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PubMed2000

Harpin-induced hypersensitive cell death is associated with altered mitochondrial functions in tobacco cells.

Mitochondria play important roles in animal apoptosis and are implicated in salicylic acid (SA)-induced plant resistance to viral pathogens. In a previous study, we demonstrated that SA induces rapid inhibition of mitochondrial electron transport and oxidative phosphorylation in tobacco cells. In the present study, we report that plant programmed cell death induced during pathogen elicitor-induced hypersensitive response (HR) is also associated with altered mitochondrial functions. Harpin, an HR elicitor produced by Erwinia amylovora, induced inhibition of ATP synthesis in tobacco cell cultures. Inhibition of ATP synthesis occurred almost immediately after incubation with harpin and preceded hypersensitive cell death induced by the elicitor. Diphenylene iodonium, an inhibitor of the oxidative burst, did not block harpin-induced inhibition of ATP synthesis or cell death, suggesting that oxidative burst was not the direct cause for these two harpin-induced processes. Unlike SA, harpin had no significant effect on total respiratory O2 uptake of treated cells. However, respiration of harpin-treated tobacco cells became very sensitive to the alternative oxidase inhibitors salicyl-hydroxamic acid and n-propyl gallate. Thus, harpin treatment resulted in reduced capacity of mitochondrial cytochrome pathway electron transport, which could lead to the observed inhibition of ATP synthesis. Given the recently demonstrated roles of mitochondria in apoptosis, this rapid inhibition of mitochondrial functions may play a role in harpin-induced hypersensitive cell death.

Adenosine Triphosphate↗
PubMed1996

Purification and characterization of linoleate 8-dioxygenase from the fungus Gaeumannomyces graminis as a novel hemoprotein.

The fungus Gaeumannomyces graminis, which causes the major root disease of wheat known as "take-all," can metabolize linoleic acid to (8R)-hydroperoxylinoleic acid. The enzyme linoleate 8-dioxygenase abstracts hydrogen and introduces molecular oxygen in an antarafacial way at C-8. We have now purified the enzyme 1000-fold to a specific activity of 1.8 micronol/min/mg of protein. Acetone powder of mycelia of G. graminis was subjected to extraction and ammonium sulfate precipitation with solubilization. The 8-dioxygenase was purified by hydrophobic interaction chromatography, size-exclusion chromatography, anion-exchange chromatography, and immobilized metal ion affinity chromatography. The active enzyme appeared to consist of four subunits since the active enzyme had an apparent molecular mass of 520 kDa determined by gel filtration, while SDS-polyacrylamide gel electrophoresis showed a protein band of 130 kDa. Spectroscopy indicated the presence of heme. The characteristic pyridine ferrohemochrome alpha-band was found at 557 nm and the beta-band at 525 nm. The purified protein showed an absorption maximum at 408 nm (gamma, Soret). The absorption maximum shifted to 429 nm after reduction with dithionite and to 421 nm after treatment of the reduced enzyme with carbon monoxide. BW A4C, a hydroxamic acid derivative, inhibited the enzyme by >90% at 10 microM. The pH optimum was 7.2-7.4, the isoelectric point was 5.2 by chromatofocusing, and the Km values were 8 microM for linoleic acid and 30 microM for oxygen. We conclude that linoleate 8-dioxygenase appears to be a tetrameric hemoprotein distinct from other fatty-acid dioxygenases.

Chromatography, Affinity↗
PubMed1996

Fitting an inhibitor into the active site of thermolysin: a molecular dynamics case study.

