Biomedical subjects
Publications and source records attributed to A Zeeck.
Five new piperidine alkaloids (1-5), named streptazones A, B(1), B(2), C, and D, were isolated from Streptomyces strains FORM5 and A1. Their structures were established on the basis of detailed MS and NMR analysis. Streptazone A (1) exhibited significant cytotoxic activity against selected human tumor cell lines.
The studies presented were designed to test the efficacy of farnesyltransferase inhibitors (FTIs) as potential chemopreventive compounds in the mouse lung tumor model, and in tumor cell lines. The compounds included manumycin, gliotoxin, dihydroepiandrosterone (DHEA), perillyl alcohol (POH), and FTI-276. Each of these compounds had the potential, based on in vitro and limited in vivo evidence, to inhibit mouse lung tumorigenesis. In vitro studies were conducted with both K-ras-transformed NIH-3T3 cells and mouse lung tumor epithelial cell lines. We utilized 2 primary mouse lung tumor models that reliably produce lung tumors with an oncogenic K-ras mutation when induded by 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone (NNK). Manumycin, gliotoxin, DHEA, and POH were administered 3 times per week peritoneally (i.p.), starting 1 week prior to carcinogen treatment, and throughout the test period (4.5 months). FTI-276 was delivered daily for 4 months by a time-release pellet method. Both the manumycin and gliotoxin treatment groups demonstrated 100% incidence and an increase in tumor multiplicity over control, of 66% and 58% increase respectively (P < .05). Although DHEA showed no significant chemopreventive effect, POH treatment demonstrated a 22% reduction in tumor incidence (P < .05) and a 58% reduction in tumor multiplicity (P < .05). Finally, FTI-276 reduced both the tumor multiplicity by 41.7% (P < .005), and the total tumor volume/burden per mouse by 79.4% (P < .0001). The apoptotic index in FTI-276-treated tumors showed an increase of 77% over control tumors (P < .05). In vitro, all compounds demonstrated growth inhibition at a dose-response manner; however, manumycin, gliotoxin, and DHEA demonstrated an initial increase in growth rate at lower doses. In summary, we have shown that POH and FTI-276 are chemopreventive in a primary mouse lung tumor model. In contrast, DHEA was not significantly chemopreventive at the dosage utilized, and treatment of an immunocompetent host with manumycin or gliotoxin demonstrated a significant increase in tumorigenicity over carcinogen control.
Streptomyces coelicolor 1A and Pseudomonas citronellolis were able to degrade synthetic high-molecular-weight poly(cis-1,4-isoprene) and vulcanized natural rubber. Growth on the polymers was poor but significantly greater than that of the nondegrading strain Streptomyces lividans 1326 (control). Measurement of the molecular weight distribution of the polymer before and after degradation showed a time-dependent increase in low-molecular-weight polymer molecules for S. coelicolor 1A and P. citronellolis, whereas the molecular weight distribution for the control (S. lividans 1326) remained almost constant. Three degradation products were isolated from the culture fluid of S. coelicolor 1A grown on vulcanized rubber and were identified as (6Z)-2,6-dimethyl-10-oxo-undec-6-enoic acid, (5Z)-6-methyl-undec-5-ene-2,9-dione, and (5Z,9Z)-6, 10-dimethyl-pentadec-5,9-diene-2,13-dione. An oxidative pathway from poly(cis-1,4-isoprene) to methyl-branched diketones is proposed. It includes (i) oxidation of an aldehyde intermediate to a carboxylic acid, (ii) one cycle of beta-oxidation, (iii) oxidation of the conjugated double bond resulting in a beta-keto acid, and (iv) decarboxylation.
The biosynthesis of cladospirone bisepoxide (1) was investigated by feeding 13C-labeled acetate to growing cultures of the fungus Sphaeropsidales sp. (strain F-24'707). 13C NMR spectral analysis demonstrated the polyketide origin of both naphthalene units. The origin of two epoxide oxygens was confirmed as from air by cultivation of the strain in an 18O2-enriched atmosphere. The [18O]incorporation pattern into palmarumycin C12 (11), the putative precursor of 1 led to the hypothesis that the carbonyl oxygen of 1 is derived from water by exchange of an oxygen atom. Inhibition of the biosynthesis of 1 with tricyclazole, an inhibitor of the 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis, confirmed the connection of both biosynthetic pathways.
Four new polyketides, named rubiginone D2 (2), 4-O-acetyl-rubiginone D2 (3), rubiginone H (6) and rubiginone I (7) were isolated from the cultures of Streptomyces sp. (strain Gö N1/5). Their structures were established by a detailed spectroscopic analysis. The absolute configuration of 3 was determined by derivatization with chiral acids (Helmchen's method). The rubiginones inhibit the growth of some Gram-positive bacteria and are cytostatically active against different tumor cell lines.
Two novel angucyclinone-type antibiotics, simocyclinones D4 and D8, were detected in the mycelium extract of Streptomyces antibioticus Tü 6040 by HPLC-diode-array and HPLC-electrospray-mass-spectrometry screening. The compounds show antibiotic activities against Gram-positive bacteria and cytostatic effects on various tumor cell lines.
