ABSTRACT
Four tachykinin (NK2) receptor inhibitors, SCH 378161 (1), SCH 217048 (2), SCH 378199 (3), and SCH 378167 (4) were isolated from the fermentation broth of a taxonomically unidentified fungus. These compounds were separated from the fermentation broth by ethyl acetate extraction. Purification and separation of the individual compounds were achieved by NK2 assay-guided fractionation using gel filtration, reverse phase chromatography and HPLC. They were identified to be a family of depsipeptides by spectroscopic and degradation studies. Compounds 1 and 3 contain proline and differ as an amide and acid whereas 2 and 4 contain pipecolic acid and differ in being an amide and acid. All of these compounds contain an identical hydroxy acid. They are selective NK2 inhibitors with Ki values ranging from 27-982 nM and demonstrate no activity at 10 microM in the NK1 and NK3 assays. In addition, compounds 1 and 2 inhibited NKA-induced increases in the concentration of intracellular Ca2+, [Ca2+]i, in a CHO cell expressing the human NK2 receptor; this inhibition was competitive in nature with pA2 values of 7.2 and 7.5, respectively. These data demonstrate that these natural products are selective and competitive receptor antagonists of the human NK2 receptor.
Subject(s)
Depsipeptides , Fungi/metabolism , Oligopeptides , Receptors, Neurokinin-2/antagonists & inhibitors , Animals , CHO Cells , Chromatography, Gel , Chromatography, Ion Exchange , Cricetinae , Fermentation , Humans , Molecular Structure , Oligopeptides/biosynthesis , Oligopeptides/chemistry , Oligopeptides/isolation & purification , Oligopeptides/pharmacology , Peptides, Cyclic/biosynthesis , Peptides, Cyclic/chemistry , Peptides, Cyclic/isolation & purification , Peptides, Cyclic/pharmacology , Radioligand Assay , Receptors, Neurokinin-2/metabolism , Spectrum AnalysisABSTRACT
A gene expression system using recombinant Autographa california nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external addition of air/O2 mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks, Sf-9 cells were adapted to grow to high cell densities (6-10 x 10(6) cells ml-1) in shake flasks in serum-free media. With these procedures, cell densities of 5 x 10(6) cells ml-1 were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the 247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell-1, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography with reproducible yields of 11-38 mg L-1 of recombinant protein and > or = 90% purity. Maximum production of this protein was observed at a cell density of 5.0 x 10(6) cells ml-1.
Subject(s)
Endopeptidases/biosynthesis , Recombinant Fusion Proteins/biosynthesis , Simplexvirus/enzymology , Viral Proteins/biosynthesis , Animals , Baculoviridae/genetics , SpodopteraABSTRACT
A novel antifungal agent, Sch 54445, was isolated from the fermentation broth of an Actinoplanes species. Sch 54445 was identified as a polycyclic xanthone related to the albofungin family of compounds on the basis of analyses of spectroscopic data. As a broad-spectrum antifungal agent, Sch 54445 exhibits highly potent activities against various yeasts and dermatophytes with MIC values approximately 0.00038 microgram/mL.
Subject(s)
Actinomycetales/metabolism , Antifungal Agents/pharmacology , Xanthenes/pharmacology , Antifungal Agents/biosynthesis , Antifungal Agents/isolation & purification , Carbohydrate Sequence , Fermentation , Fungi/drug effects , Magnetic Resonance Spectroscopy , Mass Spectrometry , Microbial Sensitivity Tests , Molecular Conformation , Molecular Sequence Data , Xanthenes/isolation & purification , Xanthenes/metabolismABSTRACT
Seven neurokinin (NK) receptor inhibitors SCH 60059 (1), SCH 60065 (2), SCH 64879 (3), SCH 60061 (4), SCH 60063 (5), SCH 60057 (7), and SCH 64878 (9) and two uncharacterized components 6 and 8, were isolated from the fermentation broth of a fungus taxonomically classified as an Acremonium sp. These compounds were isolated from the fermentation broth by ethyl acetate extraction. Purification and separation of the individual compounds were achieved by NK1 and NK2 assay-guided fractionation using gel filtration, reverse phase chromatography and HPLC. The NK active compounds were identified to be a family of polyhydroxy isoprenoid derivatives, including glycosylated members, by spectroscopic and degradation studies. Compounds 1 approximately 5 and 7 contain nine isoprene units connected in head to tail fashion and compound 9 contains fifteen isoprene units connected in a similar manner. All these compounds showed dual inhibition in NK1 and NK2 assays with IC50 values ranging from 2.5 approximately 11 microM in the NK1 assay and 6.8 approximately 16 microM in the NK2 assay.
