Irreversible Inhibitor (irreversible + inhibitor)

Distribution by Scientific Domains
Distribution within Chemistry


Selected Abstracts


Structural Modifications of (1S,3S)-3-Amino-4-difluoromethylenecyclopentanecarboxylic Acid, a Potent Irreversible Inhibitor of GABA Aminotransferase.

CHEMINFORM, Issue 31 2007
Hai Yuan
Abstract ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF. [source]


Two New Irreversible Inhibitors of Dihydrodipicolinate Synthase: Diethyl (E,E)-4-Oxo-2,5-heptadienedioate and Diethyl (E)-4-Oxo-2-heptenedioate.

CHEMINFORM, Issue 25 2005
Jennifer J. Turner
Abstract For Abstract see ChemInform Abstract in Full Text. [source]


Discovery of Selective Irreversible Inhibitors for Bruton's Tyrosine Kinase

CHEMMEDCHEM, Issue 1 2007
Zhengying Pan Dr.
A series of highly selective irreversible inhibitors for Bruton's tyrosine kinase (Btk) was developed using a structural bioinformatics approach. Their capabilities to modulate Btk's activity were characterized both in,vitro and in,vivo. Oral treatment with once-a-day dosing of compound 4 greatly inhibited disease development in a rodent rheumatoid arthritis (RA) model. [source]


The effect of thiamine supplementation on tumour proliferation

FEBS JOURNAL, Issue 15 2001
A metabolic control analysis study
Thiamine deficiency frequently occurs in patients with advanced cancer and therefore thiamine supplementation is used as nutritional support. Thiamine (vitamin B1) is metabolized to thiamine pyrophosphate, the cofactor of transketolase, which is involved in ribose synthesis, necessary for cell replication. Thus, it is important to determine whether the benefits of thiamine supplementation outweigh the risks of tumor proliferation. Using oxythiamine (an irreversible inhibitor of transketolase) and metabolic control analysis (MCA) methods, we measured an in vivo tumour growth control coefficient of 0.9 for the thiamine-transketolase complex in mice with Ehrlich's ascites tumour. Thus, transketolase enzyme and thiamine clearly determine cell proliferation in the Ehrlich's ascites tumour model. This high control coefficient allows us to predict that in advanced tumours, which are commonly thiamine deficient, supplementation of thiamine could significantly increase tumour growth through transketolase activation. The effect of thiamine supplementation on tumour proliferation was demonstrated by in vivo experiments in mice with the ascites tumour. Thiamine supplementation in doses between 12.5 and 250 times the recommended dietary allowance (RDA) for mice were administered starting on day four of tumour inoculation. We observed a high stimulatory effect on tumour growth of 164% compared to controls at a thiamine dose of 25 times the RDA. This growth stimulatory effect was predicted on the basis of correction of the pre-existing level of thiamine deficiency (42%), as assayed by the cofactor/enzyme ratio. Interestingly, at very high overdoses of thiamine, ,,2500 times the RDA, thiamine supplementation had the opposite effect and caused 10% inhibition of tumour growth. This effect was heightened, resulting in a 36% decrease, when thiamine supplementation was administered from the 7th day prior to tumour inoculation. Our results show that thiamine supplementation sufficient to correct existing thiamine deficiency stimulates tumour proliferation as predicted by MCA. The tumour inhibitory effect at high doses of thiamine is unexplained and merits further study. [source]


Cytochrome c oxidase isoform IV-2 is involved in 3-nitropropionic acid-induced toxicity in striatal astrocytes

GLIA, Issue 14 2009
Shilpee Singh
Abstract Astrocyte mitochondria play an important role for energy supply and neuronal survival in the brain. Toxic and degenerative processes are largely associated with mitochondrial dysfunction. We, therefore, investigated the effect of 3-nitropropionic acid (NPA), a mitochondrial toxin and in vitro model of Huntington's disease (HD), on mitochondrial function and viability of primary striatal astrocytes. Although NPA is known as an irreversible inhibitor of succinate dehydrogenase, we observed an increase of astrocyte ATP levels after NPA treatment. This effect could be explained by NPA-mediated alterations of cytochrome c oxidase subunit IV isoform (COX IV) expression. The up-regulation of COX isoform IV-2 caused an increased enzyme activity at the expense of elevated mitochondrial peroxide production causing increased cell death. The application of a small interfering RNA against COX IV-2 revealed the causal implication of COX isoform IV-2 in NPA-mediated elevation of oxidative stress and necrotic cell death. Thus, we propose a novel, additional mechanism of NPA-induced cell stress and death which is based on structural and functional changes of astrocyte COX and which could indirectly impair neuronal survival. © 2009 Wiley-Liss, Inc. [source]


