Structure of the human mitochondrial monoamine oxidase B: new chemical implications for neuroprotectant drug design.

Monoamine oxidase B (MAO-B) is an outer mitochondrial membrane-bound flavoenzyme that is a well-known target for antidepressant and neuroprotective drugs. The 3A resolution structure of recombinant human MAO-B originally determined was of the enzyme complexed with pargyline, an irreversible inhibitor covalently bound to the N5 atom of the flavin coenzyme. The crystal structure shows that the enzyme is dimeric. Each monomer binds to the membrane via a C-terminal transmembrane helix and by apolar loops located at various positions in the sequence. Substrate binding to the enzyme involves negotiating a loop covering a 290A3 entrance apolar cavity before reaching an apolar 420A3 substrate cavity where the flavin coenzyme is located. The 1.7A isatin-MAO-B structure allowed a detailed examination of the enzyme's active site. A novel specific reversible MAO-B inhibitor, which is found as a contaminant in polystyrene plastics (1,4-diphenyl-2-butene), binds in both the entrance and the substrate cavity. Analogous MAO-B-specific inhibitors that bind in a manner traversing both cavities include trans-trans farnesol and chlorostyrylcaffeine. The rotation of the Ile199 side chain to an "open" conformation plays an essential role in this specificity. These results form a molecular basis for the design of new human MAO-B-specific reversible inhibitors.

[Duo-Bonharen in a comparative study]. / Duo-Bonharen v komparativní studii.

In the 1995-2000 period, data of interest concerning preoperative and postoperative intraocular pressure (IOP) and the best corrected visual acuity (BCVA) in the group of eyes in which the brand name methylcellulose--Adatocel, Ocucoat, Metocel--(n=378) and Viscoat (n=526) was used during the surgery, were statistically evaluated. They were compared to those results obtained from the prospective clinical study performed in 2001-2002 (n=275) by using the new viscoelastic material Duo-Bonharen, which is the Czech product, manufactured on the basis of the hyaluronic acid. All groups were evaluated in a retrospective manner. In the comparative study, statistically significant differences in functional results of cataract surgery in favor of Duo-Bonharen were demonstrated. The cohesive type of the substance used tends to decrease the IOP, the dispersive materials tend to increase the IOP.

[Early changes of the intraocular tension after the application of Duo-Bonharen Dispersive and Cohesive]. / Casné zmeny nitroocní tenze po aplikaci Duo-Bonharen Dispersive a Cohesive.

The intraocular pressure (IOP) rise is one of the complications after the application of viscoelastic material during the cataract surgery. The maximum of the elevation occurs between the second and eighth hours after the surgery. Twenty-four hours after the surgery, the IOP decreases to normal values. Because of possible early IOP changes, a group of 52 eyes (39 patients) with Duo-Bonharen Dispersive and Cohesive used, was followed. The IOP was measured at 2, 8, and 24 hours after the surgery. Not a remarkable IOP increase was marked.

Structure and mechanism of monoamine oxidase.

Monoamine oxidases A and B (MAO A and MAO B) are mitochondrial outer membrane-bound flavoproteins that catalyze the oxidative deamination of neurotransmitters and biogenic amines. A number of mechanism-based inhibitors (MAOI's) have been developed for clinical use as antidepressants and as neuroprotective drugs. To facilitate the development of more effective and specific inhibitors, a detailed understanding of the structures and catalytic mechanisms of these enzymes is required. The recent development of high level expression systems for producing recombinant human liver MAO A and MAO B in Pichia pastoris has facilitated the determination of the three dimensional crystal structures of MAO B (up to 1.7 angstroms resolution) in complex with different reversible (isatin, 1,4-diphenyl-2-butene) and irreversible inhibitors (pargyline, N-(2-aminoethyl)-p-chlorobenzamide, and trans-2-phenylcyclopropylamine). The binding of substrates or inhibitors to MAO B involves an initial negotiation of a protein loop occurring near the surface of the membrane and two hydrophobic cavities; an "entrance" cavity and an "active site" cavity. These two cavities can either be separate or in a fused state depending on the conformation of the Ile199 side chain, which appears to function as a gate. The amine function of the bound substrate approaches the re face of the bent and "puckered" covalent FAD through an "aromatic cage" formed by two tyrosine residues that are perpendicular to the plane of the flavin ring. No amino acid residues that could function as acids or bases are found near the catalytic site. The existing structural data on MAO B support previous QSAR results and are also supportive of a proposed polar nucleophilic mechanism for MAO A and B catalysis rather than the alternatively proposed single electron transfer mechanism.

