| 16015337 |
Selective inhibition of cotranslational translocation of vascular cell adhesion molecule 1 |
10.1038/nature03670. |
Nature |
Selective inhibition of cotranslational translocation of vascular cell adhesion molecule 1
Abstract
- Increased expression of vascular cell adhesion molecule 1 (VCAM1) is associated with a variety of chronic inflammatory conditions, making its expression and function a target for therapeutic intervention. We have recently identified CAM741, a derivative of a fungus-derived cyclopeptolide that acts as a selective inhibitor of VCAM1 synthesis in endothelial cells. Here we show that the compound represses the biosynthesis of VCAM1 in cells by blocking the process of cotranslational translocation, which is dependent on the signal peptide of VCAM1. CAM741 does not inhibit targeting of the VCAM1 nascent chains to the translocon channel but prevents translocation to the luminal side of the endoplasmic reticulum (ER), through a process that involves the translocon component Sec61beta. Consequently, the VCAM1 precursor protein is synthesized towards the cytosolic compartment of the cells, where it is degraded. Our results indicate that the inhibition of cotranslational translocation with low-molecular-mass compounds, using specificity conferred by signal peptides, can modulate the biosynthesis of certain secreted and/or membrane proteins. In addition, they highlight cotranslational translocation at the ER membrane as a potential target for drug discovery.
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| 16033260 |
Synthesis and mechanism of action studies of a series of norindenoisoquinoline topoisomerase I poisons reveal an inhibitor with a flipped orientation in the ternary DNA-enzyme-inhibitor complex as determined by X-ray crystallographic analysis |
10.1021/jm050076b. |
J Med Chem |
Synthesis and mechanism of action studies of a series of norindenoisoquinoline topoisomerase I poisons reveal an inhibitor with a flipped orientation in the ternary DNA-enzyme-inhibitor complex as determined by X-ray crystallographic analysis
Abstract
- Several norindenoisoquinolines substituted with methoxy or methylenedioxy groups have been prepared and their anticancer properties evaluated in cancer cell cultures and in topoisomerase I inhibition assays. 2,3-Dimethoxy-8,9-methylenedioxy-11H-indeno[1,2-c]isoquinoline hydrochloride (14) is a strong topoisomerase I inhibitor and also displays very high cytotoxicity in the NCI cancer cell culture screen (mean graph midpoint of 50 nM). The X-ray crystal structure of norindenoisoquinoline 14 in complex with topoisomerase I and DNA has been solved, providing insight into the structure-activity relationships within this class of new anticancer agents. The number and position of the norindenoisoquinoline substituents have a significant influence on biological activity and demonstrate that substitution on the nitrogen atom is not an absolute requirement for the antitumor effect of the indenoisoquinolines. Removal of the 11-keto group from the lead compound 1 and replacement of the N-alkyllactam with an unsubstituted pyridine ring causes the indenoisoquinoline ring system to flip over in the DNA-enzyme-inhibitor ternary complex. This allows the nitrogen atom to assume the hydrogen bond acceptor role of the 11-keto group, resulting in hydrogen bonding to Arg364.
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| 16040066 |
Two cyclopeptides from the seeds of Annona cherimola |
10.1016/j.phytochem.2005.06.011. |
Phytochemistry |
Two cyclopeptides from the seeds of Annona cherimola
Abstract
- Bioassay-guided fractionation of cytotoxic of methanol extract of the seeds of Annona cherimola provided two novel cyclic peptides, cherimolacyclopeptide E (1) and cherimolacyclopeptide F (2), which exhibited significant cytotoxic activity against the KB (human nasopharyngeal carcinoma) cell culture system. The peptide 1 and 2 were elucidated by MS/MS fragmentation experiments using a Q-TOF mass spectrometer equipped with an ESI source, extensive 2D NMR analyses and chemical degradation.