Synthesis of Monosaccharide-Derived Spirocyclic Cyclopropylamines and Their Evaluation as Glycosidase Inhibitors

HELVETICA CHIMICA ACTA, Issue 9 2003
Christian Blüchel
The glucose-, mannose-, and galactose-derived spirocyclic cyclopropylammonium chlorides 1a,1d, 2a,2d and 3a,3d were prepared as potential glycosidase inhibitors. Cyclopropanation of the diazirine 5 with ethyl acrylate led in 71% yield to a 4,:,5,:,1,:,20 mixture of the ethyl cyclopropanecarboxylates 7a,7d, while the Cu-catalysed cycloaddition of ethyl diazoacetate to the exo -glycal 6 afforded 7a,7d (6,:,2,:,5,:,3) in 93,98% yield (Scheme,1). Saponification, Curtius degradation, and subsequent addition of BnOH or t- BuOH led in 60,80% overall yield to the Z- or Boc-carbamates 11a,11d and 12a,12d, respectively. Hydrogenolysis of 11a,11d afforded 1a,1d, while 12a,12d was debenzylated to 13a,13d prior to acidic cleavage of the N -Boc group. The manno - and galacto -isomers 2a,2d and 3a,3d, respectively, were similarly obtained in comparable yields (Schemes,2 and 4). Also prepared were the differentially protected manno- configured esters 24a,24d; they are intermediates for the synthesis of analogous N -acetylglucosamine-derived cyclopropanes (Scheme,3). The cyclopropylammonium chlorides 1a,1d, 2a,2d and 3a,3d are very weak inhibitors of several glycosidases (Tables,1 and 2). Traces of Pd compounds, however, generated upon catalytic debenzylation, proved to be strong inhibitors. PdCl is, indeed, a reversible, micromolar inhibitor for the ,- glucosidases from C. saccharolyticum and sweet almonds (non-competitive), the , -galactosidases from bovine liver and from E. coli (both non-competitive), the , -galactosidase from Aspergillus niger (competitive), and an irreversible inhibitor of the , -glucosidase from yeast and the , -galactosidase from coffee beans. The cyclopropylamines derived from 1a,1d or 3a,3d significantly enhance the inhibition of the ,- glucosidase from C. saccharolyticum by PdCl, lowering the Ki value from 40,,M (PdCl) to 0.5,,M for a 1,:,1 mixture of PdCl and 1d. A similar effect is shown by cyclopropylamine, but not by several other amines. [source]


Rat mast cell protease-I enhances immunoglobulin E production by mouse B cells stimulated with interleukin-4

IMMUNOLOGY, Issue 3 2001
Tsutomu Yoshikawa
Summary Mast cell chymase plays important roles in inflammation and tissue remodeling. Here we show that mast cell chymase also functions as an enhancer of immunoglobulin production. In the culture of murine spleen cells stimulated with lipopolysaccharide and interleukin-4, purified rat chymase (rat mast cell protease-I; RMCP-I), at physiological concentrations, enhanced immunoglobulin E (IgE) and IgG1 syntheses but not IgG3 synthesis. The enhancement was also evident when spleen cells depleted of T cells and macrophages were employed as responding cells. Enzymatic activity of RMCP-I was required to enhance IgE and IgG1, because two inhibitors for chymotryptic enzymes, chymostatin and Y-40613, a novel chymase inhibitor, suppressed the enhanced immunoglobulin production, and phenylmethylsulphonyl fluoride, an irreversible inhibitor for serine proteases, totally abolished the enhancing effect. Furthermore, a specific inhibitor for Zn2+ -dependent metalloproteases, GI 129471, could also completely inhibit the production of IgE and IgG1 that was enhanced by RMCP-I, suggesting that a metalloprotease also played an essential role in the immunoglobulin production. Our results together with others show that proteases from mast cell granules have important function not only in the efferent phase but also in the afferent phase of immune responses. [source]


Atrazine-induced changes in aromatase activity in estrogen sensitive target tissues

