Biotransformation of germacrane-type sesquiterpenoids by Aspergillus niger

Three germacrane-type sesquiterpenoids, (+)-germacrone-4,5-epoxide, germacrone and (+)-curdione were biotransformed by Aspergillus niger to give hydroxylated guaiane-type sesquiterpenoids together with allylic alcohols and spirolactone.     

Analysis of the mineralocorticoid receptor in rat heart with the aid of two new spirolactone derivatives

Two derivatives of spirolactone, synthesized in an effort to eliminate the obnoxious side effects of the native molecule, were employed to dissect various aspects of the MR structure and function in rat heart. The introduction of a propyl residue in position 7 of spirolactone produced a molecule (RU 26752) that exhibited an increased affinity for the agonist specific MR, and furthermore revealed an antagonist-specific MR population in the target organ heart but absent from nontarget lung and liver. The specificity for both sites increased when a methoxycarbonyl group was introduced in the 7 position (ZK 91587). RU 26752 labilized the MR at 35 degrees C but did not interfere with thermal activation assessed on DNA-cellulose and sucrose density gradients. ZK 91587 was even more effective in labilizing the MR and did not permit thermal activation at all. Whereas only one ionic species was observed with RU 26752 on DE-52 columns, two were evident with ZK 91587. Both antimineralocorticoids were bound to populations of two molecular sizes on Ultrogel columns. Thus, the nature of chemical substitution in the 7 position of spirolactone dramatically alters the receptor-mediated antisteroid action of the resulting molecule. Such differences may permit distinction between agonist versus the antagonist-specific receptor conformations, and could possibly be exploited for the eventual purification of the mineralocorticoid receptor from various organs.     

Studies on spirolactone steroid antagonists in ACTH-induced hypertension in sheep

This study investigated the anti-mineralocorticoid potency and haemodynamic effects of a series of mineralocorticoid antagonists of the spirolactone type (RU 28318, spironolactone, K-prorenoate, K-canrenoate and canrenone), for their ability to prevent the development of ACTH-induced hypertension in conscious sheep. In vivo bioassay, using aldosterone dependent changes in parotid salivary [Na+]/[K+] of sodium depleted adrenalectomized sheep, showed spironolactone was the most potent anti-mineralocorticoid tested. Infusions of the antagonists at equal doses alone for 4 days demonstrated that none affected mean arterial pressure, except for K-prorenoate which exhibited slight pressor activity. All the antagonists produced a natriuresis. Some of the steroid antagonists of the spirolactone group blocked the development of ACTH hypertension in sheep, spironolactone being the most effective. This study provides additional evidence for an essential mineralocorticoid component in ACTH-induced hypertension.     

Mineralocorticoid hormone action in plant cells.

The multiplication of Chlamydomonas reinhardtii wild type cells can be arrested by the spirolactone RU 26752 and this is fully reversible by the natural mineralocorticoid aldosterone. Evidence is presented for a 52 kDa protein that possesses functional DNA and ligand binding domains and tests positive for mineralocorticoid receptor-like activity by immuneprecipitation, macroaggregation, and photoaffinity. The regulation of trans-activation by steroid hormones in the animal world would therefore appear to be just as valid for the plant kingdom, thereby providing a new model for genetic analysis.     

A new phenanthrene with a spirolactone from Dendrobium chrysanthum and its anti-inflammatory activities

Investigation of phenolic patterns from the stems of Dendrobium chrysanthum by HPLC-PDA-MS has led to the isolation of a new phenanthrene derivative with a spirolactone ring, dendrochrysanene (1), that proved to suppress the mRNA level of TNF-alpha, IL8, IL10, and iNOS in murine peritoneal macrophages. The structure of 1 was characterized on the basis of various NMR (1H, 13C, 1H-1H COSY, HMQC, and HMBC), mass spectrometry, and X-ray crystal diffraction data.     

Evidence for an antagonist specific receptor that does not bind mineralocorticoid agonists

Kinetics of association--dissociation, competition and chromatography on two different resins, all revealed the presence of a new binding site which: specifically accepts 7-alpha-propyl spirolactone (3H-RU-26752), has little affinity for aldosterone, is present only in the target tissue (rat kidney), and is wanting in a non-target organ (liver). The presence of such sites could explain syndromes of mineralocorticoid excess where even trace amounts of an unusual aldosterone analogue, with little affinity for the classical mineralocorticoid receptor, can nevertheless produce hypertension through the intervention of an entirely new and abundant receptor system. This new molecule thus forms a novel tool to understand the nature and function of the soluble mineralocorticoid receptor in target organs.     