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| 16046462 |
Activity of aminocandin (IP960) compared with amphotericin B and fluconazole in a neutropenic murine model of disseminated infection caused by a fluconazole-resistant strain of Candida tropicalis |
10.1093/jac/dki268. |
J Antimicrob Chemother |
Activity of aminocandin (IP960) compared with amphotericin B and fluconazole in a neutropenic murine model of disseminated infection caused by a fluconazole-resistant strain of Candida tropicalis
Abstract
- To compare the activity of aminocandin (IP960), a new echinocandin with broad-spectrum in vitro activity against Aspergillus and Candida spp., with that of amphotericin B and fluconazole in a temporarily immunocompromised murine model of disseminated candidiasis.
Mice were rendered neutropenic with cyclophosphamide and infected intravenously 3 days later with a fluconazole-resistant Candida tropicalis strain. Mice were treated with intraperitoneal amphotericin B (5 mg/kg/dose), oral fluconazole (50 mg/kg/dose), intravenous aminocandin (0.1--5 mg/kg/dose) or solvent control for 9 days. Mice were observed for survival and survivors were sacrificed 11 days post-infection. Kidneys, liver, brain and lungs were removed for semi-quantitative culture.
Control mice had 90--100% mortality. After infection with C. tropicalis, aminocandin 2.5 and 5 mg/kg/day and amphotericin B yielded 80% survival; aminocandin 1 mg/kg/day yielded 70% survival; aminocandin 0.25 and 0.1 mg/kg/day yielded 30% and 20% survival, respectively; and fluconazole 50 mg/kg/day and control regimens yielded 10% and 0--10% survival, respectively. Aminocandin 2.5 and 5.0 mg/kg/day and amphotericin B were superior in reducing mortality compared with aminocandin 0.25 and 0.1 mg/kg/day, fluconazole and controls (P<0.047). The only regimen to reduce organ burdens below detectable levels was amphotericin B, which cleared 40% of mice. All organ burdens in the aminocandin 1.0, 2.5 and 5.0 mg/kg/day and amphotericin B regimens were significantly lower than other groups (P<0.02).
The data demonstrate that aminocandin at doses of >or=1.0 mg/kg/day is as effective as amphotericin B at improving survival and reducing organ burdens in this murine model of disseminated C. tropicalis.
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| 16051604 |
Cartilage oligomeric matrix protein/thrombospondin 5 supports chondrocyte attachment through interaction with integrins |
10.1074/jbc.M504778200. |
J Biol Chem |
Cartilage oligomeric matrix protein/thrombospondin 5 supports chondrocyte attachment through interaction with integrins
Abstract
- Cartilage oligomeric matrix protein/thrombospondin 5 (COMP/TSP5) is a major component of the extracellular matrix of the musculoskeletal system. Although COMP/TSP5 abnormalities are associated with several pathological conditions, its normal function remains unclear. This study was undertaken to delineate the function(s) of COMP/TSP5 in cartilage, especially regarding its interaction with chondrocytes. We show that COMP/TSP5 can support chondrocyte attachment and that the RGD sequence in COMP/TSP5 and the integrin receptors alpha5beta1 and alphaVbeta3 on the chondrocytes are involved in mediating this attachment. The interactions of COMP/TSP5 with the integrins are dependent on COMP/TSP5 conformation. Chondrocyte attachment to COMP/TSP5 in the calcium-replete conformation was inhibited by function-blocking integrin alpha5 and beta1 antibodies, suggesting the involvement of the alpha5beta1 integrin. Under this condition, a function-blocking antibody against alphaVbeta3 did not have any effect on cell attachment. On the other hand, chondrocyte attachment to reduced COMP/TSP5 was instead sensitive to alphaVbeta3 function-blocking antibodies, suggesting that COMP/TSP5 mediates attachment through chondrocyte alphaVbeta3 integrin under this condition. Cell attachment to reduced COMP/TSP5 was not inhibited by beta1 antibodies. These data indicate that COMP/TSP5 in different conformations can utilize different integrin receptors. These results are the first to demonstrate that COMP/TSP5 can mediate chondrocyte attachment through interactions with integrins. Through these interactions, COMP/TSP5 may be able to regulate cellular activities and respond to environment in the surrounding cartilage matrix.