JOURNAL OF APPLIED TOXICOLOGY, Issue 3 2008
A. C. Holloway
Abstract Atrazine (ATR) is a pesticide used widely throughout North America. Although not directly estrogenic, ATR treatment has been shown to increase aromatase activity in tumor cell lines. Thus, it is suggested that ATR can increase local tissue estrogen levels in estrogen sensitive target tissues through increased aromatase activity. Therefore the effect of ATR on aromatase activity was measured in human granulosa-lutein cell cultures, cells that abundantly express aromatase, and endometrial stromal cell (ESC) cultures, cells that do not express aromatase. Aromatase activity was quantified by the tritiated water method and the specificity of the assay was confirmed by co-incubation with 4-hydroxyandrostenedione, an irreversible inhibitor of the catalytic activity of aromatase. Aromatase activity in ATR treated (1,10 µm) granulosa-lutein cells was increased more than 2-fold compared with control cultures. There were no treatment related changes in cellular protein and thus it is suggested that the ATR-induced change in aromatase activity was not due to an increase in cell number. ATR-treatment had no effect on ESC aromatase activity at any concentration tested. Similarly, there was no effect of ATR treatment on human recombinant aromatase activity in our cell-free test system. Therefore it is concluded that µm concentrations of ATR can increase aromatase activity of human granulosa cells but not ESC and this effect is not elicited at the enzyme level. Copyright © 2007 John Wiley & Sons, Ltd. [source]


Protection against acetaminophen hepatotoxicity by clofibrate pretreatment: Role of catalase induction,

JOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 5 2002
Chuan Chen
Abstract Mice pretreated with the peroxisome proliferator clofibrate (CFB) are highly resistant to acetaminophen (APAP)-induced hepatotoxicity. The objective of the present study was to investigate whether the increase in hepatic catalase activity following CFB pretreatment plays a role in this hepatoprotection. An irreversible inhibitor, 3-amino-1,2,4-triazole (3-AT), was used to modulate catalase activity. Hepatic catalase activity in mice pretreated with CFB (500 mg/kg, i.p., for 10 days) was significantly inhibited by 3-AT (100 or 500 mg/kg, i.p.). In addition, the lower dose of 3-AT (100 mg/kg) had minimal effect on biliary and urinary excretion of APAP metabolites generated from a nontoxic dose, suggesting that APAP metabolism was not modulated by this dose of 3-AT. The mortality rate of corn-oil-pretreated mice challenged with APAP (800 mg/kg, p.o.) was significantly increased by 3-AT (100 mg/kg, i.p.) given 1 h before APAP. As expected, CFB pretreatment conferred full protection against APAP-induced hepatotoxicity. The same 3-AT treatment, however, did not abolish hepatoprotection in CFB-pretreated mice, despite the marked inhibition of hepatic catalase activity. In conclusion, these results indicate that elevated catalase activity in mice exposed to CFB does not appear to mediate the hepatoprotection, suggesting that other cellular defense mechanisms are involved. © 2002 Wiley Periodicals, Inc. J Biochem Mol Toxicol 16:227,234, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10043 [source]


Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells

JOURNAL OF NEUROCHEMISTRY, Issue 5 2007
Xingshun Xu
Abstract Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson's disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate-induced oxytosis. In this study, we explore the protective effects of necrostatin-1 (Nec-1), an inhibitor of necroptosis, on glutamate-induced oxytosis. We show that Nec-1 inhibits glutamate-induced oxytosis in HT-22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec-1 had no protective effect on free radical-induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec-1 has no antioxidant effects. Interestingly, the protective effect of Nec-1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec-1 significantly blocks the nuclear translocation of apoptosis-inducing factor (a marker of caspase-independent programmed cell death) and inhibits the integration of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (a pro-death member of the Bcl-2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec-1 prevents glutamate-induced oxytosis in HT-22 cells through GSH related as well as apoptosis-inducing factor and Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-related pathways. [source]


Induction of hypoxia inducible factor-1 attenuates metabolic insults induced by 3-nitropropionic acid in rat C6 glioma cells

JOURNAL OF NEUROCHEMISTRY, Issue 3 2005
Ya-Ting Yang
Abstract Compromised mitochondrial function in neurons and glia has been observed in several neurodegenerative disorders, including Huntington's disease and Alzheimer's disease. Chemical/hypoxic preconditioning may afford protection against subsequently more severe oxidative damages. In this study, we tested whether induction of hypoxia inducible factor-1 (HIF-1) may exert cytoprotective effects against mitochondrial dysfunction caused by 3-nitropropionic acid (3-NP) in glial cells. Preconditioning of C6 astroglial cells with cobalt chloride, mimosine (MIM), and desferrioxamine (DFO), all of which known to activate HIF-1, significantly attenuated cytotoxicity induced by 3-NP, an irreversible inhibitor of mitochondrial complex II, and antimycin A, a mitochondrial complex III inhibitor. Application of cadmium chloride capable of neutralizing cobalt-induced HIF-1 activation, HIF-specific oligodeoxynucleotide (ODN) decoy, and antisense phosphorothioate ODN against HIF-1, abolished the protective effect mediated by preconditioning with cobalt chloride. Preloading of C6 cells with SN50, PD98059, or SB202190, the respective inhibitor of nuclear factor-,B (NF-,B), p44/p42 extracellular signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (MAPK), failed to affect the protection afforded by cobalt preconditioning. Taken together, these results suggest that HIF-1 induction secondary to preconditioning with cobalt chloride or iron chelators may mediate the protective effects against metabolic insult induced by the mitochondrial inhibitor 3-NP in C6 astroglial cells. [source]


