by the x-ray diffraction method. Crystal I showed a pseudo C2-symmetric saddle-shaped rectangular conformation. Similar conformations were also observed in crystal II, where there were two crystallographically independent C2-symmetric molecules (named Mol-A and -B) per asymmetric unit. Mol-A and -B included H2O and H2O/C2H5OH solvents within their ring structures, respectively. These water and ethanol molecules were located on the crystallographic dyad axes, and were stabilized by the van der Waals contacts (including hydrogen bonds) with the polar-ring N atoms and nonpolar D-Val side-chain atoms. The conformational characteristics of ascidiacyclamide and its fluctuation/variation were discussed based on the present and previously reported x-ray results.
|
| 1644198 |
Biosynthesis of bacillomycin D by Bacillus subtilis. Evidence for amino acid-activating enzymes by the use of affinity chromatography |
10.1016/0014-5793(92)81040-s. |
FEBS Lett |
Biosynthesis of bacillomycin D by Bacillus subtilis. Evidence for amino acid-activating enzymes by the use of affinity chromatography
Abstract
- Bacillomycin D is an antifungal lipopeptide produced by B. subtilis. The formation of the peptidyl bonds of bacillomycin D occurs non-ribosomally, as demonstrated by the use of chloramphenicol, an inhibitor of protein biosynthesis. Amino acid-activating enzymes were found in B. subtilis cell lysates purified by affinity chromatography on a gel containing L-Pro, an amino acid of bacillomycin D. Presence of ATP during this purification increases the binding of enzymatic proteins and their activity. An enzyme, with an apparent molecular weight of 230 kDa, catalyzed ATP-PPi exchange reactions, which were mediated by specific amino acids, corresponding to a partial sequence of bacillomycin D.
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| 1646995 |
Oxytocin and an oxytocin agonist administered centrally decrease food intake in rats |
10.1016/0196-9781(91)90176-p. |
Peptides |
Oxytocin and an oxytocin agonist administered centrally decrease food intake in rats
Abstract
- Intracerebroventricular administration of oxytocin (OT) and an OT agonist significantly decreased food intake in a dose-related manner in fasted rats. Central administration of an OT antagonist by itself (up to doses of 8 nmol) did not potentiate deprivation-induced food intake, but pretreatment with the OT receptor antagonist prevented the expected inhibition of food intake produced by OT and the OT agonist. Once-daily ICV injections of OT led to the development of tolerance to the inhibitory effects on food intake by the third day of treatment, but daily pretreatment with the OT antagonist prevented the development of this tolerance. In addition to causing decreased food intake, ICV administration of OT significantly increased grooming behavior but produced no dyskinesias. The inhibitory effect of OT on food intake was characterized by decreased amounts of food intake but a normal pattern of ingestion. The anorexia produced was central in nature and was not associated with altered plasma levels of hormones involved in caloric homeostasis or with changes in blood glucose. The OT agonist had relatively little effect on water intake when given in doses that significantly inhibited food intake. These results support the hypothesis that specific OT receptors within the central nervous system participate in the inhibition of feeding under certain conditions in rats.
|
| 1648055 |
The outer membrane permeability-increasing action of deacylpolymyxins |
10.7164/antibiotics.44.517. |
J Antibiot (Tokyo) |
The outer membrane permeability-increasing action of deacylpolymyxins
Abstract
- The outer membrane permeability-increasing action of deacylpolymyxins was compared to the well-known potent action of polymyxin B nonapeptide (PMBN). Deacylpolymyxin B (DAPB), prepared by treating polymyxin B with polymyxin acylase, was found to be a slightly more effective permeabilizer than PMBN. As low a DAPB concentration as 1 microgram/ml sensitized Escherichia coli to the probe antibiotics (rifampin, fusidic acid, erythromycin, clindamycin, novobiocin) by factors 30-100 and Salmonella typhimurium by factors 10-100. A higher concentration (3 micrograms/ml) of DAPB elicited further sensitization. Also deacylcolistin (DAC) was found to be an effective permeabilizer.
