| 19393553 |
Improvement of FR901379 production by mutant selection and medium optimization |
10.1016/j.jbiosc.2009.01.002. |
J Biosci Bioeng |
Improvement of FR901379 production by mutant selection and medium optimization
Abstract
- FR901379 (WF11899A) is a novel echinocandin type of lipopeptide antibiotic produced by Coleophoma empetri F-11899. Micafungin (FK463) is derived from the chemical modification of deacylated FR901379. In the present paper, we performed seven generation's strain-breeding, beginning with a wild type, was performed. Selection medium for screening and production medium for high FR901379 production were designed. Sodium chloride content in the selection plate was affected to FR901379 production and shrinkage of the colony size was observed in high producing strains. As selection markers, large colony-shrinking rate and large inhibition circle in the agar-piece method using C. albicans was selected. Using CMA medium with high sodium chloride, 3 mutants, M-1 to M-3, have achieved a high FR901379 production and M-3 showed 5.0 U/mL, while 1.0 U/mL of production was achieved in wild type strain. A-2 medium supplemented with 6% of soluble starch as a carbon source and 0.6% of ammonium sulfate as nitrogen source was also further effective for mutant screening. The FR901379 production of mutant M-4 (fourth generation) increased until 16.0 U/mL. The concentration of the phosphate salt in the medium seemed to inhibit the growth so as to extend the culture period. When the A-3 medium supplemented with low concentration of phosphate salt and magnesium sulfate as a sulfate source was designed and used, mutants with improved production were successively obtained. Finally, variant strain M-7 showed 30.0 U/mL of production, which was about 30 times higher than that of the wild strain.
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| 19394223 |
Discovery of 3,5-disubstituted-1H-pyrrolo[2,3-b]pyridines as potent inhibitors of the insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase |
10.1016/j.bmcl.2008.12.110. |
Bioorg Med Chem Lett |
Discovery of 3,5-disubstituted-1H-pyrrolo[2,3-b]pyridines as potent inhibitors of the insulin-like growth factor-1 receptor (IGF-1R) tyrosine kinase
Abstract
- Exploration of the SAR around a series of 3,5-disubstituted-1H-pyrrolo[2,3-b]pyridines led to the discovery of novel pyrrolopyridine inhibitors of the IGF-1R tyrosine kinase. Several compounds demonstrated nanomolar potency in enzyme and cellular mechanistic assays.
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| 19400188 |
Synthesis of cyclic tetrapeptide CJ 15,208: a novel kappa opioid receptor antagonist |
10.1007/978-0-387-73657-0_121. |
Adv Exp Med Biol |
Synthesis of cyclic tetrapeptide CJ 15,208: a novel kappa opioid receptor antagonist
Abstract
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| 19413330 |
Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach. |
10.1021/ac9004309 |
Anal. Chem. |
Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.
Abstract
- The analysis of proteome-wide phosphorylation events is still a major analytical challenge because of the enormous complexity of protein phosphorylation networks. In this work, we evaluate the complementarity of Lys-N, Lys-C, and trypsin with regard to their ability to contribute to the global analysis of the phosphoproteome. A refined version of low-pH strong cation exchange was used to efficiently separate N-terminally acetylated, phosphorylated, and nonmodified peptides. A total of 5036 nonredundant phosphopeptides could be identified with a false discovery rate of <1% from 1 mg of protein using a combination of the three enzymes. Our data revealed that the overlap between the phosphopeptide data sets generated with different proteases was marginal, whereas the overlap between two similarly generated tryptic data sets was found to be at least 4 times higher. In this way, the parallel use of Lys-N and trypsin enabled a 72% increase in the number of detected phosphopeptides as compared to trypsin alone, whereas a trypsin replicate experiment only led to a 25% increase. Thus, when focusing solely on the trypsin and Lys-N data, we identified 4671 nonredundant phosphopeptides. Further analysis of the detected sites showed that the Lys-N and trypsin data sets were enriched in significantly different phosphorylation motifs, further evidencing that multiprotease approaches are very valuable in phosphoproteome analyses.
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| 19416919 |
Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis. |
10.1073/pnas.0812191106 |
Proc. Natl. Acad. Sci. U.S.A. |
Identification and structural basis of the reaction catalyzed by CYP121, an essential cytochrome P450 in Mycobacterium tuberculosis.