Impaired Mitochondrial Function Results in Increased Tissue Transglutaminase Activity In Situ

JOURNAL OF NEUROCHEMISTRY, Issue 5 2000
Mathieu Lesort
Abstract: Tissue transglutaminase (tTG) is a transamidating enzyme that is elevated in Huntington's disease (HD) brain and may be involved in the etiology of the disease. Further, there is evidence of impaired mitochondrial function in HD. Therefore, in this study, we examined the effects of mitochondrial dysfunction on the transamidating activity of tTG. Neuroblastoma SH-SY5Y cells stably overexpressing human tTG or mutated inactive tTG were treated with 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase. 3-NP treatment of tTG-expressing cells resulted in a significant increase of TG activity in situ. In vitro measurements demonstrated that 3-NP had no direct effect on tTG activity. However, 3-NP treatment resulted in a significant decrease of the levels of GTP and ATP, two potent inhibitors of the transamidating activity of tTG. No significant changes in the intracellular levels of calcium were observed in 3-NP-treated cells. Treatment with 3-NP in combination with antioxidants significantly reduced the 3-NP-induced increase in in situ TG activity, demonstrating that oxidative stress is a contributing factor to the increase of TG activity. This study demonstrates for the first time that impairment of mitochondrial function significantly increases TG activity in situ, a finding that may have important relevance to the etiology of HD. [source]


Effect of Exogenous and Endogenous Antioxidants on 3-Nitropionic Acid-Inducedin vivo Oxidative Stress and Striatal Lesions

JOURNAL OF NEUROCHEMISTRY, Issue 4 2000
Insights into Huntington's Disease
Abstract: 3-Nitropropionic acid (3-NP) is an irreversible inhibitor of complex II in the mitochondria. 3-NP toxicity has gained acceptance as an animal model of Huntington's disease (HD). In the present study, we confirmed that rats injected with 3-NP (20 mg/kg, i.p., daily for 4 days) exhibit increased oxidative stress in both striatum and cortical synaptosomes as well as lesions in the striatum. Synaptosomal membrane proteins from rats injected with 3-NP exhibited a decrease in W/S ratio, the relevant electron paramagnetic resonance (EPR) parameter used to determine levels of protein oxidation, and western blot analysis for protein carbonyls revealed direct evidence of increased synaptosomal protein oxidation. Treatment of rats with the brain-accessible free radical spin trap 5-diethoxyphosphoryl-5-methyl-1-pyrroline N -oxide (DEPMPO; 30 mg/kg, i.p., daily 2 h before 3-NP injection) or with N -acetylcysteine (NAC; 100 mg/kg, i.p., daily 2 h before 3-NP injection), a known glutathione precursor, before 3-NP treatments protects against oxidative damage induced by 3-NP as measured by EPR and western blot analysis for protein carbonyls. Furthermore, both DEMPMPO and NAC treatments before 3-NP administration significantly reduce striatal lesion volumes. These data suggest oxidative damage is a prerequisite for striatal lesion formation and that antioxidant treatment may be a useful therapeutic strategy against 3-NP neurotoxicity and perhaps against HD as well. [source]


GABAergic Modulation of the Expression of Genes Involved in GABA Synaptic Transmission and Stress in the Hypothalamus and Telencephalon of the Female Goldfish (Carassius auratus)

