Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase

Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100 microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100 microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1 microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone all had IC(50) values between 1 and 5 microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.     

The Impact of the Extreme Floods in July 1997 on the Ichthyocenosis of the Oder Catchment Area (Czech Republic)

Changes in the ichthyocenosis of five smaller streams of 2nd to 4th order existing in the spring section of the River Odra (Oder) catchment area, in the territory of the Czech Republic, were observed before and after the catastrophic floods of July 1997. Quantitative catches using electro-fishing were performed in June, August, and September 1997, i.e. a month before and 2 months after the floods. The maximum flow of water in the examined sites usually reached or exceeded the limit of the so-called ȁ8once in a century high waterȁ9. Extreme floods did not influence statistically the proven average readings of species diversity of ichthyocenosis in the examined sites (p = 0.5625), species variety (p = 0.7316), abundance per hectare (p = 0.3125), and biomass per hectare (p = 0.4375). A distinctive decline in abundance and also in the biomass of the ichthyocenosis took place in geomorphic reaches. Significant incisions and alterations in the routes of the streams were also observed. Statistical data of catches of key reophilous fish species from the River Oder catchment basin, all of which were affected by the floods, in the periods 1995–1996, 1997, and 1998–1999, were evaluated. Catches of the species of Salmo trutta and Thymallus thymallus in 1997 considerably increased. Only Thymallus thymallus experienced a more pronounced decrease in the years after the floods, when compared with the average readings made in the period 1995–1996. The readings of the adult abundance of Barbus barbus and Chondrostoma nasus did not prove a negative impact of the floods on the adult population of both species in the assessed years. Our findings suggest that the original fish communities may be naturally well adapted to culmination flows.     

Biological half-lives of bromide and sodium in the rat are connected and dependent on the physiological state

The parallel course of the excretion rates of sodium and bromide ions was demonstrated in adult male rats administered simultaneously with ²⁴Na-sodium chloride and ⁸²Br-bromide. These excretion rates were inversely proportional to the magnitude of sodium intake in the animals. The biological half-life of bromide, as a substitute for sodium or chloride, was investigated with the aid of the radionuclide ⁸²Br in animals situated in very different physiological states (i.e., in lactating and nonlactating female rats as well as in young rats of varying ages [2,4,6, and 10 wk of age]). The ⁸²Br radioactivity retained in mothers and in whole litters was measured in vivo at appropriate time intervals (up to 240 h) after the application of ⁸²Br-bromide to the mothers. The time-course of the changes in the ⁸²Br radioactivity of the young was calculated as the difference between the rate of ⁸²Br intake in the mother's milk and the ⁸²Br excretion through the kidneys into the urine. The rate of ⁸²Br excretion through the kidneys of the dam could be calculated also. Nonweaned young rats (12 d) had the highest half-life (269 h) and lactating dams had the lowest (44 h). The determined values demonstrated that nonweaned young apparently conserve sodium, because of its relatively low concentration in mother's milk, whereas lactating dams, because of their large food intake, waste sodium. Presented in part at the 4th International Symposium on Trace Elements in Human: New Perspectives held in Athens (Greece) on 9–11 October 2003     

Female morphology and systematic status of Philometroides cyprini (Nematoda: Philometridae), a parasite of carp

Gravid females of Philometroides were found under the scales of stock carp Cyprinus carpio L. imported from Lithuania to the Czech Republic in the spring of 1997 and kept in a quarantine pond. These specimens and those obtained from pond-cultured carp in Russia (Moscow district) were used for detailed morphological studies, including scanning electron microscopy. The results confirm that the valid name of this parasite is Philometroides cyprini (Ishii, 1931), whereas Philometra lusii Vismanis, 1962, Philometra schikhobalowae Belous, 1965, Philometra lusiana Vismanis, 1966 and Philometroides lusiana (Vismanis, 1966) Ivashkin et al., 1971 are its junior synonyms. P. cyprini, the agent of a serious disease of carp, seems to have been originally distributed in east Asia, from where it was introduced into Europe in the early 1960s.     

pH-dependent Ca2+ binding to the F0 c-subunit affects proton translocation of the ATP synthase from Synechocystis 6803