Abstract
- The gene encoding the cytochrome P450 CYP121 is essential for Mycobacterium tuberculosis. However, the CYP121 catalytic activity remains unknown. Here, we show that the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) binds to CYP121, and is efficiently converted into a single major product in a CYP121 activity assay containing spinach ferredoxin and ferredoxin reductase. NMR spectroscopy analysis of the reaction product shows that CYP121 catalyzes the formation of an intramolecular C-C bond between 2 tyrosyl carbon atoms of cYY resulting in a novel chemical entity. The X-ray structure of cYY-bound CYP121, solved at high resolution (1.4 A), reveals one cYY molecule with full occupancy in the large active site cavity. One cYY tyrosyl approaches the heme and establishes a specific H-bonding network with Ser-237, Gln-385, Arg-386, and 3 water molecules, including the sixth iron ligand. These observations are consistent with low temperature EPR spectra of cYY-bound CYP121 showing a change in the heme environment with the persistence of the sixth heme iron ligand. As the carbon atoms involved in the final C-C coupling are located 5.4 A apart according to the CYP121-cYY complex crystal structure, we propose that C-C coupling is concomitant with substrate tyrosyl movements. This study provides insight into the catalytic activity, mechanism, and biological function of CYP121. Also, it provides clues for rational design of putative CYP121 substrate-based antimycobacterial agents.
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| 19434240 |
The somatostatin 2A receptor is enriched in migrating neurons during rat and human brain development and stimulates migration and axonal outgrowth. |
10.1371/journal.pone.0005509 |
PLoS ONE |
The somatostatin 2A receptor is enriched in migrating neurons during rat and human brain development and stimulates migration and axonal outgrowth.
Abstract
- The neuropeptide somatostatin has been suggested to play an important role during neuronal development in addition to its established modulatory impact on neuroendocrine, motor and cognitive functions in adults. Although six somatostatin G protein-coupled receptors have been discovered, little is known about their distribution and function in the developing mammalian brain. In this study, we have first characterized the developmental expression of the somatostatin receptor sst2A, the subtype found most prominently in the adult rat and human nervous system. In the rat, the sst2A receptor expression appears as early as E12 and is restricted to post-mitotic neuronal populations leaving the ventricular zone. From E12 on, migrating neuronal populations immunopositive for the receptor were observed in numerous developing regions including the cerebral cortex, hippocampus and ganglionic eminences. Intense but transient immunoreactive signals were detected in the deep part of the external granular layer of the cerebellum, the rostral migratory stream and in tyrosine hydroxylase- and serotonin- positive neurons and axons. Activation of the sst2A receptor in vitro in rat cerebellar microexplants and primary hippocampal neurons revealed stimulatory effects on neuronal migration and axonal growth, respectively. In the human cortex, receptor immunoreactivity was located in the preplate at early development stages (8 gestational weeks) and was enriched to the outer part of the germinal zone at later stages. In the cerebellum, the deep part of the external granular layer was strongly immunoreactive at 19 gestational weeks, similar to the finding in rodents. In addition, migrating granule cells in the internal granular layer were also receptor-positive. Together, theses
Results strongly suggest that the somatostatin sst2A receptor participates in the development and maturation of specific neuronal populations during rat and human brain ontogenesis.
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| 19445532 |
Substrate specificity and scope of MvdD, a GRASP-like ligase from the microviridin biosynthetic gene cluster |
10.1021/cb900088r. |
ACS Chem Biol |
Substrate specificity and scope of MvdD, a GRASP-like ligase from the microviridin biosynthetic gene cluster
Abstract
- The cyanobacterial protease inhibitor microviridin K is ribosomally biosynthesized as a prepeptide (MvdE) and subsequently modified posttranslationally by double lactonization followed by lactamization. Two proteins belonging to the GRASP superfamily of ligases catalyze these ring closures. We here show that one of these ligases (MvdD) forms the lactones in a specific order, the larger ring being formed first, and that the ring size requirement for both lactonizations is stringent. However, for the first cyclization MvdD accepts alanine substitution in all C-terminal positions of the microviridin prepeptide that are not directly involved in the cross-linking, whereas the second lactonization is dependent on the presence of specific residues in MvdE. This suggests that MvdD possesses some, albeit limited, substrate tolerance that might be useful for the modification of peptides and proteins not belonging to the microviridin group of metabolites.