We used molecular dynamics computer simulation to "fly" a small flexible ligand, L-leucine hydroxamic acid, into the active site of thermolysin. The potential, which imposed no constraints between protein and ligand, produced conformations close to the crystallographically determined one. The calculations made use of the combined molecular mechanics/grid method of Luty et al. (J. Comp. Chem. 16:454-464, 1995), in which atoms of the active site are free to move whereas the rest of the protein, assumed to be rigid, is represented as points of a grid, and which also includes an implicit solvation model. The method is sufficiently fast that large sets of simulations could be carried out, enabling statistical sampling and exploration of the effect of initial position and conformation of the ligand on the probability of successful docking. In a charged catalytic Glu/uncharged ligand regime, when the initial position of the ligand was determined by random translations and rotations that kept the center of mass within 8.0 angstroms of the crystal one, none of the 20 runs placed the ligand correctly. In a second set with uncharged Glu and zwitterionic ligand, 3 of 24 similarly placed random structures flew the ligand in successfully. In a third set with the same protonation scheme as the second the starting positions had randomly determined conformations but kept the hydroxamate oxygens within 4.0 angstroms of the zinc; in this case 22 of 25 runs reoriented correctly. A diverse set of energetic, structural, and dynamic criteria was used for evaluation of the calculations. The results indicate the method to be a promising tool for the rational drug design process.

Binding Sites↗
PubMed1999

Gas chromatographic-mass spectrometric analysis of hydroxylamine for monitoring the metabolic hydrolysis of metalloprotease inhibitors in rat and human liver microsomes.

A gas chromatographic-mass spectrometric (GC-MS) method was developed for the analysis of hydroxylamine (HA) in supernatants obtained from liver microsomes. HA monitoring was used to determine the metabolic hydrolysis of two hydroxamic acid-based matrix metalloprotease inhibitors in rat and human liver microsomes. The hydrolysis of the hydroxamic acids to their corresponding carboxylic acids releases HA as a common metabolic product. HA was derivatized to acetone oxime by addition of acetone to the liver microsomal supernatant, followed by direct injection of the supernatant into the GC-MS, with detection of the oxime by selected-ion-monitoring. The method is simple, reproducible, and sensitive for the determination of the hydrolysis of hydroxamic acid compounds, where hydrolysis is the major metabolic pathway. The methodology can be used for rank ordering and selecting hydroxamic acid analogs based on their susceptibility to hydrolysis.

Animals↗
PubMed1997

Metalloproteinases regulate parietal endoderm differentiating and migrating in cultured mouse embryos.

Extracellular matrix (ECM) adhesion and proteolysis play important roles in embryonic development. In previous work (Behrendtsen et al. [1992] Development 114:447-456) we showed that gelatinase B activity is rate-limiting for trophoblast-mediated invasion and degradation of ECM in culture. In the present study, we show that metalloproteinases (MMPs) have distinct roles in migration along ECM as opposed to invasion through ECM. We investigated the role of ECM proteolysis in the differentiation and migration of parietal endoderm (PE), the first embryonic migratory cell type, adhering to ECM surfaces. Gelatinase B was the major MMP of PE; mRNA and protein were detected in PE of 7.5- and 8.5-day embryos. Using cultures of inner cell masses (ICMs) isolated from mouse blastocysts, we found that inhibitors of metalloproteinases, specifically, tissue inhibitor of metalloproteinases (TIMP)-1 and a peptide hydroxamic acid stimulated outgrowth and differentiation of PE from ICMs cultured on fibronectin, but inhibitors of plasminogen activators did not. TIMP-1 increased the number of PE cells and mean distance migrated and increased expression of the PE differentiation marker vimentin; the increase in cell number was not at the expense of other cell types. The stimulatory effect of TIMP-1 was most marked on low concentrations of substrate fibronectin, decreasing as concentrations of fibronectin increased. TIMP-1 also stimulated the outgrowth of PE in blastocyst cultures and in ICM/trophectoderm co-cultures; in ICM/trophectoderm co-cultures TIMP-1 stimulated PE differentiation on higher concentrations of fibronectin than was permissive for ICMs cultured alone. These data indicate that metalloproteinase inhibitors preserved the migration-inducing status of the ECM. We conclude that metalloproteinases have distinct roles in invasive activity through ECM barriers and migratory activity along ECM surfaces.