|
| 1653806 |
Differential inhibition of human neutrophil activation by cyclosporins A, D, and H. Cyclosporin H is a potent and effective inhibitor of formyl peptide-induced superoxide formation |
None |
J Immunol |
Differential inhibition of human neutrophil activation by cyclosporins A, D, and H. Cyclosporin H is a potent and effective inhibitor of formyl peptide-induced superoxide formation
Abstract
- Cyclosporin (Cs)A but not CsH inhibits activation of human lymphocytes. We studied the effects of CsA, CsD, and CsH on human neutrophil activation induced by chemoattractants and by various substances that circumvent receptor stimulation. CsH inhibited superoxide (O2-) formation induced by the chemotactic peptide, FMLP (30 nM), with a half-maximal effect at 40 nM. O2- formation was abolished by CsH at 1 microM. CsH increased the concentration of FMLP causing half-maximal activation of O2- formation from 30 nM to 0.8 microM and substantially reduced the stimulatory effect of FMLP at supra-maximally effective concentrations. The inhibitory effect of CsH on O2- formation was evident immediately after addition to neutrophils. CsH also markedly inhibited the increase in cytosolic Ca2+ (Ca2+i), beta-glucuronidase, and lysozyme release and aggregation stimulated by FMLP. CsA and CsD were considerably less effective than CsH to inhibit FMLP-induced O2- formation. CsA and CsD were without effect on exocytosis, rises in Ca2+i, and aggregation induced by the chemotactic peptide. Cyclosporines inhibited FMLP-induced O2- formation in an additive manner, indicating that they acted through a mechanism they had in common. Cyclosporines only slightly inhibited O2- formation and lysozyme release induced by C5a. Aggregation and rises in Ca2+i stimulated by C5a were not affected by cyclosporines, and they did not inhibit O2- formation and exocytosis induced by platelet-activating factor and leukotriene B4. Cyclosporines partially inhibited O2- formations induced by NaF and gamma-hexachlorocyclohexane. CsA marginally inhibited PMA-induced O2- formation and lysozyme release. CsA, CsD, and CsH did not inhibit arachidonic acid-induced O2- formation and its potentiation by NaF or stable guanine nucleotides in a cell-free system from DMSO-differentiated HL-60 cells. CsH partially inhibited binding of FML 3HP to formyl peptide receptors in membranes from DMSO- or dibutyryl cAMP-differentiated HL-60 cells. Our data show that: 1) cyclosporines differentially inhibit activation of human neutrophils; and 2) CsH is, indeed, not immunologically inactive but is a potent and effective inhibitor of FMLP-induced O2- formation. 3) CsH interferes with agonist binding to formyl peptide receptors and in addition, cyclosporines may also act at sites distal to chemoattractant receptors.
|
| 1653844 |
Effect of cyclo(Leu-Gly) on cyclic GMP-phosphodiesterase activity changes associated with development of tolerance to morphine-induced antinociception, catalepsy, respiratory depression and mydriasis |
None |
J Pharmacol Exp Ther |
Effect of cyclo(Leu-Gly) on cyclic GMP-phosphodiesterase activity changes associated with development of tolerance to morphine-induced antinociception, catalepsy, respiratory depression and mydriasis
Abstract
- A biochemical basis for the development of tolerance to morphine has yet to be defined. Although a number of models have been proposed, can account for complete tolerance to this drug. Previous studies in our laboratory indicated that the development of complete tolerance to certain morphine-induced behaviors (antinociception, catalepsy and respiratory depression) is associated with changes in the activity of some form(s) of phosphodiesterase with cyclic GMP as substrate (cGMP-PDE) activity in the brain areas that mediate these behaviors (periaqueductal gray, striatum and medulla). In the present study, experiments were performed in which Cyclo(Leu-Gly), a dipeptide that inhibits the development of tolerance to morphine, was administered daily (2 mg/kg) to morphine-naive rats, coadministered with morphine or coadministered with morphine to morphine-tolerant rats and the cGMP-PDE activity was measured. The development of tolerance to the effects was inhibited or reversed by administration of cyclo(Leu-Gly) and there were corresponding changes in cGMP-PDE activity in various brain regions. Differences in cGMP hydrolysis between brain regions from morphine-tolerant animals, tolerance-inhibited animals and tolerance-reversed animals strengthens the evidence for direct involvement of cGMP-PDE(s) in tolerance phenomena.