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| 19457344 |
TxXIIIA, an atypical homodimeric conotoxin found in the Conus textile venom |
10.1016/j.jprot.2009.01.021. |
J Proteomics |
TxXIIIA, an atypical homodimeric conotoxin found in the Conus textile venom
Abstract
- Venoms of predatory Conus snails are composed of several hundreds of peptide toxins. Many of these peptides display a high selectivity for particular membrane receptors such as ionic channels or G-protein coupled receptors. This property makes them very promising tools for the study of receptors and potential new drugs. Conus snails synthesize toxins under various folds, each fold related to particular pharmacological activities. Aiming the discovery of new conotoxins, we looked for toxins with original fold in the Conus textile venom by offline LC-MALDI-TOF/TOF mass spectrometry. Venom fractions were analysed by MALDI-TOF (in 2,5-dihydroxybenzoic acid) before and after the "in-solution" reduction of the disulfide bridges. Comparison of the spectra allows the classification of a large number of conotoxins according to the number of disulfide bridges. We focussed on a component at m/z 2785.7 (non-reduced)/ 1398.4 (reduced), which might represent a novel type of homodimeric toxin. The sequence TSDCCFYHNCCC was determined by De novo sequencing on the reduced species and represent a new fold. This sequence has already been described as the C-terminus part of a conotoxin scaffold IX precursor (expasy: Q9BPH1) but the power of our study resides in the fact that mass spectrometry highlights the right length of the toxin as well as its homodimeric form which could not be determined by the previous cDNA study. TxXIIIA is also the first homodimeric conotoxin with five disulfide bonds and composed of two monomers containing an odd number of cysteins.
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| 19459609 |
A thermodynamic study of ligand binding to the first three domains of the human insulin receptor: relationship between the receptor alpha-chain C-terminal peptide and the site 1 insulin mimetic peptides |
10.1021/bi900261q. |
Biochemistry |
A thermodynamic study of ligand binding to the first three domains of the human insulin receptor: relationship between the receptor alpha-chain C-terminal peptide and the site 1 insulin mimetic peptides
Abstract
- The C-terminal segment of the insulin receptor (IR) alpha-chain plays a critical role in insulin binding. This 16-residue peptide together with the central beta-sheet of the receptor L1 domain forms one of the insulin binding surfaces of the IR monomer. Here we use isothermal titration calorimetry to assay directly the binding of the IR alphaCT peptide to an IR construct (IR485) consisting of the three N-terminal domains of the receptor monomer. Our measurements show further that the binding of the IR alphaCT peptide to IR485 competes with the binding of a prototypical "Site 1" insulin mimetic peptide to the same receptor fragment. The competitive nature of their binding appears to be reflected in a previously undetected sequence similarity between the IR alphaCT peptide and the Site 1 mimetic peptide. In contrast, a prototypical "Site 2" peptide has very limited affinity for IR485. Taken together, these results complement our recent observation that there is a possible structural relationship between these mimetic peptides and insulin itself. They also add support to the view that the segment of unexplained electron density lying on the surface of the central beta-sheet of the L1 domain in the IR ectodomain crystal structure arises from the IR alphaCT peptide. Finally, we show that mutation of the critical IR alphaCT peptide residue Phe714 to alanine does not affect the peptide's affinity for IR485 and conclude that the resultant loss of insulin binding with this mutation results from loss of interaction of the phenylalanine side chain with insulin.
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| 19462049 |
Circular proteins from Melicytus (Violaceae) refine the conserved protein and gene architecture of cyclotides |
10.1039/b823020j. |
Org Biomol Chem |
Circular proteins from Melicytus (Violaceae) refine the conserved protein and gene architecture of cyclotides
Abstract
- Cyclotides are cyclic disulfide rich mini-proteins found in various Rubiaceae (coffee family), Violaceae (violet family) and Cucurbitaceae (squash family) plant species. Within the Violaceae, cyclotides have been found in numerous species of the genus Viola as well as species from two other genera, namely Hybanthus and Leonia. This is the first in-depth report of cyclotides in the genus Melicytus (Violaceae). We present the chromatographic profiles of extracts of eight Melicytus species and one Melicytus hybrid that were found to contain these circular peptides. We isolated and characterised five novel cyclotides (mra1 to mra5) from the aerial parts of a common New Zealand tree, Melicytus ramiflorus. All five peptides show the characteristics of the bracelet subfamily of cyclotides. Furthermore, we isolated 17 non-redundant cDNA clones from the leaves of Melicytus ramiflorus encoding cyclotide prepropeptides. This detailed report on the presence of cyclotides in several species of the genus Melicytus further strengthens our hypothesis that cyclotides are ubiquitous in Violaceae family plants and provides additional insight into the biochemical processing mechanisms that produce the cyclic protein backbone of this unique family of ultra-stable plant proteins.