Animals↗
PubMed1998

Two-dimensional parallel array technology as a new approach to automated combinatorial solid-phase organic synthesis

An automated, 96-well parallel array synthesizer for solid-phase organic synthesis has been designed and constructed. The instrument employs a unique reagent array delivery format, in which each reagent utilized has a dedicated plumbing system. An inert atmosphere is maintained during all phases of a synthesis, and temperature can be controlled via a thermal transfer plate which holds the injection molded reaction block. The reaction plate assembly slides in the X-axis direction, while eight nozzle blocks holding the reagent lines slide in the Y-axis direction, allowing for the extremely rapid delivery of any of 64 reagents to 96 wells. In addition, there are six banks of fixed nozzle blocks, which deliver the same reagent or solvent to eight wells at once, for a total of 72 possible reagents. The instrument is controlled by software which allows the straightforward programming of the synthesis of a larger number of compounds. This is accomplished by supplying a general synthetic procedure in the form of a command file, which calls upon certain reagents to be added to specific wells via lookup in a sequence file. The bottle position, flow rate, and concentration of each reagent is stored in a separate reagent table file. To demonstrate the utility of the parallel array synthesizer, a small combinatorial library of hydroxamic acids was prepared in high throughput mode for biological screening. Approximately 1300 compounds were prepared on a 10 μmole scale (3-5 mg) in a few weeks. The resulting crude compounds were generally >80% pure, and were utilized directly for high throughput screening in antibacterial assays. Several active wells were found, and the activity was verified by solution-phase synthesis of analytically pure material, indicating that the system described herein is an efficient means for the parallel synthesis of compounds for lead discovery. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗
PubMed2005

The Barton reaction: can a more tenable pathway be hypothesized for the formation of nitrosoalkyl derivatives?

Herein, we report that in the formation of nitrosoalkyl derivatives during the photolysis of alkyl nitrites, the formation of the intermediate alkyl alkoxy nitroxide, due to the trapping of alkyl radicals by the starting nitrite, is the key step of the entire process. In fact, these nitroxides, detectable by EPR spectroscopy, decay to the final nitroso derivatives under thermodynamic control. In light of this, the Barton reaction mechanism has been reviewed. The nitrosoalkyl derivatives, or the hydroxamic acids when steroids are involved, have now to be considered as the ending products of the entire process and not, unless a very high concentration of NO is present in the medium, the result of a direct reaction of NO with the alkyl radical, as is commonly accepted.

Journal Article↗
PubMed1994

The X-ray crystal structure of the catalytic domain of human neutrophil collagenase inhibited by a substrate analogue reveals the essentials for catalysis and specificity.

Matrix metalloproteinases are a family of zinc endopeptidases involved in tissue remodelling. They have been implicated in various disease processes including tumour invasion and joint destruction. These enzymes consist of several domains, which are responsible for latency, catalysis and substrate recognition. Human neutrophil collagenase (PMNL-CL, MMP-8) represents one of the two 'interstitial' collagenases that cleave triple helical collagens types I, II and III. Its 163 residue catalytic domain (Met80 to Gly242) has been expressed in Escherichia coli and crystallized as a non-covalent complex with the inhibitor Pro-Leu-Gly-hydroxylamine. The 2.0 A crystal structure reveals a spherical molecule with a shallow active-site cleft separating a smaller C-terminal subdomain from a bigger N-terminal domain, composed of a five-stranded beta-sheet, two alpha-helices, and bridging loops. The inhibitor mimics the unprimed (P1-P3) residues of a substrate; primed (P1'-P3') peptide substrate residues should bind in an extended conformation, with the bulky P1' side-chain fitting into the deep hydrophobic S1' subsite. Modelling experiments with collagen show that the scissile strand of triple-helical collagen must be freed to fit the subsites. The catalytic zinc ion is situated at the bottom of the active-site cleft and is penta-coordinated by three histidines and by both hydroxamic acid oxygens of the inhibitor. In addition to the catalytic zinc, the catalytic domain harbours a second, non-exchangeable zinc ion and two calcium ions, which are packed against the top of the beta-sheet and presumably function to stabilize the catalytic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2004

Impact of novel histone deacetylase inhibitors, CHAP31 and FR901228 (FK228), on adenovirus-mediated transgene expression.

