Binding of nitrogen‐containing bisphosphonates (N‐BPs) to the Trypanosoma cruzi farnesyl diphosphate synthase homodimer

Bisphosphonates (BPs) are a class of compounds that have been used extensively in the treatment of osteoporosis and malignancy‐related hypercalcemia. Some of these compounds act through inhibition of farnesyl diphosphate synthase (FPPS), a key enzyme in the synthesis of isoprenoids. Recently, nitrogen‐containing bisphosphonates (N‐BPs) used in bone resorption therapy have been shown to be active against Trypanosoma cruzi, the parasite that causes American trypanosomiasis (Chagas disease), suggesting that they may be used as anti‐trypanosomal agents. The crystal structures of TcFPPS in complex with substrate (isopentenyl diphosphate, IPP) and five N‐BP inhibitors show that the C‐1 hydroxyl and the nitrogen‐containing groups of the inhibitors alter the binding of IPP and the conformation of two TcFPPS residues, Tyr94 and Gln167. Isothermal titration calorimetry experiments suggest that binding of the first N‐BPs to the homodimeric TcFPPS changes the binding properties of the second site. This mechanism of binding of N‐BPs to TcFPPS is different to that reported for the binding of the same compounds to human FPPS. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

The moonlighting protein fructose‐1, 6‐bisphosphate aldolase of Neisseria meningitidis: surface localization and role in host cell adhesion

Fructose‐1, 6‐bisphosphate aldolases (FBA) are cytoplasmic glycolytic enzymes, which despite lacking identifiable secretion signals, have also been found localized to the surface of several bacteria where they bind host molecules and exhibit non‐glycolytic functions. Neisseria meningitidis is an obligate human nasopharyngeal commensal, which has the capacity to cause life‐threatening meningitis and septicemia. Recombinant native N. meningitidis FBA was purified and used in a coupled enzymic assay confirming that it has fructose bisphosphate aldolase activity. Cell fractionation experiments showed that meningococcal FBA is localized both to the cytoplasm and the outer membrane. Flow cytometry demonstrated that outer membrane‐localized FBA was surface‐accessible to FBA‐specific antibodies. Mutational analysis and functional complementation was used to identify additional functions of FBA. An FBA‐deficient mutant was not affected in its ability to grow in vitro, but showed a significant reduction in adhesion to human brain microvascular endothelial and HEp‐2 cells compared to its isogenic parent and its complemented derivative. In summary, FBA is a highly conserved, surface exposed protein that is required for optimal adhesion of meningococci to human cells.     

Binding of Azole Antibiotics to Staphylococcus aureus Flavohemoglobin Increases Intracellular Oxidative Stress

In this work, we report that flavohemoglobin contributes to the azole susceptibility of Staphylococcus aureus. We first observed that deletion of the flavohemoglobin gene leads to an increase in the viability of imidazole-treated S. aureus cells and that reversion to the wild-type phenotype occurs upon expression of flavohemoglobin from a multicopy plasmid. Further spectroscopic analyses showed that miconazole, the most efficient azole antibiotic against S. aureus, ligates to heme of both oxidized and reduced flavohemoglobin. The binding of miconazole to oxidized flavohemoglobin, with an association constant of 1.7 × 10⁶ M⁻¹, typical of a tight, specific binding equilibrium, results in augmentation of the superoxide production by the enzyme. These results are corroborated by in vivo studies showing that imidazole-treated S. aureus cells expressing flavohemoglobin contain a larger amount of reactive oxygen species. Moreover, it was observed that the survival of miconazole-treated S. aureus internalized by murine macrophages is higher for cells lacking flavohemoglobin. Altogether, the present data revealed that in S. aureus, flavohemoglobin enhances the antimicrobial activity of imidazoles via an increase of intracellular oxidative stress.Staphylococcus aureus is an opportunistic pathogen responsible for a large number of human infections that cause systemic diseases from a mild to life-threatening character. The increasing incidence of methicillin-resistant S. aureus (MRSA) strains observed in the past few years makes S. aureus infections a leading threat to public health, causing more deaths in the United States and Europe than human immunodeficiency virus (AIDS) (11). Like other Gram-positive bacteria, staphylococci are sensitive to imidazoles (27). Imidazoles (such as clotrimazole, miconazole, ketoconazole, and sulconazole) (Fig. ​(Fig.1)1) represent one of the major classes of azole antifungal that are useful in the treatment of infections, including cutaneous and vaginal candidiasis (8). The activity of these antifungal drugs derives primarily from inhibition of the biosynthesis of ergosterol, an essential component of the fungal plasma membrane, at the level of lanosterol 14-alpha demethylase. Furthermore, in fungi and yeast, azole treatment leads to an increase in the endogenous production of reactive oxygen species (ROS) (12, 25). For example, in Candida albicans and Saccharomyces cerevisiae, the miconazole inhibition of cytochrome c oxidase, peroxidase, and catalase has been reported to be responsible for a high level of ROS production (3, 4). It has also been reported that clotrimazole inhibition of Plasmodium falciparum hemoperoxidase leads to ROS accumulation in this protozoan pathogen (26). For S. cerevisiae, C. albicans, and Escherichia coli, the action of imidazoles was also correlated with the inhibition of the nitric oxide (NO) scavenger activity of flavohemoglobin (7).Open in a separate windowFIG. 1.Structures of the azole (imidazole; 1,2,4-triazole) antibiotics investigated.Flavohemoglobins (Hmp) are widespread among bacteria and yeast and contain three domains: C-terminal NAD- and flavin adenine dinucleotide (FAD)-binding domains, which together constitute a ferredoxin-NADP⁺ oxidoreductase-like domain, and an N-terminal globin domain, which harbors a single B-type heme. The high-spin heme contains one axial histidine and binds small molecules like NO, carbon monoxide (CO), and dioxygen (O₂). The heme can also bind bulky aromatic bases, since it is inserted in a large hydrophobic pocket (7). We observed that the binding of imidazoles to S. aureus flavohemoglobin results in an increase in the amount of deleterious reactive oxygen species produced by flavohemoglobin that contributes to the bactericidal effect of azole antibiotics toward S. aureus.     