[Duo-Bonharen--a new viscoelastic preparation. Experimental study]. / Duo-Bonharen--nové viskoelastikum. Experimentální studie.

The paper discusses preclinical tests of a new viscoelastic preparation for ophthalmosurgery. Experiments revealed that preparation Duo-Bonharen which contains hyaluronic acid prepared by bacterial fermentation is suited for clinical tests. After a 24-hour interval following administration into the anterior chamber it does not raise intraocular pressure. In experiments it has no side-effects on the eye. The dispersed form in a higher concentration (5%) adheres very well to ocular tissues and protects them adequately during surgery. The cohesive form in an optimal concentration (1.5%) can be rapidly removed from inside the eye. The properties of Duo-Bonharen in experiments are comparable with similar viscomaterials which are used. The new viscoelastic material Duo-Bonharen appears suitable for clinical tests in particular for cataract surgery.

Cyclic AMP-dependent protein kinase phosphorylates group III metabotropic glutamate receptors and inhibits their function as presynaptic receptors.

Recent evidence suggests that the functions of presynaptic metabotropic glutamate receptors (mGluRs) are tightly regulated by protein kinases. We previously reported that cAMP-dependent protein kinase (PKA) directly phosphorylates mGluR2 at a single serine residue (Ser843) on the C-terminal tail region of the receptor, and that phosphorylation of this site inhibits coupling of mGluR2 to GTP-binding proteins. This may be the mechanism by which the adenylyl cyclase activator forskolin inhibits presynaptic mGluR2 function at the medial perforant path-dentate gyrus synapse. We now report that PKA also directly phosphorylates several group III mGluRs (mGluR4a, mGluR7a, and mGluR8a), as well as mGluR3 at single conserved serine residues on their C-terminal tails. Furthermore, activation of PKA by forskolin inhibits group III mGluR-mediated responses at glutamatergic synapses in the hippocampus. Interestingly, beta-adrenergic receptor activation was found to mimic the inhibitory effect of forskolin on both group II and III mGluRs. These data suggest that a common PKA-dependent mechanism may be involved in regulating the function of multiple presynaptic group II and group III mGluRs. Such regulation is not limited to the pharmacological activation of adenylyl cyclase but can also be elicited by the stimulation of endogenous G(s)-coupled receptors, such as beta-adrenergic receptors.

High-level expression of human liver monoamine oxidase B in Pichia pastoris.