BACKGROUND: Histone deacetylase inhibitors (HDIs) are known to enhance adenovirus (Ad)-mediated transgene expression. Recently, novel HDIs, including cyclic hydroxamic-acid-containing peptide 31 (CHAP31) and FR901228 (FK228), have been developed. METHODS: The effects of these two novel HDIs on Ad-transduced or endogenous gene expression were investigated. Acetylation of core histones and the expression of the coxsackie and adenovirus receptor (CAR) in HDI-treated cells were examined using Western blot and a quantitative reverse transcription polymerase chain reaction (TaqMan RT-PCR), respectively. Their in vivo effect on adenoviral gene expression was investigated in BALB/c mice. RESULTS: Both compounds enhanced and prolonged Ad-mediated beta-galactosidase expression more effectively than did trichostatin A, a classic HDI. The same effect was observed in Ad-transduced heat shock protein 72 (HSP72), but not in hyperthermia-induced endogenous expression of HSP72, suggesting that the effect is specific for transduced gene expression. Hyperacetylation of core histones induced by HDIs was considered responsible for the augmentative effects of gene expression. Intravenous administration of either CHAP31 or FR901228 enhanced beta-galactosidase expression in mice infected with AdLacZ. CONCLUSIONS: CHAP31 and FR901228 amplified Ad-mediated transgene expression. The enhancement of transgene expression by HDIs may result in fewer vector doses for necessary gene expression, helping to alleviate disadvantages caused by Ad vectors. This could be a useful tool in overcoming current limitations of gene therapy using adenovirus vectors.

Acetylation↗
PubMed1992

Steroidal nonspecific esterase metabolism of N-hydroxy-2-acetylaminofluorene: evidence for selective activation by the cellular reductant NADPH.

The significance of the nonspecific esterases of human mononuclear leukocytes (HMLs) in arylamine carcinogenesis is suggested by data showing that the metabolically formed hydroxamic acid derivative of 2-acetylaminofluorene, N-hydroxy-2-acetylaminofluorene, is a substrate for this class of enzymes. A viable cell assay for the nonspecific esterases using alpha-naphthyl acetate as substrate is described, and data showing this activity to be sensitive to already known substrates for HML esterases as measured by three previously described assays are presented. All four assays of the same esterase activity are shown to be highly sensitive to up- and down-regulation by addition of NADPH or NADP to viable HML cultures. Selective activation of a purified rabbit nonspecific esterase by NADPH, but not by the other cellular reductants, NADH and glutathione, was demonstrated. Cytosols prepared from normal human tissue samples of liver, breast, colon, and brain were also activated by the presence of NADPH. These data do not indicate that steroidal nonspecific esterases are redox-modulated by the presence of mixed disulfides in their structure. Instead, they support the direct and specific influence of NADPH as a widespread activator of esterase activity by a mechanism not yet understood.

Animals↗
PubMed2003

Utilisation of electrospray time-of-flight mass spectrometry for solving complex fragmentation patterns: application to benzoxazinone derivatives.

In this paper we describe the application of electrospray time-of-flight mass spectrometry (ESI-TOFMS) to structural elucidation of the fragment ions formed from a range of natural and synthetic allelochemical derivatives. The extensive mass spectrometric characterisation of ten non-glucosylated benzoxazinone derivatives using this method is described here for the first time. The analytes include six naturally occurring 1,4-benzoxazin-3(4H)-one derivatives, including the hydroxamic acids DIMBOA [2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one] and DIBOA [2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one], lactams HBOA [2-hydroxy-2H-1,4-benzoxazin-3(4H)-one] and HMBOA [2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one], benzoxazolinones BOA [benzoxazolin-2(3H)-one] and MBOA [6-methoxy-benzoxazolin-2(3H)-one] and four synthetic variations, 2'H-DIBOA [4-hydroxy-2H-1,4-benzoxazin-3(4H)-one], 2'OMe-DIBOA [2-methoxy-4-hydroxy-2H-1,4-benzoxazin-3(4H)-one], 2'H-HBOA [2H-1,4-benzoxazin-3(4H)-one] and 2'OMe-HBOA [2-methoxy-2H-1,4-benzoxazin-3(4H)-one]. Assignments of the mass spectral fragments were aided by elemental composition calculation results, comparison of structural analogues and background literature, and acquired knowledge regarding feasible structures for the compounds. The influence of substituents on the chemical reactivity of the compounds with respect to the observed MS behaviour over varying nozzle potentials is addressed and, through comparison of the structural analogues, generic fragmentation patterns have also been identified.