Pesticide exposure: the hormonal function of the female reproductive system disrupted?

Some pesticides may interfere with the female hormonal function, which may lead to negative effects on the reproductive system through disruption of the hormonal balance necessary for proper functioning. Previous studies primarily focused on interference with the estrogen and/or androgen receptor, but the hormonal function may be disrupted in many more ways through pesticide exposure. The aim of this review is to give an overview of the various ways in which pesticides may disrupt the hormonal function of the female reproductive system and in particular the ovarian cycle. Disruption can occur in all stages of hormonal regulation: 1. hormone synthesis; 2. hormone release and storage; 3. hormone transport and clearance; 4. hormone receptor recognition and binding; 5. hormone postreceptor activation; 6. the thyroid function; and 7. the central nervous system. These mechanisms are described for effects of pesticide exposure in vitro and on experimental animals in vivo. For the latter, potential effects of endocrine disrupting pesticides on the female reproductive system, i.e. modulation of hormone concentrations, ovarian cycle irregularities, and impaired fertility, are also reviewed. In epidemiological studies, exposure to pesticides has been associated with menstrual cycle disturbances, reduced fertility, prolonged time-to-pregnancy, spontaneous abortion, stillbirths, and developmental defects, which may or may not be due to disruption of the female hormonal function. Because pesticides comprise a large number of distinct substances with dissimilar structures and diverse toxicity, it is most likely that several of the above-mentioned mechanisms are involved in the pathophysiological pathways explaining the role of pesticide exposure in ovarian cycle disturbances, ultimately leading to fertility problems and other reproductive effects. In future research, information on the ways in which pesticides may disrupt the hormonal function as described in this review, can be used to generate specific hypotheses for studies on the effects of pesticides on the ovarian cycle, both in toxicological and epidemiological settings.     

Trypanosoma cruzi contains major pyrophosphate stores, and its growth in vitro and in vivo is blocked by pyrophosphate analogs

High field (31)P nuclear magnetic resonance spectroscopy showed that inorganic pyrophosphate (P(2)O(7)(4-)) is more abundant than ATP in Trypanosoma cruzi, the causative agents of Chagas' disease. These results were confirmed by specific analytical assays, which showed that in epimastigotes, the concentrations of inorganic pyrophosphate and ATP were 194.7 +/- 25.9 and 37.6 +/- 5.5 nmol/mg of protein, respectively, and for the amastigote form, the corresponding concentrations were 358.0 +/- 17.0 and 36.0 +/- 1.9 nmol/mg of protein. High performance liquid chromatographic analysis of perchloric acid extracts of epimastigotes labeled for 3 h with (32)P-orthophosphate showed a significant incorporation of the precursor into inorganic pyrophosphate. Inorganic pyrophosphate was not uniformly distributed in T. cruzi but was shown by (31)P-NMR and chemical analysis to be particularly associated with acidocalcisomes, organelles shown previously to contain large amounts of phosphorus and various elements. Electron microscopy analysis of pyrophosphatase-treated permeabilized epimastigotes showed disappearance of the electron density of the acidocalcisomes. Nonmetabolizable analogs of pyrophosphate, currently used for the treatment of bone resorption disorders, selectively inhibited the proliferation of intracellular T. cruzi amastigotes and produced a profound suppression in the number of circulating trypomastigotes in mice with an acute infection of T. cruzi, offering a potentially new route to chemotherapy.     