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| 19464172 |
Nascent structure-activity relationship study of a diastereomeric series of kappa opioid receptor antagonists derived from CJ-15,208 |
10.1016/j.bmcl.2009.04.105. |
Bioorg Med Chem Lett |
Nascent structure-activity relationship study of a diastereomeric series of kappa opioid receptor antagonists derived from CJ-15,208
Abstract
- Cyclic tetrapeptide c[Phe-pro-Phe-trp] 2, a diastereomer of CJ-15,208 (1), was identified as a potent dual kappa/mu opioid receptor antagonist devoid of delta opioid receptor affinity against cloned human receptors: K(i) (2)=3.8nM (kappa), 30nM (mu); IC(50) ([(35)S]GTPgammaS binding)=140nM (kappa), 21nM (mu). The d-tryptophan residue rendered 2 ca. eightfold and fourfold more potent at kappa and mu, respectively, than the corresponding l-configured tryptophan in the natural product 1. Phe analogs 3-10, designed to probe the effect of substituents on receptor affinity and selectivity, possessed K(i) values ranging from 14 to 220nM against the kappa opioid receptor with mu/kappa ratios of 0.45-3.0. An alanine scan of 2 yielded c[Ala-pro-Phe-trp] 12, an analog equipotent to 2. Agents 2 and 12 were pure antagonists in vitro devoid of agonist activity. Ac-pro-Phe-trp-Phe-NH(2)16 and Ac-Phe-trp-Phe-pro-NH(2)17 two of the eight possible acyclic peptides derived from 1 and 2, were selective, modestly potent mu ligands: K(i) (16)=340nM (mu); K(i) (17)=360nM (mu).
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| 19476492 |
Dual modulation of prothrombin activation by the cyclopentapeptide plactin |
10.1111/j.1742-4658.2009.06976.x. |
FEBS J |
Dual modulation of prothrombin activation by the cyclopentapeptide plactin
Abstract
- Plactin, a family of cyclopentapeptides, enhances fibrinolytic activity by elevating the activity of cellular urokinase-type plasminogen activator (u-PA), a protease involved in a variety of extracellular proteolytic events. Factor(s) in the blood plasma is an absolute requirement for this plactin activity. In this study, we found that plactin promoted plasma cofactor-dependent conversion of inactive single-chain u-PA to active two-chain u-PA on U937 cells. Using plactin-affinity chromatography, we identified prothrombin as one of the plasma cofactors. In incubations of U937 cells with prothrombin and Xa, plactin increased the formation of thrombin, which cleaved single-chain u-PA to afford the inactive two-chain form. Thrombin-cleaved two-chain u-PA was alternatively activated by cellular cystatin-sensitive peptidase activity, yielding fully active two-chain u-PA. In a purified system, plactin bound to prothrombin, altered its conformation and dually modulated factor Xa-mediated proteolytic activation of prothrombin to alpha-thrombin. Plactin inhibited the activation catalyzed by Xa in complex with Va, Ca(2+) and phospholipids (prothrombinase), whereas the activations catalyzed by nonmembrane-associated Xa were enhanced markedly by plactin. Plactin inhibited in vitro plasma coagulation, which involved prothrombinase formation. Plactin did not cause prothrombin activation or thrombosis in normal mice at doses that produced a protective effect in a thrombin-induced pulmonary embolism mouse model. Therefore, the dual modulation of prothrombin activation by plactin may be interpreted as leading to anticoagulation under physiological coagulating conditions.