A flow-injection chemiluminescence method for the determination of human serum albumin, using the reaction of fluorescein-human serum albumin-sodium hypochlorite by the enhancement effect of the cationic surfactant cetyltrimethylammonium bromide.

The interaction between surfactant and fluorescein was studied, using a fluorescence spectroscopy and flow-injection (FI) chemiluminescence (CL) method. It was found that the cationic surfactant cetyltrimethylammonium bromide (CTAB) could cause the structural transformation of fluorescein from the quinone to the spirolactone form, and greatly enhance the CL intensity of the fluorescein-human serum albumin (HSA) complex. Based on this finding, a rapid and sensitive FI-CL method was developed for the determination of HSA. Under the optimum conditions, the proposed method has a linear range of 0.05-24.0 microg/mL, with a detection limit of 0.03 microg/mL for HSA (3sigma). The relative standard deviation (RSD) of 1.2 microg/mL HSA (n = 8) is 0.8%. The method was applied to the determination of protein content in urine samples, with satisfactory results. Density functional theory was used to study the mechanism of surfactant-enhanced CL intensity of the fluorescein-HSA complex.     

Biological spectrum of two spirolactone derivatives with some observations on anti-fertility activity.

Bioassays were conducted to determine various endocrinological properties of two spirolactone derivatives, 4',5'-dihydrospiro-[estr-4-ene-17,2'(3'H)-furan]-3-one (Compound I) and dispiro[cyclopropane-1,6'-estr-4'-ene-17',2"(3"h)-furan]-3'-one (Compound II). They proved to be very potent estrogen antagonists in immature mice and castrate ewes and neither exhibited inherent estrogenicity. They were moderately active as progestins and gonadotropin inhibitors. Compound II possessed slight androgenic activity and was more active orally than I. Compound I underwent a limited amount of testing in normally cycling rhesus monkeys. It was found to increase viscosity of the cervical mucus and, in oral doses of 0.5 or 2.0 mg/day, prevented pregnancy. One animal given 0.5 mg/day did become pregnant in her first treatment cycle, probably before a drug effect had been established. The results obtained indicate both compounds have potential utility as low-dose oral contraceptives.     

Spectroscopic studies on heme d in the visible and infrared

Heme d has been isolated from the terminal oxidase complex of Escherichia coli strain MR43L/F152 and purified by high-pressure liquid chromatography. The infrared spectrum indicated that carbonyls in the chlorin skeleton of this isolated heme existed as carboxylic acids. Earlier work on the iron-free chlorin had demonstrated the presence of a spirolactone substituent. This may have arisen from a cyclization reaction from a dicarboxylic acid, diol precursor. Although the free heme in extracts can exist as a diol, this does not prove that the diol as such is the precise form in the enzyme complex. Visible and fluorescence spectra are reported for a variety of derivatives and complexes of heme d to establish a spectral library that may be used to prove the presence of this structure in other enzymes or cells. Association constants have been measured for complexes of heme d with cyanide, imidazole, and pyridine and are contrasted to available data for protoheme.     

Inhibition of type 2 17beta-hydroxysteroid dehydrogenase by estradiol derivatives bearing a lactone on the D-ring: structure-activity relationships

The peripheral conversion of steroid precursors into biologically active forms can be a major source of steroid synthesis, and these steroids support the growth of hormone-dependent diseases. The 17beta-hydroxysteroid dehydrogenase (17beta-HSD) enzyme family is involved in the biosynthesis of active steroids and its inhibition constitutes an interesting approach for treating estrogen- and androgen-dependent cancers. We previously found that a compound formed by the introduction of a spiro-gamma-lactone at position 17 of estradiol (E2) produces a significant inhibition of type 2 17beta-HSD. To optimize the inhibitory potency of such compounds, we synthesized a series of estradiol derivatives bearing a lactone on the D-ring and tested their ability to inhibit the type 2 17beta-HSD transformation of 4-androstenedione into testosterone. The results of our structure-activity relationship study determined the importance of the 17beta-orientation of the oxygen atom. Indeed, the 17beta-O-isomer of spiro-gamma-lactone-E2 is a much more potent inhibitor than the 17alpha-O-analog (respectively 85 and 9% of inhibition at 1 microM). The carbonyl function is essential since the percentage of inhibition shifts from 85 to 30%, 15, or 3%, when the carbonyl group is transformed into a hydroxyl, a methoxy or a methylene (cycloether) group, respectively. Our results lead us to realize the importance of the spirolactone versus the C17beta-O/C16beta lactone (respectively 32 and 2% of inhibition at 0.1 microM, for the same size of lactone ring). The optimal size for the spirolactone was also established to be six members. All the types of substituents (methyl, dimethyl, allyl, propyl, and methoxycarbonyl) that we added on the spiro-delta-lactone moiety decreased the inhibitory activity, suggesting steric restrictions for the space that can be occupied in proximity of the spiro-delta-lactone functionality. 17-(Spiro-delta-lactone)-E2, compound 6, was thus the most potent inhibitor of type 2 17beta-HSD with a K(i) value of 29 +/- 5 nM. This compound reversibly inhibits type 2 17beta-HSD in a non-competitive manner.     