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| 16075425 |
Structure of the antimicrobial, cationic hexapeptide cyclo(RRWWRF) and its analogues in solution and bound to detergent micelles |
10.1002/cbic.200500095. |
Chembiochem |
Structure of the antimicrobial, cationic hexapeptide cyclo(RRWWRF) and its analogues in solution and bound to detergent micelles
Abstract
- Antimicrobial, cationic peptides are abundant throughout nature as part of many organisms' defence against microorganisms. They exhibit a large variety of sequences and structural motifs and are thought to act by rupturing the bacterial membrane. Several models based on biophysical experiments have been proposed for their mechanism of action. Here we present the NMR-determined structure of the cyclic, cationic antimicrobial peptide cyclo(RRWWRF) both free in aqueous solution and bound to detergent micelles. The peptide has a rather flexible but ordered structure in water. A distinct structure is formed when the peptide is bound to a detergent micelle. The structures in neutral and negatively charged micelles are nearly identical but differ from that in aqueous solution. The orientation of the amino acid side chains creates an amphipathic molecule with the peptide backbone forming the hydrophilic part. The orientation of the peptide in the micelle was determined by using NOEs and paramagnetic agents. The peptide is oriented mainly parallel to the micelle surface in both detergents. Substitution of the arginine and tryptophan residues is known to influence the antimicrobial activity. Therefore the structure of the micelle-bound analogues cyclo(RRYYRF), cyclo(KKWWKF) and cyclo(RRNalNalRF) were also determined. They exhibit remarkable similarities in backbone conformation and side-chain orientation. The structure of these peptides allows the side-chain properties to be correlated to biological activity.
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| 16075448 |
Total synthesis of halipeptins: isolation of halipeptin D and synthesis of oxazoline halipeptin analogues |
10.1002/chem.200500624. |
Chemistry |
Total synthesis of halipeptins: isolation of halipeptin D and synthesis of oxazoline halipeptin analogues
Abstract
- The isolation from the marine sponge Leiosella cf. arenifibrosa and structural elucidation of halipeptin D (5), a relative of the previously isolated halipeptins A-C (1-3), is described along with the total synthesis of a number of oxazoline analogues (7 a-d and epi-7 c-d). The developed synthetic strategy provides a flexible entry into the various isomers of the polyketide domain of the halipeptins and improvises for a late stage construction of the oxazoline ring after a macrolactamization process which secures the required macrocycle.
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| 16083266 |
Extended Range Proteomic Analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of beta-casein and epidermal growth factor receptor (EGFR) |
10.1021/pr050113n. |
J Proteome Res |
Extended Range Proteomic Analysis (ERPA): a new and sensitive LC-MS platform for high sequence coverage of complex proteins with extensive post-translational modifications-comprehensive analysis of beta-casein and epidermal growth factor receptor (EGFR)
Abstract
- We have developed a new and sensitive LC-MS platform, Extended Range Proteomic Analysis (ERPA), which is able to achieve very high sequence coverage and comprehensive characterization of post-translational modifications in complex proteins. This new platform provides advantages of both the top-down and bottom-up proteomic approaches by combining (i) digestion of the protein with an enzyme, such as Lys-C, which cuts less frequently than trypsin, leading to on average a higher molecular weight peptide size, (ii) high-performance LC separation of the resulting fragments, (iii) a new data acquisition strategy using the LTQ-FTMS, a hybrid mass spectrometer that couples a linear ion trap with a Fourier transform ion cyclotron resonance (FTICR) cell, for analysis of peptides in the range of 0.5 to 10 kDa, and (iv) new data analysis methods for assigning large peptide structures and determining the site of attachment of post-translational modifications as well as structural features from the accurate precursor mass together with MS(2) and MS(3) fragmentations. The LC retention of the Lys-C fragments is increased, relative to a tryptic digest, due to the generally greater hydrophobicity of the larger peptides, a result that is particularly important for peptides containing hydrophilic modifications such as glycosylation and phosphorylation. Furthermore, additional positively charged arginine and lysine residues in the Lys-C fragments enhance the sensitivity of the post-translationally modified phospho- and glycopeptides by at least 10-fold relative to tryptic fragments. In typical operation, the FTICR cell provides a survey scan with the high mass resolution (> 100 000) and accurate mass (<2 ppm) to characterize the higher charge-state precursor ions of the larger peptides. In parallel, the linear ion trap provides MS(2) and MS(3) fragmentation spectra, with a scan speed sufficiently fast for on-line LC-MS. Together, these data provide multiple means to determine or enhance the confidence of assignment of large or complicated peptide. Using ERPA, we demonstrate >95% sequence coverage in the analysis of two heavily phosphorylated and glycosylated proteins, beta-casein at the 50 fmole level and the epidermal growth factor receptor (EGFR) at the 1 pmole level. In summary, the combination of digestion strategy, high-performance separation, and the hybrid LTQ-FTMS instrument enables comprehensive characterization of large proteins, including posttranslational modifications.
