ABSTRACT
We have isolated four related compounds named phepropeptins A, B, C, and D, as inhibitors of proteasome proposed to regulate many cellular functions. From an NMR analysis, the phepropeptins appeared as cyclic hexapeptides, differing in the two residues of the constituent amino acids from one another, with four conserved amino acid moieties. Based on an amino acid analysis, we synthesized two possible cyclic peptides to phepropeptin B that differ in the configurations. A comparison of the properties between the natural and synthesized compounds revealed that the structure of phepropeptin B was cyclo(-L-Leu-D-Phe-L-Pro-L-Phe-D-Leu-L-Val-). The phepropeptins showed inhibition to the proteasomal chymotrypsin-like activity but not to alpha-chymotrypsin.
Subject(s)
Anti-Bacterial Agents/isolation & purification , Enzyme Inhibitors/isolation & purification , Multienzyme Complexes/antagonists & inhibitors , Peptides, Cyclic/isolation & purification , Streptomyces/metabolism , Amino Acids/analysis , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Cysteine Endopeptidases , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Fermentation , Fluorometry , Gas Chromatography-Mass Spectrometry , Magnetic Resonance Spectroscopy , Optical Rotation , Peptide Hydrolases/analysis , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/chemistry , Proteasome Endopeptidase Complex , Stereoisomerism , Streptomyces/chemistryABSTRACT
The structures of tyropeptins A and B, new proteasome inhibitors produced by Kitasatospora sp. MK993-dF2, were determined by analysis of various NMR experiments. The 1H and 13C NMR of tyropeptins were complicated due to the presence of an aldehyde group. Therefore, tyropeptins were converted to their alcohols by sodium borohydride. These alcohol derivatives gave assignable NMR spectra. The stereochemistry of tyropeptins were determined by analysis of acid hydrolysis products from tyropeptins, and further confirmed by the total synthesis. The structures of tyropeptins A and B were found to be isovaleryl-L-tyrosyl-L-valyl-DL-tyrosinal and n-butyryl-L-tyrosyl-L-leucyl-DL-tyrosinal, respectively.
Subject(s)
Dipeptides/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Cysteine Endopeptidases , Dipeptides/chemistry , Enzyme Inhibitors/chemistry , Magnetic Resonance Spectroscopy , Multienzyme Complexes/antagonists & inhibitors , Proteasome Endopeptidase Complex , StereoisomerismABSTRACT
Tyropeptins A and B, new proteasome inhibitors, were isolated from the culture broth of Kitasatospora sp. MK993-dF2. They were purified using ethyl acetate extraction, silica gel column chromatography, Sephadex LH-20 column chromatography and HPLC. Tyropeptin A inhibited the chymotrypsin-like (ChT-L) and trypsin-like (T-L) activities of 20S proteasome with IC50 values of 0.1 microg/ml and 1.5 microg/ml respectively, but did not inhibit the peptidylglutamyl-peptide hydrolyzing (PGPH) activity of 20S proteasome at a concentration of 100 microg/ml. The inhibitory activities of tyropeptin A were about two times as strong as that of tyropeptin B. Taxonomy of the producing strain is also described.
Subject(s)
Dipeptides/isolation & purification , Enzyme Inhibitors/isolation & purification , Multienzyme Complexes/antagonists & inhibitors , Animals , Cysteine Endopeptidases , Dipeptides/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Female , Mice , Mice, Inbred ICR , Proteasome Endopeptidase ComplexABSTRACT
The roles of PKC in iNOS induction by IFN-gamma have been shown in some cell types. The effect of a PKC activator, phorbol ester, in iNOS induction is thought to be due to multiple mechanisms, and it is necessary to examine the involvement of phorbol ester on IFN-gamma-induced iNOS in detail. In the present study, we investigated the mechanisms of phorbol ester on IFN-gamma-induced iNOS in RAW 264.7 cells. PMA synergistically increased iNOS activity, protein and mRNA levels in IFN-gamma-treated RAW 264.7 cells. PMA together with IFN-gamma increased iNOS mRNA without affecting the iNOS mRNA degradation, suggesting that the synergistic effect of PMA on IFN-gamma-induced iNOS mRNA production may depend on the elevation of the transcription rate rather than a prolongation of mRNA stability. The DNA binding proteins that are involved in the regulation of iNOS expression are mainly NF-kappa B and IRF-1. IRF-1 transcriptionally regulates many IFN-inducible genes such as iNOS whose promoter contains an IRF-1 binding site. PMA might modulate iNOS induction as a cosignal with IFN-gamma in RAW 264.7 cells because the synergistic effect of PMA was mediated through IRF-1, rather than NF-kappa B. Ro 31-8220, a PKC inhibitor, decreased iNOS activity, protein, mRNA levels and IRF-1 activity, indicating that the effect of PMA on iNOS induction might occur via the PKC pathway. It is evidence that PKC plays an important role in IRF-1 activation and that phorbol ester has a synergistic effect on iNOS induction through IRF-1 activation in IFN-gamma-treated RAW 264.7 cells. The synergistic effect of PMA on IFN-gamma-induced IRF-1 binding activity was observed in macrophage cell line J774 cells as well as RAW 264.7 cells, but not in thioglycollate-elicited peritoneal macrophages.