The high-level heterologous expression, purification, and characterization of the mitochondrial outer membrane enzyme human liver monoamine oxidase B (MAO B) using the methylotrophic yeast Pichia pastoris expression system are described. A 2-L culture of P. pastoris expresses approximately 1700 U of MAO B activity, with the recombinant enzyme associated tightly with the membrane fraction of the cell lysate. By a modification of the published procedure for purification of bovine liver MAO B [Salach, J. I. (1979) Arch. Biochem. Biophys. 192, 128-137], recombinant human liver MAO B is purified in a 34% yield ( approximately 200 mg from 2 L of cell culture). The isolated enzyme exhibits an M(r) of approximately 60, 000 on SDS-PAGE and 59,474 from electrospray mass spectrometry measurements, which is in good agreement with the mass predicted from the gene sequence and inclusion of the covalent FAD. One mole of covalent FAD per mole of MAO B is present in the purified enzyme and is bound by an 8alpha-S-cysteinyl(397) linkage, as identified by electrospray mass spectrometry of the isolated tryptic/chymotryptic flavin peptide. Recombinant human liver MAO B and bovine liver MAO B are shown to be acetylated at the seryl residues at their respective amino termini. The benzylamine oxidase activity of recombinant MAO B ranges from 3.0 to 3.4 U/mg and steady-state kinetic parameters for this enzyme preparation compare well with those published for the bovine liver enzyme: k(cat) = 600 min(-1), K(m)(benzylamine) = 0.50 mM, and K(m)(O(2)) = 0.33 mM. Kinetic isotope effect parameters using [alpha,alpha-(2)H(2)]benzylamine are also similar to those found for the bovine enzyme. Recombinant MAO B exhibits a (D)k(cat) = 4.7, a (D)[k(cat)/K(m)(benzylamine)] = 4.5, and a (D)[k(cat)/K(m)(O(2))] = 1.0. In contrast to bovine liver MAO B, no evidence was found for the presence of any anionic flavin radical either by UV-vis or by EPR spectroscopy in the resting form of the enzyme. These data demonstrate the successful heterologous expression of a functional, membrane-bound MAO B, which will permit a number of mutagenesis studies as structural and mechanistic probes not previously possible.

cAMP-dependent protein kinase inhibits mGluR2 coupling to G-proteins by direct receptor phosphorylation.

One of the primary physiological roles of group II and group III metabotropic glutamate receptors (mGluRs) is to presynaptically reduce synaptic transmission at glutamatergic synapses. Interestingly, previous studies suggest that presynaptic mGluRs are tightly regulated by protein kinases. cAMP analogs and the adenylyl cyclase activator forskolin inhibit the function of presynaptic group II mGluRs in area CA3 of the hippocampus. We now report that forskolin has a similar inhibitory effect on putative mGluR2-mediated responses at the medial perforant path synapse and that this effect of forskolin is blocked by a selective inhibitor of cAMP-dependent protein kinase (PKA). A series of biochemical and molecular studies was used to determine the precise mechanism by which PKA inhibits mGluR2 function. Our studies reveal that PKA directly phosphorylates mGluR2 at a single serine residue (Ser(843)) on the C-terminal tail region of the receptor. Site-directed mutagenesis combined with biochemical measures of mGluR2 function reveal that phosphorylation of this site inhibits coupling of mGluR2 from GTP-binding proteins

WD40 repeat proteins striatin and S/G(2) nuclear autoantigen are members of a novel family of calmodulin-binding proteins that associate with protein phosphatase 2A.

Protein phosphatase 2A (PP2A) is a multifunctional serine/threonine phosphatase that is critical to many cellular processes including development, neuronal signaling, cell cycle regulation, and viral transformation. PP2A has been implicated in Ca(2+)-dependent signaling pathways, but how PP2A is targeted to these pathways is not understood. We have identified two calmodulin (CaM)-binding proteins that form stable complexes with the PP2A A/C heterodimer and may represent a novel family of PP2A B-type subunits. These two proteins, striatin and S/G(2) nuclear autoantigen (SG2NA), are highly related WD40 repeat proteins of previously unknown function and distinct subcellular localizations. Striatin has been reported to associate with the post-synaptic densities of neurons, whereas SG2NA has been reported to be a nuclear protein expressed primarily during the S and G(2) phases of the cell cycle. We show that SG2NA, like striatin, binds to CaM in a Ca(2+)-dependent manner. In addition to CaM and PP2A, several unidentified proteins stably associate with the striatin-PP2A and SG2NA-PP2A complexes. Thus, one mechanism of targeting and organizing PP2A with components of Ca(2+)-dependent signaling pathways may be through the molecular scaffolding proteins striatin and SG2NA.

The FAD binding sites of human liver monoamine oxidases A and B: investigation of the role of flavin ribityl side chain hydroxyl groups in the covalent flavinylation reaction and catalytic activities.