Subject(s)
Acremonium/chemistry , Butadienes/chemistry , Butadienes/isolation & purification , Hemiterpenes , Neurokinin-1 Receptor Antagonists , Pentanes , Receptors, Neurokinin-2/antagonists & inhibitors , Acremonium/growth & development , Butadienes/pharmacology , Culture Media , Fermentation , Hydrolysis , Receptors, Neurokinin-1/drug effects , Receptors, Neurokinin-2/drug effectsSubject(s)
Antifungal Agents/isolation & purification , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Candida albicans/drug effects , Diterpenes/chemistry , Diterpenes/isolation & purification , Diterpenes/pharmacology , Fermentation , Glycosides/chemistry , Glycosides/isolation & purification , Glycosides/pharmacology , Molecular StructureABSTRACT
The response of Clostridium acetobutylicum ATCC 4259 to the stresses produced by a temperature upshift from 28 degrees C to 45 degrees C and by exposure of the organisms to 0.1% n-butanol or to air was examined by analysis of pulse-labeled proteins. The stress response was the induction of the synthesis of a number of proteins, some of which were elicited by the three forms of stress. Eleven heat shock proteins were identified by two-dimensional electrophoresis, as were two proteins whose synthesis was heat sensitive. In the absence of applied stress, the synthesis of four proteins was found to be associated with the growth phase in batch culture; three of these proteins had a higher rate of de novo synthesis when the cells entered the solvent production phase. One of the stress-induced proteins, hsp74, was partially purified an found to be immunologically related to Escherichia coli heat shock protein Dnak. The similarities of the proteins induced at the onset of solventogenesis and by stress suggest a relationship between the two processes.
ABSTRACT
H conductance in de-energized cells of Clostridium thermoaceticum and Clostridium acetobutylicum was determined from the rate of realkalinization of the medium after an acid pulse. In both organisms, cell membrane proton permeability was increased by fermentation end products and ionophores. In C. thermoaceticum, H conductance was increased by Na ions compared with K as counterions. In these cells, addition of Na, but not K, elicited efflux of H; H efflux was stimulated by SCN and decreased by various ionophores. We concluded that C. thermoaceticum possesses an electrogenic Na/H antiporter. In contrast, C. acetobutylicum cells did not have an electrogenic Na/H antiporter.
ABSTRACT
We investigated the intracellular physiological conditions associated with the induction of butanol-producing enzymes in Clostridium acetobutylicum. During the acidogenic phase of growth, the internal pH decreased in parallel with the decrease in the external pH, but the internal pH did not go below 5.5 throughout batch growth. Butanol was found to dissipate the proton motive force of fermenting C. acetobutylicum cells by decreasing the transmembrane pH gradient, whereas the membrane potential was affected only slightly. In growing cells, the switch from acid to solvent production occurred when the internal undissociated butyric acid concentration reached 13 mM and the total intracellular undissociated acid concentration (acetic plus butyric acids) was at least 40 to 45 mM. Similar values were obtained when cultures were supplemented with 50 mM butyric acid initially or when a phosphate-buffered medium was used instead of an acetate-buffered medium. To measure the induction of the enzymes involved in solvent synthesis, we determined the rates of conversion of butyrate to butanol in growing cells. The rate of butanol formation reached a maximum in the mid-solvent phase, when the butanol concentration was 50 mM. Although more solvent accumulated later, de novo enzyme synthesis decreased and then ceased.
ABSTRACT
Spirochaeta aurantia M1 cells were grown in a chemostat under conditions of energy and carbon source limitation. The chemotactic responses of the chemostat-grown cells were compared with those of S. aurantia cells grown in batch culture in the presence of excess energy and carbon source. Chemotactic responses were measured by determining the number of cells that entered a capillary tube containing a solution of attractant. S. aurantia cells grown in the chemostat under energy and carbon source limitation exhibited enhanced chemotactic responses and detected lower concentrations of attractant, as compared with cells grown in batch culture. The chemotactic response toward an attractant was specifically enhanced when that attractant was the growth-limiting energy and carbon source. The medium used contained either D-glucose or D-xylose as the sole energy and carbon source. Cells had the greatest chemotactic response toward glucose when grown at a dilution rate (D) of 0.045 h-1 under glucose limitation and toward xylose when grown at D = 0.06 h-1 under xylose limitation. When cells were grown under glucose limitation (D = 0.045 h-1), they sensed concentrations of attractant (glucose) ca. 1,000 times lower than those sensed by batch-grown cells. A similar enhancement of sensing ability (toward xylose) was observed in cells grown under xylose limitation. The results indicated that S. aurantia cells are able to regulate their chemosensory system in response to nutrient limitation. Maximum enhancement of chemotaxis occurs in cells growing at very low concentrations of energy and carbon source. Most likely, this property provides the spirochetes with competitive advantages when the availability of nutrients becomes severely limited in their habitats.