|
| 1657150 |
Human substance P receptor (NK-1): organization of the gene, chromosome localization, and functional expression of cDNA clones |
10.1021/bi00108a006. |
Biochemistry |
Human substance P receptor (NK-1): organization of the gene, chromosome localization, and functional expression of cDNA clones
Abstract
- The gene for the human substance P receptor (NK-1) was cloned using cDNA probes made by the polymerase chain reaction from primers based on the rat sequence. The gene spans 45-60 kb and is contained in five exons, with introns interrupting at sites homologous to those in the NK-2 receptor gene. Analysis of restriction digests of genomic DNA from mouse/human cell hybrids indicates the NK-1 receptor is a single-copy gene located on human chromosome 2. Polymerase chain reaction using primers based on the 5' and 3' ends of the coding sequence was used to generate full-length cDNAs from human lung and from IM9 lymphoblast cells. When transfected into COS-7 cells, the NK-1 receptor binds 125I-BHSP with a Kd of 0.35 +/- 0.07 nM and mediates substance P induced phosphatidylinositol metabolism. The receptor is selective for substance P; the relative affinity for neurokinin A and neurokinin B is 100- and 500-fold lower, respectively. Human IM9 lymphoblast cells express relatively high levels of the NK-1 receptor, and Northern blot analysis indicates modulation of mRNA levels by glucocorticoids and growth factors, suggesting that this cell line may be useful as a model for studying the control of NK-1 receptor gene expression.
|
| 1657804 |
Oxytocin antagonists with changes in the Asn5 position shed light on hormone-oxytocin receptor interactions |
10.1111/j.1399-3011.1991.tb01406.x. |
Int J Pept Protein Res |
Oxytocin antagonists with changes in the Asn5 position shed light on hormone-oxytocin receptor interactions
Abstract
- Since oxytocin agonists and antagonists have different structure-activity relationships, we have investigated the stereostructural and stereoelectronic requirements of the Asn5 residue in oxytocin antagonists by the synthesis of four analogues of the potent, prolonged acting oxytocin antagonist Pen1,D-Phe2,Thr4,Orn8-oxytocin (I) in which Asn5 was replaced respectively with Thr (II), Leu5 (III), Asp5 (IV) and Tyr5 (V). These analogues had pA2 values in the antioxytocic in vitro rat uterine assay of 7.23 (I), 7.16 (II), 6.67 (III), 7.21 (IV), and 6.76 (IV), respectively. All were also found to be weakly potent in the in vivo anti-vasopressor assay in the rat. These studies demonstrate very different structural and stereoelectronic requirements for oxytocin agonists and antagonists when they interact with the oxytocin uterine receptor.
|
| 1659396 |
Isolation and characterisation of the human lung NK-1 receptor cDNA |
10.1016/s0006-291x(05)81181-7. |
Biochem Biophys Res Commun |
Isolation and characterisation of the human lung NK-1 receptor cDNA
Abstract
- Functional cDNA clones for human NK-1 receptor were isolated from human lung RNA using the polymerase chain reaction (PCR). We have screened a human cosmid library and isolated a clone which appeared to contain the entire NK-1 receptor gene. From the published rat NK-1 receptor cDNA sequence we designed primers within the protein coding sequence, but outwards towards both the 5' and 3' ends of the putative human protein sequence. By this method we derived DNA sequence from the 3' end of the human gene. In order to determine the 5' end of the gene we used a PCR based method called Rapid Amplification of cDNA Ends (RACE). From the derived human sequences amplimers were designed upstream of the ATG initiation codon and downstream of the stop codon. The entire cDNA was obtained by RNA-PCR from human lung RNA. The sequence obtained was 407 amino acids in length, encoding an open-reading frame that was highly homologous to the rat NK-1 receptor cDNA (89%). The entire human cDNA was then cloned into a mammalian expression vector and mRNA was synthesized by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesized mRNA. The most potent of the three tachykinin peptides tested was Substance P. The human NK-1 receptor gene has been mapped to chromosome 2 using the polymerase chain reaction to specifically amplify the human sequence in hamster/human hybrid DNA and also in mouse/human monochromosome hybrids.