It was shown before (Wooten, D. C., and Dilley, R. A. (1993) J. Bioenerg. Biomembr. 25, 557–567; Zakharov, S. D., Li, X., Red'ko, T. P., and Dilley, R. A. (1996) J. Bioenerg. Biomembr. 28, 483–493) that pH dependent reversible Ca²⁺ binding near the N- and C-terminal end of the 8 kDa subunit c modulates ATP synthesis driven by an applied pH jump in chloroplast and E. coli ATP synthase due to closing a proton gate proposed to exist in the F₀ H⁺ channel of the F₀F₁ ATP synthase. This mechanism has further been investigated with the use of membrane vesicles from mutants of the cyanobacterium Synechocystis 6803. Vesicles from a mutant with serine at position 37 in the hydrophilic loop of the c-subunit replaced by the charged glutamic acid (strain plc 37) has a higher H⁺/ATP ratio than the wild type and therefore shows ATP synthesis at low values of _H ⁺. The presence of 1 mM CaCl₂ during the preparation and storage of these vesicles blocked acid–base jump ATP formation when the pH of the acid side (inside) was between pH 5.6 and 7.1, even though the pH of the acid–base jump was thermodynamically in excess of the necessary energy to drive ATP formation at an external pH above 8.28. That is, in the absence of added CaCl₂, ATP formation did occur under those conditions. However, when the base stage pH was 7.16 and the acid stage below pH 5.2, ATP was formed when Ca²⁺ was present. This is consistent with Ca²⁺ being displaced by H⁺ ions from the F₀ on the inside of the thylakoid membrane at pH values below about 5.5. Vesicles from a mutant with the serine of position 3 replaced by a cysteine apparently already contain some bound Ca²⁺ to F₀. Addition of 1 mM EGTA during preparation and storage of those vesicles shifted the otherwise already low internal pH needed for onset of ATP synthesis to higher values when the external pH was above 8. With both strains it was shown that the Ca²⁺ binding effect on acid–base induced ATP synthesis occurs above an internal pH of about 5.5. These results were corroborated by ⁴⁵Ca²⁺- ligand blot assays on organic solvent soluble preparations containing the 8 kDa F₀ subunit c from the S-3-C mutant ATP synthase, which showed ⁴⁵Ca²⁺ binding as occurs with the pea chloroplast subunit III. The phosphorylation efficiency (P/2e), at strong light intensity, of Ca²⁺ and EGTA treated vesicles from both strains were almost equal showing that Ca²⁺ or EGTA have no other effect on the ATP synthase such as a change in the proton to ATP ratio. The results indicate that the Ca²⁺ binding to the F₀ H⁺ channel can block H⁺ flux through the channel at pH values above about 5.5, but below that pH protons apparently displace the bound Ca²⁺, opening the CF₀ H⁺ channel between the thylakoid lumen and H⁺ conductive channel.     

Effect of acute hyperketonemia on the cerebral uptake of ketone bodies in nondiabetic subjects and IDDM patients

Using R-beta-[1-(11)C]hydroxybutyrate and positron emission tomography, we studied the effect of acute hyperketonemia (range 0.7-1.7 micromol/ml) on cerebral ketone body utilization in six nondiabetic subjects and six insulin-dependent diabetes mellitus (IDDM) patients with average metabolic control (HbA(1c) = 8.1 +/- 1.7%). An infusion of unlabeled R-beta-hydroxybutyrate was started 1 h before the bolus injection of R-beta-[1-(11)C]hydroxybutyrate. The time course of the radioactivity in the brain was measured during 10 min. For both groups, the utilization rate of ketone bodies was found to increase nearly proportionally with the plasma concentration of ketone bodies (1.0 +/- 0.3 micromol/ml for nondiabetic subjects and 1.3 +/- 0.3 micromol/ml for IDDM patients). No transport of ketone bodies from the brain could be detected. This result, together with a recent study of the tissue concentration of R-beta-hydroxybutyrate in the brain by magnetic resonance spectroscopy, indicate that, also at acute hyperketonemia, the rate-limiting step for ketone body utilization is the transport into the brain. No significant difference in transport and utilization of ketone bodies could be detected between the nondiabetic subjects and the IDDM patients.     

Characterization of a purified beta-fructofuranosidase from Bifidobacterium infantis ATCC 15697

AIMS: To characterize the beta-fructofuranosidase of Bifidobacterium infantis ATCC 15697 and to compare it with other bacterial beta-fructofuranosidases. METHODS AND RESULTS: The beta-fructofuranosidase of B. infantis ATCC 15697 was purified 46.8 times over the crude extract by anion exchange chromatography, ultrafiltration and gel filtration. The sequence of 15 amino acid residues of the NH2 terminal was determined. This enzyme was a monomeric protein (Mr 70 kDa) with beta-fructofuranosidase and invertase activities. The isoelectric point was pH 4.3, the optimum pH 6.0 and pKas (4.5 and 7.2) of two active groups were obtained. The activities were inhibited by Hg2+ and p-chloromercuribenzoic acid (pCMB). The optimal temperature was 37 degrees C and activities were unstable at 55 degrees C. beta-fructofuranosidase activity was more efficient than that of invertase with Vm/Km ratios of 0.65 and 0.025 x 10-3 l min(-1) mg(-1), respectively. The enzyme catalyses the hydrolysis of fructo-oligosaccharides, sucrose and inulin at relative velocities of 100, 10 and 6, respectively. CONCLUSIONS: The enzyme of B. infantis ATCC 15697 is an exo-inulinase which has beta-fructofuranosidase and invertase activities. This protein was different from the beta-fructofuranosidase of another strain of B. infantis (B. infantis JCM no. 7007). SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of bacterial beta-fructofuranosidases, especially from bifidobacteria, has been gained.     

Increase of free cysteine and citric acid in plant cells exposed to cobalt ions

Cobalt complexation was investigated in a suspension cell culture of the cobalt hyperaccumulator Crotalaria cobalticola. C. cobalticola cells were more tolerant towards cobalt ions than the suspension cells of the non-accumulators Rauvolfia serpentina and Silene cucubalus. While the concentration of various compounds increased in cells of C. cobalticola challenged with cobalt ions, phytochelatin biosynthesis was not induced. Instead, the exposure to cobalt ions resulted in the increase of citrate and cysteine in cells. Size exclusion chromatography demonstrated the co-elution of cobalt and cysteine in C. cobalticola cell extracts. A significant increase in cysteine was observed also in cells of R. serpentina and S. cucubalus when they were exposed to cobalt ions. These results suggest that free cysteine is involved in cobalt ion complexation in plant cells.