Affinity of corticosteroids for mineralocorticoid and glucocorticoid receptors of the rabbit kidney: effect of steroid substitution

Corticosteroid derivatives coupled in the C3, C7 or C17 position with a long aliphatic chain were synthesized in order to select a suitable ligand for the preparation of a biospecific affinity adsorbent for mineralocorticoid receptor purification. The affinity of these derivatives for mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) was explored in rabbit kidney cytosol. In this model, aldosterone bound to a single class of receptors with high affinity (Kd 1 nM) and mineralocorticoid specificity. RU26988, a highly specific ligand for GR, did not compete for these sites. The C7 and C17 positions were found to be of crucial importance in the steroid's interaction with the mineralocorticoid receptors, since the linkage of a long side chain in these positions induced complete loss of affinity. Hence, deoxycorticosterone no longer bound to MR after 17 beta substitution with a 9-carbon aliphatic chain. This loss of affinity was not observed for glucocorticoids. The 17 beta nonylamide derivative of dexamethasone still competed for GR. Increasing the length of the C7 side of the spirolactone SC26304 suppressed its affinity for MR. Finally, C3 was an appropriate position for steroid substitution. The 3-nonylamide of carboxymethyloxime deoxycorticosterone bound to MR but not to GR, and therefore constitutes a suitable ligand for the preparation of a mineralocorticoid adsorbent.     

Comparison of the feeding deterrent activity of some sesquiterpene lactones and a lignan lactone towards selected insect storage pests

Four sesquiterpene lactones isolated from Eurpatorium cannabinum, Homogyne alpina and Petasites albus (all Compositae), together with two of their adducts, as well as one lignan lactone of dibenzylbutanolide type from Libocedrus yateensis (Cupressaceae), were tested for their feeding deterrent activity against the adults of Sitophilus granarius and Tribolium confusum, as well as against the larvae of Tribolium confusum and Trogoderma granarium. The strongest deterrent activity against all species was exhibited by the lignan lactone yatein and the sesquiterpene spirolactone bakkenolide A. Both may be included to the class of very good insect feeding deterrents. The comparison presented here showed that the conjugated α-exomethylene moiety of the lactone ring was not the decisive factor for the deterrent activity in the investigated test model.     

Mutants that alter the covalent structure of catalase hydroperoxidase II from Escherichia coli.

The three-dimensional structures of two HPII variants, V169C and H392Q, have been determined at resolutions of 1.8 and 2.1 A, respectively. The V169C variant contains a new type of covalent bond between the sulfur atom of Cys(169) and a carbon atom on the imidazole ring of the essential His(128). This variant enzyme has only residual catalytic activity and contains heme b. The chain of water molecules visible in the main channel may reflect the organization of the hydrogen peroxide substrates in the active enzyme. Two alternative mechanisms, involving either compound I or free radical intermediates, are presented to explain the formation of the Cys-His covalent bond. The H392Q and H392E variants exhibit 75 and 25% of native catalytic activity, respectively. The Gln(392) variant contains only heme b, whereas the Glu(392) variant contains a mixture of heme b and cis and trans isomers of heme d, suggesting of a role for this residue in heme conversion. Replacement of either Gln(419) and Ser(414), both of which interact with the heme, affected the cis:trans ratio of spirolactone heme d. Implications for the heme oxidation mechanism and the His-Tyr bond formation in HPII are considered.     