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| 16094865 |
Enterocin AS-48RJ: a variant of enterocin AS-48 chromosomally encoded by Enterococcus faecium RJ16 isolated from food |
10.1016/j.syapm.2005.01.007. |
Syst Appl Microbiol |
Enterocin AS-48RJ: a variant of enterocin AS-48 chromosomally encoded by Enterococcus faecium RJ16 isolated from food
Abstract
- The bacteriocinogenic strain RJ16 isolated from goat cheese has been identified as Enterococcusfaecium by species-specific PCR, DNA-rRNA hybridization and rDNA sequencing. Purified bacteriocin from strain RJ16 is a carboxypeptidase A-resistant peptide with a molecular mass (7125 Da) very close to the cyclic peptide enterocin AS-48. Bacteriocin from strain RJ16 and AS-48 show identical antibacterial spectra, although the former is slightly less active on strains of Listeria monocytogenes and Bacillus cereus. Producer strains show cross-immunity. PCR amplification of total DNA from strain RJ16 with primers for the AS-48 structural gene and sequencing of the amplified fragment revealed an almost identical sequence (99.5%), except for a single mutation that predicts the change of Glu residue at position 20 of AS-48 to Val. Therefore, bacteriocin produced by E. faecium RJ16 should be considered a variant of AS-48, which we call AS-48RJ. PCR amplification revealed that strain RJ16 contains the complete as-48. gene cluster. Hybridization with probes for as-48 gene cluster revealed a chromosomal location of as-48 genes in strain RJ16, being the first example of a chromosomal location of this bacteriocin trait. Strain RJ16 produced enzymes of interest in food processing (esterase, esterase lipase and phytase activities), and did not decarboxylate amino acids precursors for biogenic amines. Strain RJ16 did not exhibit haemolytic or gelatinase activities, and PCR amplification revealed the lack of genes encoding for known virulence determinants (aggregation substance, collagen adhesin, enterococcal surface protein, endocarditis antigens, as well as haemolysin and gelatinase production). Strain RJ16 was resistant to ciprofloxacin (MIC > 2 mgl(-1)) and levofloxacin (MIC > 4 mgl(-1)) and showed intermediate resistance to nitrofurantoin and erythromycin, but was sensitive to ampicillin, penicillin, streptomycin, gentamicin, rifampicin, chloramphenicol, tetracycline, quinupristin/dalfopristin, vancomycin and teicoplanin. Altogether, results from this study suggest that this broad-spectrum bacteriocin-producing strain may have a potential use in food preservation.
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| 16097433 |
Establishment of an in vitro model using NR8383 cells and Mycobacterium bovis Calmette-Guerin that mimics a chronic infection of Mycobacterium tuberculosis |
None |
In Vivo |
Establishment of an in vitro model using NR8383 cells and Mycobacterium bovis Calmette-Guerin that mimics a chronic infection of Mycobacterium tuberculosis
Abstract
- Mycobacterium tuberculosis infection affects one-third of the world's population and causes the death of three million people each year. To clarify details of M. tuberculosis survival strategies, it is important to establish a suitable in vitro model that mimics a chronic infection in alveolar macrophages by M. tuberculosis. For this reason, we established a new in vitro model using a rat alveolar macrophage cell line, NR8383.
