ABSTRACT
Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.
Subject(s)
Aspergillus niger/metabolism , alpha 1-Antitrypsin/metabolism , Amino Acid Sequence , Aspergillus niger/genetics , Biomass , Bioreactors , Carbohydrate Conformation , Carbohydrate Metabolism , Carbohydrates/chemistry , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Glycosylation , Humans , Hydrogen-Ion Concentration , Peptide Mapping , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/metabolism , Polysaccharides/chemistry , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Temperature , alpha 1-Antitrypsin/chemistry , alpha 1-Antitrypsin/genetics , alpha 1-Antitrypsin/isolation & purificationABSTRACT
OBJECTIVES: Assessment of antifungal activity of a compound isolated from the marine sponge Dysidea herbacea against the fungal pathogens Candida (primarily C. albicans) and Aspergillus (primarily A. fumigatus) species, and investigations of the possible mode of activity of the compound. METHODS: Freeze dried sponges were extracted with EtOAc-MeOH. Bioassay guided separation was used to identify the active compound. Antifungal activity was assessed in vitro by a modified NCCLS technique. For determination of the possible mode of activity of the compound we tested the effect on fungal cellular morphology (light, scanning and transmission electron microscopy) and possible site of activity in the fungal cells, such as cell membrane (ion leakage kinetics) as well as toxicity (cytotoxicity tests). RESULTS AND CONCLUSIONS: The active compound was determined to be 3,5-dibromo-2-(3,5-dibromo-2-methoxyphenoxy) phenol. This compound exhibited in vitro activity against the tested fungal pathogens. The experiments on the mode of activity revealed that there are significant changes in fungal cell morphology, as demonstrated by scanning and transmission electron microscopy. The compound, apparently, affects the fungal cell membrane, expressed primarily in leakage of potassium ions from the fungal cells. Two other bromo diphenyl ethers were also found to be active. Further experiments in in vivo models are planned.
Subject(s)
Aspergillus/drug effects , Candida albicans/drug effects , Marine Biology , Phenols/pharmacology , Porifera/chemistry , Tissue Extracts/pharmacology , Animals , Aspergillus/ultrastructure , Candida albicans/ultrastructure , Microscopy, Electron, Scanning , Microscopy, Electron, Scanning Transmission , Tissue Extracts/chemistry , Tissue Extracts/isolation & purificationABSTRACT
Six new eunicellin diterpenes designated klyxumines A and B (1, 2) and epoxycladines A-D (3-6) were isolated from Klyxum flaccidum and Cladiella kashmani, collected in Kenya. The structures of the compounds were elucidated by interpretation of MS, COSY, HMQC, HMBC, and NOESY data.
Subject(s)
Anthozoa/chemistry , Diterpenes/chemistry , Diterpenes/isolation & purification , Animals , Kenya , Molecular StructureABSTRACT
Three novel compounds, designated kitungolides A (1), B (2), and C (3), were isolated from a soft coral of a new genus collected at Kitungamwe, Kenya. The three new compounds are of a unique heterotricyclic skeleton. The structures and relative stereochemistry of the compounds were elucidated by interpretation of MS, COSY, HMQC, HMBC, and NOESY experiments. [structure: see text]
Subject(s)
Anthozoa/chemistry , Diterpenes/isolation & purification , Pyrones/isolation & purification , Animals , Anthozoa/classification , Molecular ConformationABSTRACT
[structure: see text] Two novel compounds, barrenazine A (1) and B (2), were isolated from an unidentified tunicate collected at Barren Islands, Madagascar. The two new compounds are of an unprecedented heterocyclic skeleton, namely 1,3,4,6,8,9-hexahydrodipyridino[3,4-b:3',4'-e]pyrazine. The structures of the two alkaloids were elucidated by interpretation of MS, COSY, HMQC, HMBC, NOESY, and (15)N-HMBC data. Barrenazine A exhibits mild cytotoxicity against LOVO-DOX colon carcinoma (with a GI(50) value of 0.9 g/mL)
Subject(s)
Antineoplastic Agents/isolation & purification , Urochordata/chemistry , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Drug Screening Assays, Antitumor , Tumor Cells, CulturedABSTRACT
Two novel cyclic hexapeptides, didmolamides A and B, were isolated from the compound ascidian Didemnum molle collected in Madagascar. The structure of the two peptides was elucidated by interpretation of MS, COSY, HMQC, and HMBC data. The absolute configuration of all amino acids was determined to be l using Marfey's method for HPLC.
Subject(s)
Amino Acids/chemistry , Peptides, Cyclic/isolation & purification , Urochordata/chemistry , Animals , Chromatography, High Pressure Liquid , Drug Screening Assays, Antitumor , Humans , Inhibitory Concentration 50 , Madagascar , Mass Spectrometry , Molecular Structure , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Tumor Cells, Cultured/drug effectsABSTRACT
Halichondramine (1), a new tetracyclic alkylbipiperidine alkaloid, has been isolated from the marine sponge Halichondria sp., collected in the Dahlak archipelago (the Red Sea), Eritrea. The structure of halichondramine was elucidated by interpretation of MS, COSY, HMQC, HMBC, TOCSY, and HSQC-TOCSY data.