JOURNAL OF NEUROENDOCRINOLOGY, Issue 5 2005
C. J. Martyniuk
Abstract GABA is one of the most abundant neurotransmitters in the vertebrate central nervous system and is involved in neuroendocrine processes such as development, reproduction, feeding and stress. To examine the effect of GABA on gene expression in the brain, we used a cDNA macroarray containing 26 genes involved in GABA synaptic transmission (GABA receptor subunits, GABA transporters), reproduction (gonadotrophin-releasing hormone isoforms and oestrogen receptor ,), feeding (neuropeptide Y and cholecystokinin), and stress [corticotrophin-releasing factor (CRF)]. To elevate GABA levels in the brain, we injected female goldfish with gamma-vinyl GABA (300 µg/g of body weight) (24 h), an irreversible inhibitor of the enzyme GABA transaminase (GABA-T). We found that increased levels of GABA in the hypothalamus resulted in a 2.2-fold down-regulation of GABAA receptor ,4 subunit mRNA. In the telencephalon, we found that increased GABA levels resulted in a 1.5-fold increase of CRF mRNA and a 1.8-fold decrease of GABAA receptor ,2 subunit mRNA. Increasing GABA in the hypothalamus and telencephalon of the goldfish did not significantly affect the mRNA abundance of genes involved in GABA synthesis (glutamic acid decarboxylase isoforms) and degradation (GABA-T), feeding, or reproduction. Our preliminary study suggests that the regulation of GABA receptor subunit mRNA expression by GABA may be a conserved evolutionary mechanism in vertebrates to modulate GABAergic synaptic transmission. [source]


In Vivo use of the P450 inactivator 1-aminobenzotriazole in the rat: Varied dosing route to elucidate gut and liver contributions to first-pass and systemic clearance

JOURNAL OF PHARMACEUTICAL SCIENCES, Issue 6 2006
Timothy J. Strelevitz
Abstract The small intestine is regarded as an absorptive organ in the uptake of orally administered drugs, but also has the ability to metabolize drugs by both phase 1 and phase 2 reactions. The amount of drug that reaches the systemic circulation can be reduced by both intestinal and hepatic metabolism. 1-Aminobenzotriazole (ABT) is an irreversible inhibitor of cytochrome P450s. Through in vivo and in vitro studies, ABT has been evaluated for its utility in studying intestinal metabolism in rats. Rats have been exposed to ABT through varied routes of administration followed by p.o. and i.v. administration of midazolam (MDZ), a CYP3A substrate. The MDZ bioavailablity in rats dosed orally and in rats dosed intravenously with ABT is 58.5% and 0.7%, respectively (%F,=,2.3% w/o ABT). The approximately 80-fold difference between the two groups suggests the majority of the extraction occurs in the intestine following an oral dose. To further study the utility of ABT, the antihistamine fexofenadine (Fex), which is not significantly metabolized and is a substrate for the uptake and efflux transporters, OATP and P-gp, was tested in rat. There was no change in oral or systemic exposure of Fex when animals were predosed with ABT, suggesting that ABT does not affect these transporters. These findings may lead to a better understanding of the interdependent role of absorption and metabolism and the specificity of ABT. This method should have utility in drug discovery for the identification of factors limiting oral bioavailability. © 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:1334,1341, 2006 [source]


Increased vigabatrin entry into the brain by polysorbate 80 and sodium caprate

JOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 2 2001
D. Dimitrijevic
The effects of a non-ionic surfactant, polysorbate 80, and the sodium salt of the saturated fatty acid, sodium caprate (C10), as potential brain absorption enhancers for vigabatrin were studied. Vigabatrin is an enzyme-activated irreversible inhibitor of ,-aminobutyric acid (GABA) transaminase that increases brain and cerebrospinal GABA concentrations in animals and man. Before intravenous administration, a range of concentrations of the surfactants were tested using erythrocyte lysis or the red blood cell lysis test to establish the non-toxic concentration range. Vigabatrin was dissolved in 0.1% polysorbate 80 and 0.1% sodium caprate and administered intravenously in doses of 4 mL kg,1 to male Wistar rats (230,250 g; n = 3). Rats were killed 2 h after drug and surfactant administration and the brains were immediately removed and homogenized in 0.4m perchloric acid. Selected ion monitoring electrospray mass spectrometry was used to determine the concentration of vigabatrin and GABA directly from the perchloric acid extract of the rat brain. This method was developed to increase the speed and efficiency of the analysis by removing the need for complex extraction and derivatization procedures while retaining the specificity of the mass spectrometer as a detector. The stability of both vigabatrin and GABA in perchloric acid was established by monitoring their pseudo molecular ions in standard solutions at timed intervals over 24 h. Although the detection level for vigabatrin and GABA was at least 50 pg, only GABA was detected in rat brain. Vigabatrin caused a small increase in whole brain GABA. However, GABA levels were higher in the samples with vigabatrin + enhancer than in the samples where vigabatrin alone was administered. One-way analysis of variance indicated a significant effect of the surfactants on GABA levels (F (5,17) = 11.86, P < 0.01) and vigabatrin absorption was presumed. The rectal temperature of the rats is lowered by the presence of vigabatrin in the brain. Vigabatrin alone decreased rectal temperature by 6%. When given with either polysorbate 80 or sodium caprate, the extent of temperature lowering was significantly greater (P < 0.001). There was no significant difference after 2 h between polysorbate 80 + vigabatrin, and sodium caprate + vigabatrin. [source]