|
| 1660465 |
Extracellular domain-IgG fusion proteins for three human natriuretic peptide receptors. Hormone pharmacology and application to solid phase screening of synthetic peptide antisera. |
10.1016/s0021-9258(18)54463-x |
J. Biol. Chem. |
Extracellular domain-IgG fusion proteins for three human natriuretic peptide receptors. Hormone pharmacology and application to solid phase screening of synthetic peptide antisera.
Abstract
- The natriuretic peptide receptors (NPRs) are a family of three cell surface glycoproteins, each with a single transmembrane domain. Two of these receptors, designated NPR-A and NPR-B, are membrane guanylyl cyclases that synthesize cGMP in response to hormone stimulation. The third receptor, NPR-C, has been reported to function in the metabolic clearance of ligand and in guanylyl cyclase-independent signal transduction. We engineered three chimeric proteins consisting of the natriuretic peptide receptor extracellular domains fused to the Fc portion of human IgG-gamma 1. These molecules provide material for detailed studies of the human receptor's extracellular domain structure and interaction with the three human natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and type-C natriuretic peptide (CNP). The homodimeric fusion proteins, designated A-IgG, B-IgG, and C-IgG, were secreted from Chinese hamster ovary cells and purified by protein-A affinity chromatography. We present here the primary characterization of these fusion proteins as represented by the intrinsic hormone affinities measured by saturation binding and competition assays. The dissociation constant of 125I-ANP for A-IgG was 1.6 pM and for C-IgG, 1.2 pM. The dissociation constant of 125I-Y0-CNP (CNP with addition of tyrosine at the amino terminus) for B-IgG was 23 pM. The rank order of potency in competitive binding for A-IgG was ANP greater than BNP much greater than CNP, whereas for B-IgG the ranking was CNP much greater than ANP greater than BNP. For C-IgG, we observed ANP greater than CNP greater than or equal to BNP. These data demonstrate that the receptor-IgG fusion proteins discriminate among the natriuretic peptides in the same manner as the native receptors and provide a basis for future structural studies with these molecules. The purified fusion proteins have a variety of potential applications, one of which we illustrate by a solid phase screening assay in which rabbit sera from a series of synthetic-peptide immunizations were titered for receptor reactivity and selectivity.