The role of ribityl side chain hydroxyl groups of the flavin moiety in the covalent flavinylation reaction and catalytic activities of recombinant human liver monoamine oxidases (MAO) A and B have been investigated using the riboflavin analogue: N(10)-omega-hydroxypentyl-isoalloxazine. Using a rib5 disrupted strain of Saccharomyces cerevisiae which is auxotrophic for riboflavin, MAO A and MAO B were expressed separately under control of a galactose inducible GAL10/CYC1 promoter in the presence of N(10)-omega-hydroxypentyl-isoalloxazine as the only available riboflavin analogue. Analysis of mitochondrial membrane proteins shows both enzymes to be expressed at levels comparable to those cultures grown on riboflavin and to contain covalently bound flavin. Catalytic activities, as monitored by kynuramine oxidation, are equivalent to (MAO A) or 2-fold greater (MAO B) than control preparations expressed in the presence of riboflavin. Although N(10)-omega-hydroxypentyl-isoalloxazine is unable to support growth of riboflavin auxotrophic S. cerevisiae, it is converted to the FMN level by yeast cell free extracts. The FMN form of the analogue is converted to the FAD level by the yeast FAD synthetase, as shown by expression of the recombinant enzyme in Escherichia coli. These data show that the ribityl hydroxyl groups of the FAD moiety are not required for covalent flavinylation or catalytic activities of monoamine oxidases A and B. This is in contrast to the suggestion based on mutagenesis studies that an interaction between the 3'-hydroxyl group of the flavin and the beta-carbonyl of Asp(227) is required for the covalent flavinylation reaction of MAO B (Zhou et al., J. Biol. Chem. 273 (1998) 14862-14868).

Stability of benzo[a]pyrene DNA adducts in rat tissues during their long-term storage at -20 degrees C or -80 degrees C.

The aim of this study was to examine whether the storage of tissues at -80 degrees C or -20 degrees C affects the benzo[a]pyrene (B[a]P)-derived DNA adduct pattern and levels in rat tissues. Three rats were treated orally with a single dose of 100 mg B[a]P/kg b.w. and killed 24 h later. White blood cells (WBC) were isolated from the fresh blood. Livers, lungs and hearts were immediately removed, dissected into small fragments and were pooled for each organ. Pooled samples were proportionally divided into 7 aliquots. DNA from the first aliquot was immediately isolated (time 0). The other aliquots were frozen and stored at -20 degrees C or at -80 degrees C. DNA was isolated from the frozen samples at 1, 5 and 10 months later. 32P-postlabeling analysis was performed at the beginning and at the end of study with the whole set of samples. Two B[a]P-derived adducts were detected in all tissues but with different intensities for different tissue types. One of the adducts was found predominantly in WBC (approximately 85%) and liver (approximately 68%), while heart and lung accounted only for approximately 43% and approximately 39%, respectively. This adduct was tentatively identified as benzo[a]pyrene diol-epoxide-N2 adduct (BPDE-N2-dG) based on TLC and HPLC analyses of 32P-postlabeled adducts. The highest total DNA adduct level (sum of 2 spots) was found in lung (4.90 adducts/10(8) nucleotides) compared with heart, liver and WBC (3.55, 2.37 and 2.32 adducts/10(8) nucleotides, respectively). The analysis of variance provided evidence that storage of tissues at -20 degrees C or at -80 degrees C up to 10 months did not significantly affect B[a]P DNA adduct levels and patterns in the rat lung, heart and liver. Our study indicates that properly stored tissues can be used for DNA adduct analysis with confidence.

Bactericidal activity of a synthetic peptide (CG 117-136) of human lysosomal cathepsin G is dependent on arginine content.

The importance of individual amino acids in mediating the broad-spectrum bactericidal action of a 20-mer amphipathic, cationic peptide (CG 117-136) of human lysosomal cathepsin G was determined by using a single amino acid replacement strategy. This strategy revealed an important role for arginine because loss of any of the four arginine residues in CG 117-136 due to substitution with alanine, citrulline, or lysine residues resulted in a reduction of its bactericidal activity against both Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 33593. However, the replacement of a single alanine residue in CG 117-136 with arginine, but not glutamic acid, enhanced the activity of CG 117-136 against both P. aeruginosa and S. aureus. The importance of certain bulky, nonpolar amino acids for the bactericidal activity of CG 117-136 was also evident, since their substitutions by alanine diminished bactericidal activity. Accordingly, contributions of hydrophobic amino acids and structural considerations of the guanidinium side chain of arginine are major determinants in the broad-spectrum antimicrobial action of CG 117-136.