CONTRASTING EFFECTS OF METHIONINE SULFOXIMINE ON UPTAKE AND ASSIMILATION OF AMMONIUM IN ULVA INTESTINALIS (CHLOROPHYCEAE),

JOURNAL OF PHYCOLOGY, Issue 4 2004
Neill G. Barr
Ammonium is assimilated in algae by the glutamine synthetase (GS),glutamine:2-oxoglutarate aminotransferase pathway. In addition to the assimilation of external ammonium taken up across the cell membrane, an alga may have to reassimilate ammonium derived from endogenous sources (i.e. nitrate reduction, photorespiration, and amino acid degradation). Methionine sulfoximine (MSX), an irreversible inhibitor of GS, completely inhibited GS activity in Ulva intestinalis L. after 12 h. However, assimilation of externally derived ammonium was completely inhibited after only 1,2 h in the presence of MSX and was followed by production of endogenous ammonium. However, endogenous ammonium production in U. intestinalis represented only a mean of 4% of total assimilation attributable to GS. The internally controlled rate of ammonium uptake (Vi) was almost completely inhibited in the presence of MSX, suggesting that Vi is a measure of the maximum rate of ammonium assimilation. After complete inhibition of ammonium assimilation in the presence of MSX, the initial or surge (Vs) rate of ammonium uptake in the presence of 400 ,M ammonium chloride decreased by only 17%. However, the amount that the rate of ammonium uptake decreased by was very similar to the uninhibited rate of ammonium assimilation. In addition, the decrease in the rate of ammonium uptake in darkness (in the absence of MSX) in the presence of 400 ,M ammonium chloride matched the decrease in the rate of ammonium assimilation. However, in the presence of 10 ,M ammonium chloride, MSX completely inhibited ammonium assimilation but had no effect on the rate of uptake. [source]


Complete inhibition of fibrinolysis by sustained carboxypeptidase B activity: the role and requirement of plasmin inhibitors

JOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 6 2007
J. B. WALKER
Summary.,Background:,The antifibrinolytic effect of activated thrombin-activatable fibrinolysis inhibitor (TAFIa) and carboxypeptidase B (CPB) displays threshold behavior. When CPB was used to simulate conditions mimicking continuous TAFIa activity, it affected the lysis of plasma clots differently to clots formed from a minimal fibrinolytic system comprising fibrinogen, plasminogen and ,2 -antiplasmin. Whereas CPB saturably prolonged clot lysis in the purified system, the effect of CPB did not appear saturable in plasma clots. Methods:,To rationalize this difference, we investigated the effects of ,2 -antiplasmin, ,2 -macroglobulin, antithrombin and aprotinin on CPB-mediated antifibrinolysis. Results:,CPB alone prolonged fibrinolysis in a saturable manner and the efficacy of CPB increased with decreasing tissue-type plasminogen activator (t-PA) concentration. The inhibitors by themselves did not halt fibrinolysis and the potency of each inhibitor in the absence of CPB mirrored their solution-phase plasmin inhibitory potentials: ,2 -antiplasmin , aprotinin >> ,2 -macroglobulin >> antithrombin. With both CPB and inhibitor present, a synergistic effect was observed. The antifibrinolytic sensitivity to CPB was related to the plasmin inhibitory potential of the inhibitor. Conclusions:,Fibrinolysis could be completely inhibited by ,2 -antiplasmin, ,2 -macroglobulin and antithrombin, but not aprotinin, in the presence of CPB, and occurred only when the irreversible inhibitor or pool of inhibitors were in excess of plasminogen. Western blot analysis indicated that the CPB-mediated shutdown of fibrinolysis was a result of plasminogen consumption prior to clot lysis. The CPB concentration required for fibrinolytic shutdown was dependent on t-PA concentration and the inhibitory potential of the irreversible inhibitor pool. [source]