Basic characteristics, including phagocytotic ability and production of nitrogen oxide and tumor necrosis factor in response to several stimuli, of NR8383 cells were compared with those of primary alveolar macrophages. The course after phagocytosis of live or killed M. bovis bacilli Calmette-Guerin (BCG) was examined over 21 days using NR8383 cells as the host.
The characteristics that have been examined to date were nearly the same for both primary alveolar macrophage and NR8383 cells. Live BCG phagocytosed by NR8383 cells had successfully begun to grow in the cells within 7 days, while killed BCG were almost completely destroyed by 21 days.
BCG-infected NR8383 cells are potentially a suitable in vitro model that mimics a chronic infection with M tuberculosis.
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| 16098199 |
Characterization of D-amino-acid-containing excitatory conotoxins and redefinition of the I-conotoxin superfamily |
10.1111/j.1742-4658.2005.04830.x. |
FEBS J |
Characterization of D-amino-acid-containing excitatory conotoxins and redefinition of the I-conotoxin superfamily
Abstract
- Post-translational isomerization of l-amino acids to d-amino acids is a subtle modification, not detectable by standard techniques such as Edman sequencing or MS. Accurate predictions require more sequences of modified polypeptides. A 46-amino-acid-long conotoxin, r11a, belonging to the I-superfamily was previously shown to have a d-Phe residue at position 44. In this report, we characterize two related peptides, r11b and r11c, with d-Phe and d-Leu, respectively, at the homologous position. Electrophysiological tests show that all three peptides induce repetitive activity in frog motor nerve, and epimerization of the single amino acid at the third position from the C-terminus attenuates the potency of r11a and r11b, but not that of r11c. Furthermore, r11c (but neither r11a nor r11b) also acts on skeletal muscle. We identified more cDNA clones encoding conopeptide precursors with Cys patterns similar to r11a/b/c. Although the predicted mature toxins have the same cysteine patterns, they belong to two different gene superfamilies. A potential correlation between the identity of the gene superfamily to which the I-conotoxin belongs and the presence or absence of a d-amino acid in the primary sequence is discussed. The great diversity of I-conopeptide sequences provides a rare opportunity for defining parameters that may be important for this most stealthy of all post-translational modifications. Our results indicate that neither the chemical nature of the side chain nor the precise vicinal sequence around the modified residue seem to be critical, but there may be favored loci for isomerization to a d-amino acid.
|
| 16104770 |
Simple assay for analyzing five microcystins and nodularin in fish muscle tissue: hot water extraction followed by liquid chromatography-tandem mass spectrometry |
10.1021/jf050257m. |
J Agric Food Chem |
Simple assay for analyzing five microcystins and nodularin in fish muscle tissue: hot water extraction followed by liquid chromatography-tandem mass spectrometry
Abstract
- A simple, specific, and sensitive procedure for determining six cyanotoxins, that is, microcystins RR, LR, YR, LA, and LW and nodularin, in fish muscle tissue is presented. This method is based on the matrix solid-phase dispersion technique with heated water as extractant followed by liquid chromatography (LC)-tandem mass spectrometry (MS) equipped with an electrospray ion source. Target compounds were extracted from tissue by 4 mL of water acidified to pH 2 and heated at 80 degrees C. After acidification and filtration, 0.2 mL of the aqueous extract was injected in the LC column. MS data acquisition was performed in the multireaction monitoring mode, with at least two precursor ion > product ion transitions selected for each target compound. Analyte recovery ranged between 61 and 82% and was not substantially affected by either the analyte concentrations or the type of fish. The xcellent recovery of some of the microcystins was traced to binding of these compounds to protein phosphatases in fish tissue occurring during sample treatment. The existence of covalently bound microcystins in fish has been evidenced by several studies. Compared to an older sample preparation procedure, this one extracted larger amounts of the analytes in a simpler and much more rapid way. On the basis of a signal-to-noise ratio of 10, limits of quantification were estimated to range between 1.6 and 4.0 ng/g. The effects of temperature and volume of the extractant on the analyte recovery were studied.