Genotoxic and embryotoxic effects of 5-bromodeoxyuridine in the chick embryo.

Chick embryos were exposed intra-amniotically to the thymidine analog bromodeoxyuridine (BrdU) in order to study its embryotoxic and genotoxic effects. Teratogenic effects were observed at doses of BrdU which failed to produce mitotic inhibition, clastogenic effects or any significant increase in sister chromatid exchanges. Clastogenic effects and depressed cell proliferation were observed only at high embryolethal doses. Thus, BrdU-induced teratogenicity was independent of genotoxic effects manifested at chromosomal level. On the contrary, a significant increase of DNA single strand breaks was detected even 24 hours after the administration of teratogenic dose. BrdU incorporation in the DNA does not appear to prevent embryonic cells from mitotic proliferation. Whether the single strand breaks in DNA would ultimately lead to BrdU-induced teratogenesis in chick embryos remained undetermined.

Detection of single-strand breaks in DNA induced by mitoxantrone in experimental tumors after in vivo treatment.

The fluorometric assay of DNA alkaline unwinding [15] was applied to murine tumor DNA after in vivo treatment. Mitoxantrone (MX) whose cytostatic effect has been extensively investigated was tested as DNA damaging compound. The single-strand breaks of DNA (SSB) in ascitic tumors (EAT. P388) and in solid tumors (B16a, B16) were detected after intraperitoneal administrations of 1 and 10 mg/kg MX in the course of 1 to 96 hours. The maximal number of SSB was measured after 1 to 6 hours and DNA damage outlasted 24 to 96 hours in dependence on the dose of MX and on the type of tumor. A dependence of DNA damage induced by MX on the route of tumor implantation was found. Ascitic tumor P388 and EAT were more than 5 times more sensitive to the effects of MX than solid melanomas. The SSB correlated well with the intracellular concentration of MX in the EAT cells.

Interaction of aflatoxin B1 with the rat monooxygenase system and relationships to its acute toxic effect.

The effect of aflatoxin B1 (AFB1) on the liver microsomal and nuclear mixed function oxidase system (MFO) of adult male rats was studied at oral doses of 1 and 3 mg/kg. At first both doses increased the activities and concentrations of all P450 components followed. The maximum values were observed between 24 and 48 h and were dose- and enzyme-dependent. After 72 h the values dropped to 15-55% of the initial values. Inhibition of the MFO system lasted even after 120 h, when a trend to return to the normal values was already noticeable. We assume that AFB1 acts as an inductor of the monooxygenase system and the reactive electrophilic intermediate(s) bind irreversibly to or autocatalytically inactivate P450.

Radioprotective effect of Aspergillus flavus.

A study designed to determine the extent and intensity of radioprotective effects of the mold conidium A. flavus in white mice revealed a higher survival rate with irradiation doses in the range of LD70-LD90; with irradiation with supralethal doses there is no difference in survival by stimulated and control mice. The radioprotective effect is independent of whether the strain A. flavus had been isolated from environment with enhanced radioactivity or not. It depends on a structure of the wall of conidia and not on the toxin produced by mold cells.

Time dependence of radioprotective effects of A. flavus conidia.

No essential difference in radioprotection results when conidia of A. flavus are applied to mice within a time lapse of a few minutes up to 48 hours prior to irradiation. When applied 72 hr before irradiation, they fail to affect mouse resistance. Application of conidia provide no protection to mice irradiated for 18-20 hr at the dose rate of 1 R/min; similarly ineffective are the chemoprotectors cistafos and mexamine. A combined administration of conidia and the two protective substances increases survival in mice and prolongs the mean survival period.