The antifungal and antibacterial properties of the crude extract from Streptomyces sp. (strain Gö 40/10) encouraged us to perform a detailed analysis of its secondary metabolite pattern by chemical screening, which revealed the presence of at least 30 different compounds. Ten of the isolated 18 metabolites proved to be new. Remarkable are hydrogenated (3, 4) and glucosylated (5, 6, 7) 14-membered macrolides derived from cineromycin B, two gamma-butyrolactones (8, 9), the so far unknown naphthomycin K (14) and the collinolactones A and B. The constitution and relative stereochemistry of these metabolites were deduced from spectroscopic data. Finally the concept of our one strain/many compounds (OSMAC) method for exploring new microbial secondary metabolites is discussed.
The biosynthesis of polyketomycin was investigated by feeding 13C-labeled acetate and propionate to the growing cultures of Streptomyces diastatochromogenes Tü 6028. 13C NMR spectral analysis demonstrated the polyketide origin of the aglycone and the dimethylsalicyloyl moieties. The O-methyl group and 6-CH3 of the aglycone as well as 3B-CH3 of L-axenose and 3C-CH3 of the salicyloyl residue were labeled by feeding L-[methyl-13C]methionine. Both deoxysugars emerged from D-glucose. The biosynthesis of the aglycone and the assembly of the glycoside are discussed. The polyketomycin producing strain may be a candidate for further exploration in combinatorial biosynthesis.
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The kdpFABC operon of Escherichia coli consists of the four structural genes kdpF, kdpA, kdpB, and kdpC. Expression of the kdpF gene was demonstrated using minicells of E. coli. In addition, it was shown that the KdpF subunit remains associated with the purified complex. Although KdpF is not essential in vivo, the purified complex lacking KdpF exhibits hardly any K(+)-stimulated ATPase activity. This clearly demonstrates that the KdpF subunit is stabilizing the transport complex. Charge translocation by the purified Kdp-ATPase was measured with the potential-sensitive dye DiSC3(5) using proteoliposomes. Upon addition of ATP a fluorescence quench was observed indicating the buildup of a negative potential inside the proteoliposomes. Using the Kdp-ATPase derived from a mutant strain, in which the K(m) value for K+ (1,2 mM) was almost identical to that of Rb+ (1.4 mM), the same fluorescence quench was observed when K+ or Rb+ were present in the lumen of the proteoliposomes. These data clearly indicate that the Kdp-ATPase transports K+ in an electrogenic manner. In order to identify the binding site(s) for the inhibitor concanamycin A within the Kdp complex, concanamycin A was synthesized. Using this compound labeling of KdpA and KdpB, but not of KdpC, could be shown with the purified complex. When everted vesicles were used only KdpB could be labeled.
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Chemical screening using thin layer chromatography and various staining reagents offers the opportunity to visualize a nearly complete picture of a microbial secondary metabolite pattern (metabolic finger-print). This approach can be used advantageously for both, the detection of so-called "talented" strains, and for qualifying microbial strain collections, especially as a fundamental step of efficiently applied biological high-throughput assays. Based on their metabolic finger-print, microbial isolates can be classified in: (i) non-producing organisms, which gave no indication of the formation of secondary metabolites up to a defined detection limit, (ii) organisms of narrow productivity, which produce one or two secondary metabolites as main products with a restricted dependence to alteration of the culture conditions, and (iii) talented organisms, which are able to synthesize an array of structurally different secondary metabolites. As an example, the talented strain, Streptomyces griseoviridis (FH-S 1832), was studied in detail. Investigations in its taxonomical characterization, fermentation, as well as the isolation and purification procedures leading to 14-membered macrocyclic lactones of the cineromycin-type (cineromycin B and three new congeners) and to the musacins A to F are reported. Musacin C exhibits anthelminthic and weak antiviral activities.
A detailed analysis of the secondary metabolite pattern produced by Streptomyces griseoviridis (strain FH-S 1832) using a chemical screening method resulted in the detection, isolation and structure elucidation of new 14-membered lactones of the cineromycin B-type [dehydrocineromycin B (5), oxycineromycin B (7), and 2,3-dihydrocineromycin B (8)], as well as new gamma-lactones related to nigrosporalactone and 4,5-dihydroxy-octa-2,6-dienoic acid esters named musacins A to F (10, 13 approximately 15, 17, 18 and 21). The constitution of these metabolites were deduced from spectroscopic data as well as chemical transformations. The configuration of musacin D (10) was determined by derivatization with chiral acids (Helmchen's method).
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The influence of increased dissolved O2 concentrations (DOC) on cell growth and production of the secondary metabolite manumycin by a strain of Streptomyces parvulus (Tü 64) was investigated in a stirred tank fermentor. DOC is given as the O2 partial pressure (po2) in the gas phase in an equilibrium state with the liquid phase. Growth of S. parvulus was not influenced up to DOC equivalent to po2 = 1260 mbar. At po2 = 2205 mbar the maximum biomass concentration was lowered by 40%. Production of manumycin was markedly influenced by DOC and reached the maximal concentration at po2 = 315 mbar. At increased DOC three new metabolites were observed. Two of them, 64p-A and 64p-B, were identified as carboxamides, which represent the branched side chain of the manumycin molecule and a derivative with a shorter chain length. The third metabolite, 64p-C, was a manumycin derivative containing an aromatic ring system. Feeding of glycerol during the production phase increased the total yield and showed a similar effect of DOC. Since DOC has significant regulation effects on product formation and selectivity, it should be used as a major parameter in development strategies of aerobic microbial processes.