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| 19489598 |
Desmethoxymajusculamide C, a cyanobacterial depsipeptide with potent cytotoxicity in both cyclic and ring-opened forms |
10.1021/np9001674. |
J Nat Prod |
Desmethoxymajusculamide C, a cyanobacterial depsipeptide with potent cytotoxicity in both cyclic and ring-opened forms
Abstract
- Cytotoxicity-guided fractionation of the organic extract from a Fijian Lyngbya majuscula led to the discovery of desmethoxymajusculamide C (DMMC) as the active metabolite. Spectroscopic analysis including 1D and 2D NMR, MS/MS, and chemical degradation and derivatization protocols were used to assign the planar structure and stereoconfiguration of this new cyclic depsipeptide. DMMC demonstrated potent and selective anti-solid tumor activity with an IC(50) = 20 nM against the HCT-116 human colon carcinoma cell line via disruption of cellular microfilament networks. A linear form of DMMC was generated by base hydrolysis, and the amino acid sequence was confirmed by mass spectrometry. Linearized DMMC was also evaluated in the biological assays and found to maintain potent actin depolymerization characteristics while displaying solid tumor selectivity equivalent to DMMC in the disk diffusion assay. A clonogenic assay assessing cytotoxicity to HCT-116 cells as a function of exposure duration showed that greater than 24 h of constant drug treatment was required to yield significant cell killing. Therapeutic studies with HCT-116 bearing SCID mice demonstrated efficacy at the highest dose used (%T/C = 60% at 0.62 mg/kg daily for 5 days).
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| 19494351 |
Naturally processed peptides spanning the HPA-1a polymorphism are efficiently generated and displayed from platelet glycoprotein by HLA-DRB3*0101-positive antigen-presenting cells |
10.1182/blood-2009-04-211839. |
Blood |
Naturally processed peptides spanning the HPA-1a polymorphism are efficiently generated and displayed from platelet glycoprotein by HLA-DRB3*0101-positive antigen-presenting cells
Abstract
- In neonatal alloimmune thrombocytopenia, almost all human platelet antigen (HPA)-1b1b mothers who produce anti-HPA-1a antibody through carrying an HPA-1a fetus are human histocompatibility leukocyte antigen (HLA)-DRB3*0101 positive. It is predicted that the HPA-1a Leu(33) polymorphism forms part of an HLA-DRB3*0101-restricted T-helper epitope, and acts as an anchor residue for binding this class II molecule. However, it is not known whether any corresponding peptides are naturally processed and presented from platelet glycoprotein. In this study, peptides displayed by a homozygous HLA-DRB3*0101 antigen-presenting cell line were identified after pulsing with recombinant HPA-1a (Leu(33) plexin-semaphorin-integrin domain). The peptides were eluted from HLA-DR molecules, fractionated by high performance liquid chromatography, and analyzed by tandem mass spectrometry. A "nested set" of naturally presented HPA-1a-derived peptides, each containing the Trp(25)-Leu(33) core epitope, was identified, with the most abundant member being the 16-mer Met(22)-Arg(37). These peptides may provide the basis for novel treatments to tolerize the corresponding T-helper response in women at risk of neonatal alloimmune thrombocytopenia.
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| 19505367 |
Protective effect of JBP485 on concanavalin A-induced liver injury in mice |
10.1211/jpp.61.06.0009. |
J Pharm Pharmacol |
Protective effect of JBP485 on concanavalin A-induced liver injury in mice
Abstract
- Cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) was first isolated from Laennec (hydrolysate of human placenta). We thought it valuable to clarify the anti-hepatitis molecular mechanism of JBP485 to develop a new oral anti-hepatitis drug.
We investigated the hepatoprotective effect of JBP485 on immune-mediated, concanavalin A (Con A)-induced liver injury in mice. Mice were administered JBP485 before and after injection of Con A (10 mg/kg). Eight hours after Con A, the cytosolic enzyme activity (alanine aminotransferase, lactate dehydrogenase) in serum, and the enzyme activity or concentration (superoxide dismutase, maleic dialdehyde, myeloperoxidase, nitric oxide) in liver homogenate were determined. The liver slices were investigated to observe changes in histology. The effect of JBP485 on level of tumour necrosis factor-alpha (TNF-alpha) and intercellular adhesion molecule-1 (ICAM-1) in liver were detected by immunohistochemistry. Hepatocyte DNA fragmentation was assayed by agarose gel electrophoresis and the transcription of the genes bax and bcl-2 in hepatocytes was determined by reverse transcription-polymerase chain reaction.
Con A increased the cytosolic and liver homogenate enzyme activity, and the concentrations of ICAM-1 and TNF-alpha, which were significantly inhibited by JBP485 administration. Also, the increase in DNA fragmentation and decrease in bcl-2/bax mRNA induced by Con A administration were significantly inhibited by JBP485.
These results indicated that immune-mediated liver damage can be prevented by JBP485, and that this is mainly associated with immunomodulatory effects on T cells and adhesion molecules, antioxidation, and inhibition of apoptosis.
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