Benzene Derivatives↗
PubMed1977

New synthesis of 1,4,2-dioxazoles and their pharmacological properties.

A series of 3-substituted 5-methoxycarbonyl-5-methoxycarbonylmethyl-1,4,2-dioxazoles was prepared by addition of hydroxamic acids to acetylene esters. Eleven of these previously unknown compounds were submitted to general pharmacological screening, and several displayed modest CNS depressant activity.

Aggression↗
PubMed1995

Recombinant porcine collagenase: purification and autolysis.

Collagenase is a member of the matrix metalloproteinase family whose members are all capable of degrading extracellular matrix components. The mature form of porcine collagenase has been expressed in Escherichia coli using the pAX5 expression vector. The fusion protein consists of beta-galactosidase at the N-terminus joined to a collagen hinge region and a blood-coagulation factor Xa cleavage site linked to an active form of collagenase. Recombinant collagenase was biologically active in the form of a fusion protein; this was cleaved with factor Xa to yield collagenase with the authentic N terminus (phenylalanine) found in vivo and purified in a single step on a peptide hydroxamic acid affinity column. On purification the recombinant porcine collagenase undergoes autolysis at a number of different bonds in the region connecting the active site domain with the C-terminal hemopexin-like domain. This may represent a loop region of poor secondary structure, making it susceptible to relatively nonspecific cleavage. The N-terminal fragment retains a reduced level of collagenolytic activity, along with that against casein and gelatin.

Amino Acid Sequence↗
PubMed1999

A fluorescence resonance energy transfer method for measuring the binding of inhibitors to stromelysin.

A sensitive fluorescence resonance energy transfer method was developed for the direct measurement of the dissociation constants of stromelysin inhibitors. The method is applied to the thiadiazole class of stromelysin inhibitors and it takes advantage of the fact that, upon binding to the active site of enzyme, the thiadiazole ring, with its absorbance centered at 320 nm, is able to quench the fluorescence of the tryptophan residues surrounding the catalytic site. The changes in fluorescence are proportional to the occupancy of the active site: Analysis of the fluorescence versus inhibitor concentration data yields dissociation constants that are in agreement with the corresponding competitive inhibitory constants measured by a catalytic rate assay. The affinity of nonthiadiazole inhibitors of stromelysin-such as hydroxamic acids and others-can be determined from the concentration-dependent displacement of a thiadiazole of known affinity. Using this displacement method, we determined the affinities of a number of structurally diverse inhibitors toward stromelysin. Since the three tryptophan residues located in the vicinity of the active site of stromelysin are conserved in gelatinase and collagenase, the method should also be applicable to inhibitors of other matrix metalloproteinases.

Catalytic Domain↗
PubMed1995

Stabilisation of purified human collagenase by site-directed mutagenesis.

During purification, human fibroblast collagenase breaks down into two major forms, an N-terminal 22000/25000-Mr fragment and a C-terminal 27000-Mr fragment; the most likely mechanism being autolysis. The cleavage site has been identified (Pro269- Ile270) and in an attempt to obtain full-length human collagenase (i.e., Mr 42570), this cleavage site and another potential cleavage site (Ala258- Ile259) have been mutated by PCR- directed mutagenesis: Ile270Ser and Ile259Leu. The mutated cDNA was then cloned into the expression vector, pGEX2T, and expressed in Escherichia coli as a fusion protein with glutathione-S-transferase (GST). After cleavage with factor Xa, the mutated collagenase was purified on a peptide hydroxamic acid affinity column. The mutated recombinant collagenase is stable, remains full length and retains the ability to cleave collagen.