Subject(s)
DNA-Binding Proteins/biosynthesis , Interferon-gamma/pharmacology , Nitric Oxide Synthase/biosynthesis , Phorbol Esters/pharmacology , Phosphoproteins/biosynthesis , Animals , Cell Line , Drug Synergism , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Interferon Regulatory Factor-1 , Mice , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type II , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , RNA, Messenger/analysisSubject(s)
Agaricales/metabolism , Antinematodal Agents/pharmacology , Caenorhabditis elegans/drug effects , Fumarates/pharmacology , Heterocyclic Compounds, 2-Ring/pharmacology , Agaricales/growth & development , Animals , Antinematodal Agents/chemistry , Antinematodal Agents/isolation & purification , Antinematodal Agents/metabolism , Fumarates/chemistry , Fumarates/isolation & purification , Fumarates/metabolism , Heterocyclic Compounds, 2-Ring/chemistry , Heterocyclic Compounds, 2-Ring/isolation & purification , Heterocyclic Compounds, 2-Ring/metabolism , Magnetic Resonance Spectroscopy , Methacrylates , Molecular StructureABSTRACT
Three new sesquiterpenoid aromatic esters designated melleolides K (1), L (2) and M (3) were isolated from the cultured mycelia of Armillariella mellea (Vahl. ex Fr.) Karst. Structures of these compounds were determined on the basis of various NMR spectral data, chemical transformations and X-ray analysis. Compounds 1, 2 and 3 showed antimicrobial activities.
Subject(s)
Agaricales/chemistry , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Sesquiterpenes/chemistry , Sesquiterpenes/pharmacology , Animals , Anti-Bacterial Agents , Anti-Infective Agents/isolation & purification , Crystallography, X-Ray , Fungi/drug effects , Gram-Positive Bacteria/drug effects , Lethal Dose 50 , Magnetic Resonance Spectroscopy , Mice , Microbial Sensitivity Tests , Molecular Structure , Sesquiterpenes/isolation & purification , Toxicity TestsABSTRACT
Ubiquitin synergistically augmented the production of tumor necrosis factor alpha (TNF-alpha) in the presence of lipopolysaccharide (LPS) in murine macrophage cell line RAW 264.7. To investigate the mechanism of this augmentation, we analyzed the effect of ubiquitin during TNF-alpha mRNA synthesis and degradation, and TNF-alpha degradation on RAW 264.7 cells stimulated by LPS. It is found that ubiquitin augmented TNF-alpha mRNA synthesis. Ubiquitin did not affect the degradation of TNF-alpha mRNA and TNF-alpha. In the presence of LPS, extracellular accumulation of TNF-alpha by ubiquitin was twice than those by LPS, but intracellular accumulation of TNF-alpha produced by ubiquitin with LPS or by LPS had no difference. These data indicate that ubiquitin might induce TNF-alpha accumulation mainly by up-regulation of the TNF-alpha gene transcription. Although extracellular functions of ubiquitin remain largely unknown, we postulate that ubiquitin might be involved in the modulatory mechanisms of immune response.
Subject(s)
Macrophages/drug effects , Tumor Necrosis Factor-alpha/biosynthesis , Ubiquitins/pharmacology , Animals , Cell Line , Dose-Response Relationship, Drug , Drug Synergism , Lipopolysaccharides/pharmacology , Macrophage Activation , Macrophages/metabolism , Mice , RNA, Messenger/biosynthesis , Tumor Necrosis Factor-alpha/geneticsABSTRACT
The structures of decatromicins A and B that strongly inhibit the growth of MRSA were elucidated by the analysis of various NMR experiments. The planar structure was determined by 1H, 13C, COSY, HMQC and HMBC NMR spectra. The relative configuration of aglycone was elucidated by NOESY experiments and the absolute structure was determined by application of the modified Mosher's method. The absolute structure of glycosyl moiety was determined by X-ray analysis of the O-(p-bromobenzoyl) derivative.