L-histidine decarboxylase as a probe in studies on histamine

THE CHEMICAL RECORD, Issue 6 2002
Takehiko Watanabe
Abstract Because the Falck-Hillarp formaldehyde fluorescence method, which was superbly applied to identify catecholaminergic and serotonergic neurons, is not applicable to histamine, the first author (T.W.) developed an antibody to L-histidine decarboxylase (HDC) for identification of the histaminergic neuron system in the brain. The anti-HDC antibody was of great use for mapping the location and distribution of this histaminergic neuron system. (S)-,-fluoromethylhistidine, a specific and potent irreversible inhibitor of HDC, was also very useful in studies on functions of the neuron system. The activity of HDC is increased by various agents, treatments, and physiological conditions. We found new compounds that increased HDC activity (i.e., tetradecanoylphobol acetate (TPA), other tumor promoters, and staphylococcal enterotoxin A); and using mast cell-deficient mutant (W/Wv) mice, we obtained evidence that this increase occurred in macrophages. To further characterize the mechanism of increases in HDC activity, the second author (H.O.) cloned human HDC cDNA and a human HDC gene. In studies on the regulation mechanism of the HDC gene, which is expressed only in limited types of cells such as mast cells, enterochromaffin-like cells in the stomach, cells in the tuberomammillary nucleus of the brain, and macrophages, CpG islands in the promoter region of the HDC gene were found to be demethylated in cells expressing the gene, whereas they are methylated in other cells that do not express the HDC gene. In collaboration with many other researchers, we developed HDC knockout mice. The resulting research is producing a lot of interesting findings in our laboratory as well as in others. In summary, HDC has been and will be useful in studies on functions of histamine. © 2002 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 2: 369,376, 2002: Published online in Wiley InterScience (www.interscience.wiley.com) DOI 10.1002/tcr.10036 [source]


Structure of human semicarbazide-sensitive amine oxidase/vascular adhesion protein-­1

ACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2005
Joakim Nilsson
Semicarbazide-sensitive amine oxidase (SSAO) belongs to a ubiquitous family of copper-containing amine oxidases (CuAOs). SSAO is also known as vascular adhesion protein-­1 (VAP-1) and has been identified as one of the adhesion molecules involved in the leukocyte-extravasation process. The structure of a truncated soluble form of human SSAO has been solved and refined to 2.5,Å. As expected, SSAO is a homodimer with a fold typical of the CuAO family. The topaquinone (TPQ) cofactor and a copper ion characteristic of CuAOs are present in the active site, with the TPQ in the active `off-copper' conformation. The structure reveals that a leucine residue (Leu469) located adjacent to the active site could function as a gate controlling its accessibility. An RGD motif is displayed on the surface, where it could be involved in integrin binding and possibly play a role in the shedding of SSAO from the membrane. Carbohydrate moieties are observed at five of six potential N-glycosylation sites. Carbohydrates attached to Asn232 flank the active-site entrance and might influence substrate specificity. The structure of an adduct of SSAO and the irreversible inhibitor 2-hydrazinopyridine has been solved and refined to 2.9,Å resolution. Together, these structures will aid efforts to identify natural substrates, provide valuable information for the design of specific inhibitors and direct further studies. [source]


SERCA activity is required for timely progression through G1/S

CELL PROLIFERATION, Issue 1 2001
V. R. Simon
Changes in intracellular Ca2+ correlate with specific events in the cell cycle. Here we investigated the role of Ca2+ in the G1 phase. HEK 293 cells were arrested in mitosis and subjected to short-term treatments that alter Ca2+ homeostasis prior to their release into G1. Treatment with thapsigargin (TG), an irreversible inhibitor of the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) lengthened the G1 phase. Moreover, TG treatment also resulted in a dramatic alteration in cellular morphology and attachment and in the reduction of MAPK activity and lower levels of cyclin D1 and cyclin E proteins. Treatments with reagents that transiently increase or decrease cytosolic Ca2+ or that temporarily inactivate SERCA did not alter any of the above parameters. Cells expressing a TG-resistant form of SERCA progressed normally through the G1/S transition after TG treatment. These results suggest that long-term SERCA inactivation affects cell cycle-dependent events and compromises progression through G1/S. [source]


Multiple Pathways for the Irreversible Inhibition of Steroid Sulfatase with Quinone Methide-Generating Suicide Inhibitors

CHEMBIOCHEM, Issue 9 2009
Vanessa Ahmed
Abstract Unexpected inhibition: 2- and 4-mono- and difluoromethyl estrone sulfate derivatives are suicide inhibitors of steroid sulfatase (STS). Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles, whereas the main inhibition pathway of the 4-difluoromethyl derivative is a result of decomposition of the initial quinone methide to an aldehyde that acts as potent, almost irreversible inhibitor. [source]


2,4-Dioxa-7-aza-, 2,4-Dioxa-8-aza-, and 2,4-Dioxa-9-aza-3-phosphadecalins as Rigid Acetylcholine Mimetics: Syntheses and Characterization