Identification of centaurin-alpha2: a phosphatidylinositide-binding protein present in fat,heart and skeletal muscle

We describe here the cloning, expression and characterisation of centaurin-alpha2 from a rat adipocyte cDNA library. The centaurin-alpha2 cDNA contains an open reading frame, which codes for a protein of 376 amino acids with predicted mass of 43.5 kDa. Centaurin-alpha2 shares 51-59% identity with centaurin-alpha1 proteins and has the same domain organisation, consisting of a predicted N-terminal ArfGAP domain followed by two successive pleckstrin homology domains. Despite the sequence similarity, there are a number of notable differences between the previously characterised centaurin-alpha1 proteins and the newly described centaurin-alpha2: (i) in vitro lipid binding experiments with centaurin-alpha2 do not reveal the same selectivity for phosphatidylinositol 3,4,5-trisphosphate over phosphatidylinositol 4,5-bisphosphate that has been shown for centaurin-alpha; (ii) unlike centaurin-alpha1 which is expressed mainly in the brain, centaurin-alpha2 has a broad tissue distribution, being particularly abundant in fat, heart and skeletal muscle; (iii) in contrast to centaurin-alpha1 which is found in both membrane and cytosolic fractions, endogenous centaurin-alpha2 is exclusively present in the dense membrane fractions of cell extracts, suggesting a constitutive membrane association. Insulin stimulation, which stimulates phosphatidylinositol 3,4,5-trisphosphate production, does not alter the subcellular distribution of centaurin-alpha2 between adipocyte membrane fractions. This observation is consistent with the lack of specificity of centaurin-alpha2 for phosphatidylinositol 3,4,5-trisphosphate over phosphatidylinositol 4,5-bisphosphate.     

Magic-angle spinning (31)P NMR spectroscopy of condensed phosphates in parasitic protozoa: visualizing the invisible

We report the results of a solid-state (31)P nuclear magnetic resonance (NMR) spectroscopic investigation of the acidocalcisome organelles from Trypanosoma brucei (bloodstream form), Trypanosoma cruzi and Leishmania major (insect forms). The spectra are characterized by a broad envelope of spinning sidebands having isotropic chemical shifts at approximately 0, -7 and -21 ppm. These resonances are assigned to orthophosphate, terminal (alpha) phosphates of polyphosphates and bridging (beta) phosphates of polyphosphates, respectively. The average polyphosphate chain length is approximately 3.3 phosphates. Similar results were obtained with whole L. major promastigotes. (31)P NMR spectra of living L. major promastigotes recorded under conventional solution NMR conditions had spectral intensities reduced with respect to solution-state NMR spectra of acid extracts, consistent with the invisibility of the solid-state phosphates. These results show that all three parasites contain large stores of condensed phosphates which can be visualized by using magic-angle spinning NMR techniques.     

Measurement of intact sulfate and glucuronide phytoestrogen conjugates in human urine using isotope dilution liquid chromatography-tandem mass spectrometry with [13C(3)]isoflavone internal standards

A method has been developed for the analysis of phytoestrogens and their conjugates in human urine using liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Stable isotopically labeled [13C(3)]daidzein and [13C(3)]genistein were synthesized and used as internal standards for isotope dilution mass spectrometry. Free aglycons and intact glucuronide, sulfate, diglucuronide, disulfate, and mixed sulfoglucuronide conjugates of isoflavones and lignans were observed in naturally incurred urine samples. Sample pretreatment was not necessary, other than addition of internal standards and pH adjustment. Urine was injected directly onto the analytical column. The limits of detection were generally <50ng/ml, precision was generally <10% CV for conjugates. Total hydrolyzed daidzein and genistein were measured against quality assurance urine sample and were accurate to within 12%. The accuracy of conjugate measurement can not be ascertained, as no reference samples are available. The mean sum of daidzein and its conjugates was within 20% of the hydrolyzed value. Concentrations of the free aglycons of up to 22% of genistein and 18% of daidzein were observed. The average pattern was ca. 54% 7-glucuronide, 25% 4(')-glucuronide, 13% monosulfates, 7% free daidzein, 0.9% sulfoglucuronides, 0.4% diglucuronide, and <0.1% disulfate. Selective enzymatic deconjugation with glucuronidase and mixed glucuronidase/sulfatase were used to validate the accuracy of the quantitation of the intact daidzein conjugates. There were no apparent sex differences, or conditioning effects on the conjugation profile of isoflavones after chronic dosing.     

Desulphurisation of benzothiophene and dibenzothiophene by actinomycete organisms belonging to the genus Rhodococcus, and related taxa

Desulphurising enzymes remove the sulphur moiety from an organosulphur molecule leaving the carbon skeleton intact. Two kinds of desulphurisation reaction are recognised. The dibenzothiophene (DBT)-specific pathway desulphurises DBT to inorganic sulphite and 2- hydroxybiphenyl (HBP), and the benzothiophene (BTH)-specific pathway desulphurises BTH to 2-(2-hydroxyphenyl)ethan 1-al (HPEal) and probably inorganic sulphite. The DBT-desulphurisation pathway was originally identified in Rhodococcus erythropolis strain IGTS8 (ATCC 53968), and the BTH-desulphurisation pathway in Gordonia sp. strain 213E (NCIMB 40816). These organisms do not further metabolise the organic product of desulphurisation.In this article current knowledge of the biochemistry and genetics of the desulphurisation enzymes is reviewed. The need for separate, DBT- and BTH-specific desulphurisation routes is rationalised in terms of the chemical differences between the two compounds. The desulphurisation pathway is compared with other microbial DBT- degrading enzyme systems. Finally some comments are made concerning the application of desulphurisation enzymes for fuel desulphurisation and on the relevance of these enzymes to the ecology of the mycolata (sensu Chun et al, 1996).