|
| 1661721 |
Phalloidin attenuates postischemic neutrophil infiltration and increased microvascular permeability |
10.1152/jappl.1991.71.4.1261. |
J Appl Physiol (1985) |
Phalloidin attenuates postischemic neutrophil infiltration and increased microvascular permeability
Abstract
- The aim of this study was to determine whether phalloidin (1 microM) or antamanide (1 microM), cyclic peptides that stabilize dense peripheral band and stress fiber F-actin in endothelium, would attenuate the increase in microvascular permeability induced by 4 h of ischemia and 30 min of reperfusion (I/R) in the isolated canine gracilis muscle. Changes in microvascular permeability (1 - sigma) were assessed by determining the solvent drag reflection coefficient for total plasma proteins (sigma) in muscles subjected to 4.5 h of continuous perfusion (nonischemic controls), I/R alone, I/R + phalloidin, or I/R + antamanide. Muscle neutrophil content was assessed by determination of myeloperoxidase (MPO) activity in tissue samples obtained at the end of the experiments. Fluorescent detection of nitrobenzoxadiazole-phallicidin in endothelial cell monolayers confirmed that phalloidin enters these cells. I/R was associated with marked increases in microvascular permeability and muscle neutrophil content (1 - sigma = 0.45 +/- 0.07; MPO = 8.9 +/- 0.5 units/g) relative to control (4.5 h continuous perfusion) preparations (1 - sigma = 0.12 +/- 0.03; MPO = 0.5 +/- 0.8 unit/g). These I/R-induced changes were largely prevented by administration of phalloidin (1 - sigma = 0.19 +/- 0.02; MPO = 0.8 +/- 0.4 U/g) or antamanide (1 - sigma = 0.07 +/- 0.11; MPO = 0.9 +/- 0.3 unit/g) at reperfusion. Similar results were obtained when phalloidin was administered before ischemia (1 - sigma = 0.24 +/- 0.04; MPO = 1.2 +/- 1.0 units/g). Although antamanide decreased superoxide production (by approximately 60%) and adherence to plastic (by approximately 75%) by activated neutrophils in vitro, phalloidin failed to alter these aspects of granulocyte function.(ABSTRACT TRUNCATED AT 250 WORDS)
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| 1666394 |
Periodate oxidation products derived from methylated alpha-amanitin: evidence for distinct aldehydic and non-aldehydic forms |
10.1111/j.1399-3011.1991.tb01521.x. |
Int J Pept Protein Res |
Periodate oxidation products derived from methylated alpha-amanitin: evidence for distinct aldehydic and non-aldehydic forms
Abstract
- Amatoxins are cyclic octapeptides which can be purified from various mushroom species. They have found widespread use due to their potent inhibition of eukaryotic RNA polymerase II. In the course of our efforts to prepare additional semisynthetic derivatives of the amatoxin, alpha-amanitin, we examined the products formed through the periodate oxidation of 6'-O-methyl-alpha-amanitin. Periodate oxidation under conditions of near-neutral pH yielded two chemically similar, yet chromatographically separable, products. On the basis of their proton NMR spectra and their lack of reactivity with sodium chlorite these products did not contain a free aldehydic group. However, both forms were interconvertible in aqueous neutral solution and could be converted to the same product, 6'-O-methyldemethyl-gamma-amanitin, through reduction with sodium borohydride. Periodate oxidation under mildly acidic conditions generated a single product which has properties of a free aldehyde. It exhibited a proton NMR spectrum with signals characteristic of an aliphatic aldehyde and was readily oxidized to the carboxylic acid with sodium chlorite. Conditions have been defined to synthesize the free aldehyde derivative of 6'-O-methyl-alpha-amanitin in generous yield to provide a precursor for oxidation and further derivatization."
|
| 1667253 |
Effect of protein binding of daptomycin on MIC and antibacterial activity |
10.1128/AAC.35.12.2505. |
Antimicrob Agents Chemother |
Effect of protein binding of daptomycin on MIC and antibacterial activity
Abstract
- A higher rate of clinical failures in patients treated with daptomycin (2 mg/kg of body weight, given once daily) compared with rates in patients treated with conventional regimens caused early termination of this comparative clinical trial. One explanation for these failures could be that daptomycin is highly protein bound and that the concentration of the unbound active drug is too low for antibacterial activity. To assess this explanation, we studied the binding of daptomycin to proteins by using an ultrafiltration method. pH (7.0 to 7.4), temperature (25 or 37 degrees C), or daily freezing and thawing over 2 months had no effect on binding of daptomycin to proteins. We found that daptomycin was bound to albumin (90%) at 4 g/100 ml. Binding of daptomycin was not concentration dependent (2.5 to 80 micrograms/ml). In human serum samples spiked with daptomycin, average binding was 94% +/- 2.4%. In 6 subjects given an intravenous infusion of daptomycin (3 mg/kg), average binding was 90% +/- 2.1%. Susceptibility studies showed that a concentration in serum 20 times the unbound concentration was needed to equal the MIC of the total drug. These results indicate that daptomycin is highly bound (90 to 94%) to albumin and that clinical failure to daptomycin can in part be explained by the low concentration of the unbound drug.
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