Spirobiflavonoid stereoisomers from the endangered conifer Glyptostrobus pensilis and their protein tyrosine phosphatase 1B inhibitory activity

Six spirobiflavonoid stereoisomers including two new ones, spiropensilisols A (1) and B (2), were isolated from a mass-limited trunk barks of Glyptostrobus pensilis, an endangered conifer endemic to China. The new structures featuring a benzofuran-containing spirolactone and their absolute configurations were determined by extensive spectroscopic methods. All the isolates showed significant inhibitory activities against the human protein tyrosine phosphatase 1B (PTP1B) enzyme, a potential therapeutic target for diabetes and obesity, with IC₅₀ values ranging from 3.3 to 17.1 µM. A preliminary SAR analysis with assistance of the molecular modeling approach was performed for the most potent compound (i.e., 1), to understand the nature of interactions governing the binding mode of spirobiflavonids within the active site of the PTP1B enzyme.     

Effects of Three Commonly-used Diuretics on the Urinary Proteome

bstract Biomarker is the measurable change associated with a physiological or pathophysiological process. Unlike blood which has mechanisms to keep the internal environment homeostatic,rine is more likely to reflect changes of the body. As a result, urine is likely to be a better biomarker ource than blood. However, since the urinary proteome is affected by many factors, including iuretics, careful evaluation of those effects is necessary if urinary proteomics is used for biomarker iscovery. Here, we evaluated the effects of three commonly-used diuretics(furosemide, F; hydrochlorothiazide,H; and spirolactone, S) on the urinary proteome in rats. Urine samples were collected before and after intragastric administration of diuretics at therapeutic doses and the roteomes were analyzed using label-free liquid chromatography–tandem mass spectrometry LC–MS/MS). Based on the criteria of P 6 0.05, a fold change P2, a spectral count P5, and false ositive rate(FDR) 61%, 14 proteins(seven for F, five for H, and two for S) were identified by rogenesis LC–MS. The human orthologs of most of these 14 proteins are stable in the healthy uman urinary proteome, and ten of them are reported as disease biomarkers. Thus, our results uggest that the effects of diuretics deserve more attention in future urinary protein biomarker tudies. Moreover, the distinct effects of diuretics on the urinary proteome may provide clues to     

Mechanistic studies on the base-catalyzed transformation of neocarzinostatin chromophore: roles of bulged DNA

Nucleic acid bulges have been implicated in a number of biological processes and are specific cleavage targets for the enediyne antitumor antibiotic neocarzinostatin chromophore (NCS-chrom) in a base-catalyzed, radical-mediated reaction. Studies designed to elucidate the detailed mechanism of the base-catalyzed activation of NCS-chrom and to evaluate the roles of bulged DNA in its activation are described. They show that nucleobases in the DNA bulge are not required to form an effective bulge pocket but enhance the binding of the wedge-shaped activated drug molecule. Analysis of solvent deuterium isotope effects on NCS-chrom degradation and DNA cleavage efficiency experiments suggests that the spirolactone biradical 6 is a relatively stable species and that intramolecular quenching of the C2 radical of 6 to form the biologically active cyclospirolactone radical 7a occurs first (pathway a in Scheme 2), leaving the C6 radical to abstract the hydrogen atom from the DNA deoxyribose and to form the cyclospirolactone 8. Binding of the activated drug at the bulge site is required, but not sufficient, for efficient 8 formation, whereas cleavage of bulged DNA is not essential. Efficient generation of 8, but inefficient DNA damage generation, comes mainly from the likely high off-rate of 7a binding. The finding that thymidine 5'-carboxylic acid-ended oligonucleotide fragment can be formed in the reaction suggests that the process of DNA cleavage is rather slow and that sequential oxidations of the target 5'-carbon are possible. Study of the effect of solvent (methanol) concentration on NCS-chrom degradation indicates that bulged DNA acts to assist the intramolecular quenching of the radical at C2 by C8' ' of the naphthoate moiety by excluding solvent from the binding pocket, thus preventing the formation of spirolactones 9, and by blocking radical polymerization. Because in the absence or near absence of solvent methanol 8 formation does not reach even 10% that formed in the presence of bulged DNA, it is possible that the DNA bulge also induces a conformational change in the drug to promote the intramolecular reaction.     

Preparation and antitrypanosomal activity of secochiliolide acid derivatives

Secochiliolide acid (1) isolated from the Patagonian shrub Nardophyllum bryoides, was used as a scaffold for the preparation of a series of nine derivatives. Compound 1 and its derivatives were tested against Trypanosoma cruzi epimastigotes grown in liquid media. It was first observed that secochiliolide acid (1) inhibited the proliferation of the parasites, with an IC₅₀ of 2 μg/mL. Six of the synthesized derivatives were also active with IC₅₀’s between 2 and 7 μg/mL which are comparable to that of the commercial drug benznidazole (2.5 μg/mL). These results indicate that the carboxyl group is not essential for the bioactivity of 1, while the presence of the tetrasubstituted exocyclic double bond seems to be important. Moreover, the presence of the furan and spirolactone rings is not essential for the bioactivity per se, but is important in combination with other structural fragments present in the molecule.     