Subject(s)
Actinomycetales/metabolism , Anti-Bacterial Agents/chemistry , Magnetic Resonance SpectroscopyABSTRACT
New antibiotics designated decatromicins A and B were isolated from the culture broth of Actinomadura sp. MK73-NF4. They were purified by butyl acetate extraction, silica gel column chromatography, silica gel TLC and Sephadex LH-20 column chromatography. Decatromicins A and B inhibited growth of Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA).
Subject(s)
Actinomycetales/classification , Anti-Bacterial Agents/isolation & purification , Macrolides/isolation & purification , Actinomycetales/metabolism , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Macrolides/chemistry , Macrolides/pharmacology , MiceABSTRACT
A new antibiotic designated polyketomycin was isolated from the culture broth of Streptomyces sp. MK277-AF1. It was purified by ethyl acetate extraction, Sephadex LH-20 column chromatography and centrifugal partition chromatography (CPC). It inhibited growth of Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). Its MICs were less than 0.2 microgram/ml. Polyketomycin exhibited cytotoxic activity against nine tumor cell lines at concentrations of 0.9-5.2 micrograms/ml.
Subject(s)
Anti-Bacterial Agents , Streptomyces/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/pharmacology , Glyoxylates/chemistry , Glyoxylates/isolation & purification , Glyoxylates/pharmacology , Methicillin Resistance , Microbial Sensitivity Tests , Staphylococcus aureus/drug effects , Streptomyces/classificationABSTRACT
A new antibiotic, polyketomycin, was isolated from the culture broth of Streptomyces sp. MK277-AF1. The structure was determined by various NMR spectroscopies, X-ray crystallographic analysis and degradation experiments.
Subject(s)
Anti-Bacterial Agents/chemistry , Glyoxylates/chemistry , Streptomyces/chemistry , Magnetic Resonance SpectroscopyABSTRACT
A new antibiotic designated nothramicin was isolated from the culture broth of Nocardia sp. MJ896-43F17 which was closely related to Nocardia brasiliensis. It was isolated by the Diaion HP-20 column chromatography, butyl acetate extraction and purified by HPLC. It inhibited the growth of mycobacteria at the concentration of 1.56 approximately 25 microg/ml. Nothramicin was revealed to be a new member of anthracycline antibiotics by the various spectroscopies.
Subject(s)
Anthracyclines , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/isolation & purification , Animals , Antibiotics, Antineoplastic/pharmacology , Lethal Dose 50 , Mice , Microbial Sensitivity Tests , Molecular Structure , NocardiaABSTRACT
Antibacterial antibiotics, diperamycin (1) was produced in the culture broth of Streptomyces griseoaurantiacus MK393-AF2. Various spectroscopic analyses of 1 suggested that 1 belonged to a member of cyclic hexadepsipeptide antibiotic. Antibiotic 1 had potent inhibitory activity against various Gram-positive bacteria including Enterococcus seriolicida and methicillin-resistant Staphylococcus aureus.
Subject(s)
Anti-Bacterial Agents/pharmacology , Peptides , Streptomyces/metabolism , Animals , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/chemistry , Antibiotics, Antineoplastic/biosynthesis , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacology , Antimicrobial Cationic Peptides , Chemical Phenomena , Chemistry, Physical , Drug Screening Assays, Antitumor , Enterococcus/drug effects , Fermentation , Gram-Positive Bacteria/drug effects , Humans , Magnetic Resonance Spectroscopy , Methicillin Resistance , Mice , Mice, Inbred ICR , Microbial Sensitivity Tests , Microscopy, Electron, Scanning , Spectrometry, Mass, Fast Atom Bombardment , Staphylococcus aureus/drug effects , Streptomyces/classification , Streptomyces/ultrastructure , Tumor Cells, CulturedABSTRACT
Vegetable oils were investigated to evaluate their potential to act as the sole carbon source for production of cephamycin C in shake and jar-fermentor cultures. Soybean oil was the best carbon source for cephamycin C production. Bioautography and HPLC analyses showed that cephamycin C was exclusively produced even when soybean oil was used as the sole carbon source. The optimal pH and initial concentration of soybean oil was 7.5 and 7 g/l, respectively. Both pH and the pH-control agent affected cephamycin C production, and among phosphoric acid, acetic acid and sulfuric acid, phosphoric acid was associated with the best production. Soybean oil was slowly consumed after the soluble nitrogen source was consumed. When the initial soybean oil concentration was 7 g/l, cephamycin C production was maximal, 2.0 g/l, which was twice as high as that from starch. The product yield from soybean oil was 4.7 times higher than that from starch. These results show that vegetable oils, which are cheaper than other carbon sources, could be used as the sole carbon source in the production of antibiotics.