Alanine↗
PubMed2002

A functional role for a flexible loop containing Glu182 in the class II fructose-1,6-bisphosphate aldolase from Escherichia coli.

Class II fructose 1,6-bisphosphate aldolases (FBP-aldolases) catalyse the zinc-dependent, reversible aldol condensation of dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (G3P) to form fructose 1,6-bisphosphate (FBP). Analysis of the structure of the enzyme from Escherichia coli in complex with a transition state analogue (phosphoglycolohydroxamate, PGH) suggested that substrate binding caused a conformational change in the beta5-alpha7 loop of the enzyme and that this caused the relocation of two glutamate residues (Glu181 and Glu182) into the proximity of the active site. Site-directed mutagenesis of these two glutamate residues (E181A and E182A) along with another active site glutamate (Glu174) was carried out and the mutant enzymes characterised using steady-state kinetics. Mutation of Glu174 (E174A) resulted in an enzyme which was severely crippled in catalysis, in agreement with its position as a zinc ligand in the enzyme's structure. The E181A mutant showed the same properties as the wild-type enzyme indicating that the residue played no major role in substrate binding or enzyme catalysis. In contrast, mutation of Glu182 (E182A) demonstrated that Glu182 is important in the catalytic cycle of the enzyme. Furthermore, the measurement of deuterium kinetic isotope effects using [1(S)-(2)H]DHAP showed that, for the wild-type enzyme, proton abstraction was not the rate determining step, whereas in the case of the E182A mutant this step had become rate limiting, providing evidence for the role of Glu182 in abstraction of the C1 proton from DHAP in the condensation direction of the reaction. Glu182 lies in a loop of polypeptide which contains four glycine residues (Gly176, Gly179, Gly180 and Gly184) and a quadruple mutant (where each glycine was converted to alanine) showed that flexibility of this loop was important for the correct functioning of the enzyme, probably to change the microenvironment of Glu182 in order to perturb its pK(a) to a value suitable for its role in proton abstraction. These results highlight the need for further studies of the dynamics of the enzyme in order to fully understand the complexities of loop closure and catalysis in this enzyme.

Amino Acid Substitution↗
PubMed1987

The in vitro/in vivo comparative metabolism of 4-aminobiphenyl using isolated hepatocytes.

The metabolism of 4-aminobiphenyl by isolated hepatocytes from various species was compared with urinary metabolite profiles in the same species. Radioactive compounds in concentrates of ether extracts from hepatocytes or urine following hydrolysis were analysed by TLC and reversed phase HPLC in conjunction with radioactivity monitoring and synthetic standards. The major metabolites from hepatocytes and in urine were 4-acetamidobiphenyl, 3-hydroxy-4-aminobiphenyl 4'-hydroxy-4-aminobiphenyl and 4'-hydroxy-4-acetamidobiphenyl. Oxidation of the amine nitrogen gave hydroxylamino, nitroso and nitro compounds. Minor metabolites were 2'-hydroxy amine and amide, the hydroxamic acid and the oxamic acid. The urinary metabolite profiles correlated well with those from hepatocytes for each species.

Aminobiphenyl Compounds↗
PubMed1987

Interleukin-1 induces chondrocyte protease production: the development of collagenase inhibitors.

Supernatants from the P388D1 macrophage cell line as well as human interleukin-1 (IL-1) stimulated primary rabbit articular chondrocytes to produce collagen- (C-ase) and proteoglycan- (PG-ase) degrading proteases. The P388D1 derived factor had a molecular weight of 16,000-20,000 and a pI of 4.5-5.0. Both protease activities were metal dependent and inhibited by EDTA, phenanthroline, and alpha 2-macroglobulin but not by PMSF, TLCK, pepstatin, or alpha 1-antitrypsin. Size exclusion chromatography indicated the molecular weights for latent PG-ase and C-ase were 44,000-56,000 and 34,000-44,000, respectively. Chemical synthesis efforts produced two classes of C-ase inhibitors--thiols and hydroxamic acids. The former had IC50 values of 10(-5)-10(-6) M while the latter approached 10(-7) M.

Animals↗