HELVETICA CHIMICA ACTA, Issue 10 2004
Stefan Furegati
Phosphorylation of suitable piperidine precursors yielded a series of novel decalin-type O,N,P-heterocycles. The title compounds, P(3)-axially and P(3)-equatorially X-substituted, cis- and trans- configurated 2,4-dioxa-7-aza-, 2,4-dioxa-8-aza-, and 2,4-dioxa-9-aza-3-phosphabicyclo[4.4.0]decane 3-oxides (X=Cl, F, 4-nitrophenoxy, and 2,4-dinitrophenoxy), are configuratively fixed and conformationally constrained P-analogues of acetylcholine and as such represent acetylcholine (7-aza and 9-aza isomers) or , -homo-acetylcholine mimetics (8-aza isomers). Being irreversible inhibitors of acetylcholinesterase (AChE), the compounds are considered to be suitable probes for the investigation of the stereochemical course of the inhibition reaction by 31P-NMR spectroscopy. Moreover, the design of these mimetics will enable studies of molecular interactions with AChE, in particular, the recognition conformation of acetylcholine. [source]


Specific reactions of S -nitrosothiols with cysteine hydrolases: A comparative study between dimethylargininase-1 and CTP synthetase

PROTEIN SCIENCE, Issue 8 2007
Oliver Braun
Abstract S-Transnitrosation is an important bioregulatory process whereby NO+ equivalents are transferred between S -nitrosothiols and Cys of target proteins. This reaction proceeds through a common intermediate R,S,N(O,),S,R, and it has been proposed that products different from S -nitrosothiols may be formed in protein cavities. Recently, we have reported on the formation of such a product, an N -thiosulfoximide, at the active site of the Cys hydrolase dimethylargininase-1 (DDAH-1) upon reaction with S -nitroso- l -homocysteine (HcyNO). Here we have addressed the question of whether this novel product can also be formed with the endogenously occurring S -nitrosothiols S -nitroso- l -cysteine (CysNO) and S -nitrosoglutathione (GSNO). Further, to explore the reason responsible for the unique formation of an N -thiosulfoximide in DDAH-1 we have expanded these studies to cytidine triphosphate synthetase (CTPS), which shows a similar active site architecture. ESI-MS and activity measurements showed that the bulky GSNO does not react with both enzymes. In contrast, S-nitrosylation of the active site Cys occurred in DDAH-1 with CysNO and in CTPS with CysNO and HcyNO. Although kinetic analysis indicated that these compounds act as specific irreversible inhibitors, no N -thiosulfoximide was formed. The reasons likely responsible for the absence of the N -thiosulfoximide formation are discussed using molecular models of DDAH-1 and CTPS. In tissue extracts DDAH was inhibited only by HcyNO, with an IC50 value similar to that of the isolated protein. Biological implications of these studies for the function of both enzymes are discussed. [source]


3-Fluoro-2,4-dioxa-3-phosphadecalins as Inhibitors of Acetylcholinesterase.

CHEMISTRY & BIODIVERSITY, Issue 3 2009
A Reappraisal of Kinetic Mechanisms, Diagnostic Methods
Abstract A systematic survey of the acetylcholine-mimetic 2,4-dioxa-3-phosphadecalins as irreversible inhibitors of acetylcholinesterase revealed hitherto overlooked properties as far as the kinetic mechanisms of interaction are concerned. As a support to past and future work in this field, we describe the kinetics of eight reaction schemes that may be found in irreversible enzyme modification and compare them with two mechanism of reversible, slow-binding inhibition. The relevant kinetic equations and their associated graphical representations are given for all mechanisms, and concrete examples illustrate their practical use. Since irreversible inhibition is a time-dependent phenomenon, kinetic analysis is greatly facilitated by fitting the appropriate integrated rate equations to reaction-progress curves by nonlinear regression. This primary scrutiny provides kinetic parameters that are indispensable tools for diagnosing the kinetic mechanism and for calculating inhibition constants. Numerical integration of sets of differential equations is an additional useful investigation tool in critical situations, e.g., when inhibitors are unstable and/or act as irreversible modifiers only temporarily. [source]


Discovery of Selective Irreversible Inhibitors for Bruton's Tyrosine Kinase

CHEMMEDCHEM, Issue 1 2007
Zhengying Pan Dr.
A series of highly selective irreversible inhibitors for Bruton's tyrosine kinase (Btk) was developed using a structural bioinformatics approach. Their capabilities to modulate Btk's activity were characterized both in,vitro and in,vivo. Oral treatment with once-a-day dosing of compound 4 greatly inhibited disease development in a rodent rheumatoid arthritis (RA) model. [source]