Comparison of the chemistry and diversity of endophytes isolated from wild-harvested and greenhouse-cultivated yerba mansa (Anemopsis californica)

With this study, we explored the identity and chemistry of fungal endophytes from the roots of yerba mansa [Anemopsis californica (Nutt.) Hook. & Arn. (Saururaceae)], a botanical traditionally used to treat infection. We compared the diversity of fungal endophytes isolated from a wild-harvested A. californica population, and those from plants cultivated for one year in a greenhouse environment. The wild-harvested population yielded thirteen fungal strains (eleven unique genotypes). Of the extracts prepared from these fungi, four inhibited growth of Staphylococcus aureus by >25% at 20 μg/mL, and three inhibited growth of Pseudomonas aeruginosa by ≥20% at 200 μg/mL. By comparison, A. californica roots after one year of cultivation in the greenhouse produced only two unique genotypes, neither of which displayed significant antimicrobial activity. The fungus Chaetomium cupreum isolated from wild-harvested A. californica yielded a new antimicrobial spirolactone, chaetocuprum (1). An additional 14 known compounds were identified using LC–MS dereplication of the various fungal endophytes. This study provides new insights into the identity and chemistry of A. californica fungal endophytes, and demonstrates the importance of considering growing conditions when pursuing natural product drug discovery from endophytic fungi.     

Induction of citrate synthase by aldosterone in the rat kidney

Summary The possible induction of renal citrate synthase (E.C. 4.1.3.7), by aldosterone was evaluated in the adrenalectomized rat. Three hours after administration of aldosterone (0.8 g/100 g body wt), renal cortical and medullary citrate synthase activity was significantly increased as reported previously by Kinne and Kirsten (Kinne, R., Kirsten, R. 1968.Pfleugers Arch. 300:244). In contrast, no change in this activity was detected in the renal papilla or the liver, under the same conditions. Kinetic analysis revealed that injection of aldosterone had no effect on theK _m s for acetyl-CoA and oxalacetate but augmentedV _max of renal medullary citrate synthase activity by 40%. The aldosterone-dependent increase in medullary citrate synthase activity was proportionate to the associated increase in the quantity of antiserum (specific for citrate synthase) required for half-maximal immuno-precipitation.The possibility that aldosterone induced the synthesis of citrate synthase was evaluated in two sets of experiments. In the first set, adrenalectomized rats were injected intraperitoneally with either aldosterone (0.8 g/100 g body wt) or the diluent, and simultaneously with³H or³⁵S methionine (500 Ci/rat). The isotopes were reversed in about half of the experiments. Three hours after the injection, renal citrate synthase was isolated by ATP-sepharose column chromatography and immuno-precipitation with the specific antiserum. Aldosterone augmented methionine incorporation into renal citrate synthase by 55% but had no effect on incorporation into the hepatic enzyme. In the second set, adrenalectomized rats were injected with either aldosterone (0.8 g/100 g body wt) or the diluent, the kidneys were removed 1 hr later and medullary slices were incubated in either³H-or³⁵S-methionine at 20° for 2 hr. Mitochondrial citrate synthase was isolated either by ATP-sepharose column chromatography and immuno-precipitation, or by polyacrylamide gel electrophoresis. Aldosterone increased methionine incorporation into the immuno-precipitates by 30% and into the enzyme peak resolved by polyacrylamide gel electrophoresis by 43%. The latter increase was eliminated by prior administration of either actinomycin D (70–80 g/100 g body wt) or spirolactone (SC-26304) (80 g/100 g body wt). An equimolar dose of dexamethasone (0.8 g/100 g body wt) had no effect on the isotope ratio associated with citrate synthase activity in the polyacrylamide gels.     

肥皂草毒蛋白分离纯化及性质研究

从肥皂草里分离到三种核糖体失活蛋白(ribosome inactivating proteins, RIPs): SO3a、SO3b,SO6.并测定SO3a,SO3b的相对分子质量分别约为22 500、19 400, 等电点都为8.4左右. 进行了氨基酸组成分析.用激光拉曼光谱初步预测SO3a和SO3b的二级结构含量.用反相毛细管色谱发现SO6含有两个组分,而Stripe等报道SO6为单一峰.