|
| 16109938 |
The heat shock genes dnaK, dnaJ, and grpE are involved in regulation of putisolvin biosynthesis in Pseudomonas putida PCL1445 |
10.1128/JB.187.17.5967-5976.2005. |
J Bacteriol |
The heat shock genes dnaK, dnaJ, and grpE are involved in regulation of putisolvin biosynthesis in Pseudomonas putida PCL1445
Abstract
- Pseudomonas putida PCL1445 produces two cyclic lipopeptides, putisolvins I and II, which possess surfactant activity and play an important role in biofilm formation and degradation. In order to identify genes and traits that are involved in the regulation of putisolvin production of PCL1445, a Tn5luxAB library was generated and mutants were selected for the lack of biosurfactant production using a drop-collapsing assay. Sequence analysis of the Tn5luxAB flanking region of one biosurfactant mutant, strain PCL1627, showed that the transposon had inserted in a dnaK homologue which is located downstream of grpE and upstream of dnaJ. Analysis of putisolvin production and expression studies indicate that dnaK, together with the dnaJ and grpE heat shock genes, takes part in the positive regulation (directly or indirectly) of putisolvin biosynthesis at the transcriptional level. Growth of PCL1445 at low temperature resulted in an increased level of putisolvins, and mutant analyses showed that this requires dnaK and dnaJ but not grpE. In addition, putisolvin biosynthesis of PCL1445 was found to be dependent on the GacA/GacS two-component signaling system. Expression analysis indicated that dnaK is positively regulated by GacA/GacS.
|
| 16116083 |
The Smk1p MAP kinase negatively regulates Gsc2p, a 1,3-beta-glucan synthase, during spore wall morphogenesis in Saccharomyces cerevisiae. |
10.1073/pnas.0502324102 |
Proc. Natl. Acad. Sci. U.S.A. |
The Smk1p MAP kinase negatively regulates Gsc2p, a 1,3-beta-glucan synthase, during spore wall morphogenesis in Saccharomyces cerevisiae.
Abstract
- Spore formation in Saccharomyces cerevisiae involves the sequential deposition of multiple spore wall layers between the prospore membranes that surround each meiotic product. The Smk1p mitogen-activated protein (MAP) kinase plays a critical role in spore formation, but the proteins that interact with Smk1p to regulate spore morphogenesis have not been described. Using mass spectrometry, we identify Gsc2p as a Smk1p-associated protein. Gsc2p is a 1,3-beta-glucan synthase subunit involved in synthesizing an inner spore wall layer. We find that 1,3-beta-glucan synthase activity is elevated in smk1 mutants, suggesting that SMK1 negatively regulates GSC2. Although deposition of the two inner spore wall layers is normal in smk1 mutants, deposition of the outer layers is aberrant. However, eliminating GSC2 activity restores normal deposition of the third spore wall layer in smk1 mutants, indicating that negative regulation of GSC2 by SMK1 is important for spore wall deposition. Our findings suggest a model for the coordination of spore wall layer deposition in which Smk1p facilitates the transition between early and late phases of spore wall deposition by inhibiting a spore wall-synthesizing enzyme important for early phases of spore wall deposition.
|
| 16124786 |
Hirsutide, a cyclic tetrapeptide from a spider-derived entomopathogenic fungus, Hirsutella sp |
10.1021/np0501536. |
J Nat Prod |
Hirsutide, a cyclic tetrapeptide from a spider-derived entomopathogenic fungus, Hirsutella sp
Abstract
- The entomopathogenic fungus Hirsutella sp., isolated from an infected spider, was found to produce the new cyclotetrapeptide hirsutide (1), cyclo-(L-NMe-Phe-L-Phe-L-NMe-Phe-L-Val), along with the known cytochalasin Q (2), using a cytotoxicity-guided isolation procedure. The structure of 1 was elucidated using one- and two-dimensional NMR experiments, mass spectrometry, and Marfey's method for analyzing the configuration of the amino acids.
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