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PubMed2001

The aminopeptidase from Aeromonas proteolytica can function as an esterase.

The aminopeptidase from Aeromonas proteolytica (AAP) can catalyze the hydrolysis of L-leucine ethyl ester ( L-Leu-OEt) with a rate of 96 +/- 5 s-1 and a Km of 700 microM. The observed turnover number for L-Leu-OEt hydrolysis by AAP is similar to that observed for peptide hydrolysis, which is 67 +/- 5 s-1. The k(cat) values for the hydrolysis of L-Leu-OEt and L-leucine- p-nitroanilide ( L- pNA) catalyzed by AAP were determined at different pH values under saturating substrate concentrations. Construction of an Arrhenius plot from the temperature dependence of AAP-catalyzed ester hydrolysis indicates that the rate-limiting step does not change as a function of temperature and is product formation. The activation energy ( Ea) for the activated ES ester complex is 13.7 kJ mol-1, while the enthalpy and entropy of activation at 25 degrees C calculated over the temperature range 298-338 K are 11.2 kJ mol-1 and -175 J K-1 mol-1, respectively. The free energy of activation at 25 degrees C was found to be 63.4 kJ mol-1. The enthalpy of ionization was also measured and was found to be very similar for both peptide and ester substrates, yielding values of 20 kJ mol-1 for L-Leu-OEt and 25 kJ mol-1 for L- pNA. For peptide and L-amino acid ester cleavage reactions catalyzed by AAP, and 6.07, respectively. Proton inventory data suggest that two protons are transferred in the rate-limiting step of ester hydrolysis while only one is transferred in peptide hydrolysis. The combination of these data with the available X-ray crystallographic, kinetic, spectroscopic, and thermodynamic data for AAP provides new insight into the catalytic mechanism of AAP.

Aminopeptidases↗
PubMed1993

The hepatobiliary disease marker serum alanine aminopeptidase predominantly comprises an isoform of the haematological myeloid differentiation antigen and leukaemia marker CD-13/gp150.

In clinical chemistry, determination of serum alanine aminopeptidase (AAP) levels has been found to be useful for detecting or confirming biliary obstructions from either intra- or extra-hepatic disorders. In haematology, 'gp150' is a surface-expressed protein molecule, recognised by monoclonal antibodies belonging to the so-called 'Cluster of Differentiation' (CD-) 13, which has independently been found to be a useful marker of myeloid leukaemia in addition to providing potential prognostic value. The current report links these two independent research streams and provides evidence that the hepatobiliary disease marker, serum AAP, predominantly comprises a circulating isoform of 'gp150'. Thus, a (CD-13) monoclonal antibody raised to, and specifically reactive with, cell surface myeloid 'gp150' is able to specifically and almost completely block serum (or plasma) AAP activity otherwise observed in its absence. This holds true for serum (or plasma) derived both from normal individuals or from patients suffering hepatic dysfunction, with or without associated biliary obstruction. In the case of patients with obstructive jaundice, raised levels of AAP are observed, which fall to near normal levels following preincubation with this monoclonal antibody. In addition, data are presented to support the view of varying isoforms of AAP within flowing blood. Finally, preliminary data is provided on AAP activity in cases of leukaemia. These studies should thus prove of use to clinical laboratories investigating the involvement of AAP activity in various pathological processes.

Aminopeptidases↗
PubMed1986

Influences of glutathione status on different cytocidal responses of monolayer rat hepatocytes exposed to aflatoxin B1 or acetaminophen.

In short-term primary monolayer cultures of rat hepatocytes, aflatoxin B1 (AFB1) causes a characteristic prelethal cytomorphological response in which peripheral attached cytoplasm contracts segmentally to form finger-like blebs. This response precedes lethal injury as detected by release of lactate dehydrogenase (LDH) into culture medium. We compared the influences of various modifiers of cellular glutathione (GSH) status on cytocidal responses of Fischer 344 rats hepatocytes exposed to AFB1 or acetaminophen (AAP), a hepatotoxin which does not produce segmental cytoplasmic contraction. N-Acetylcysteine (4 mM) reduced the degree of LDH release by AAP (4 to 16 mM) but was not protective against cell killing by AFB1, although it slightly reduced the percentage of hepatocytes with segmental cytoplasmic contraction at 6 hr. BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) at 40 microM markedly inhibited glutathione reductase and also strongly potentiated cell killing by AAP but did not significantly influence segmental cytoplasmic contraction or LDH release in response to AFB1. Diethylmaleate (40 to 160 microM), a depletor of hepatocellular GSH, and buthionine-D,L-sulfoximine (4 mM), an inhibitor of GSH synthesis, each did not alter hepatocyte killing by AFB1 but were strong potentiators of toxicity of AAP. AAP inhibited glutathione reductase but AFB1 did not. Total GSH concentrations at 6 and 18 hr were reduced by AAP and to a lesser extent by AFB1 in comparison with control cultures. These findings demonstrate that, in contrast to AAP toxicity, the characteristic mode of hepatocyte killing by AFB1 in monolayer cultures is substantially independent of induced alterations in GSH. These results indicate that GSH-dependent detoxification mechanisms do not play a major role in removing necrogenic metabolites of AFB1 in Fischer 344 rat hepatocytes. They further suggest that prelethal responses of AFB1-injured hepatocytes are not affected by GSH-dependent cytoprotective mechanisms.

Acetaminophen↗
PubMed1991

Oltipraz-induced amelioration of acetaminophen hepatotoxicity in hamsters. I. Lack of dependence on glutathione.

These studies were designed to test the hypothesis that oltipraz (OTP) provided protection against AAP intoxication in a sensitive species, the hamster; and further, to show that the sparing effect was related to the marked increase in hepatic reduced glutathione (GSH) levels. Dose-response and time-course experiments demonstrated that maximal increases in liver GSH occurred at 48 hr after an oltipraz dose of approximately 2.0 mmol/kg (po). Accompanying greater GSH levels were increased glutathione disulfide (GSSG) levels. Decreased indices of the oxidation state of glutathione and of hepatic pyridine nucleotides indicated a greater share of glutathione existed as GSH and that increased reducing equivalents were present, respectively. Additionally, glutathione disulfide reductase activity was greater in OTP-treated groups. Glutathione S-transferase activities were only marginally increased. OTP treatment did not elicit observable hepatotoxicity, whereas AAP (2.6 mmol/kg, ip) resulted in a reproducible model of liver damage. OTP-treated groups were protected from AAP-induced toxicity, as shown by decreased plasma appearance of liver enzymes and unremarkable histopathology. However, the degree of liver GSH depletion by AAP was fourfold greater in non-OTP treated groups compared to those which had received the dithiolthione. To test the importance of increased hepatic GSH, the biosynthesis of glutathione was interrupted. Buthionine sulfoximine (BSO) treatment decreased hepatic GSH, the biosynthesis of glutathione was interrupted. Buthionine sulfoximine (BSO) treatment decreased hepatic GSH content to 50% of control in hamsters which either had or had not received OTP. The groups receiving BSO and AAP incurred 83% lethality, while no lethality, unremarkable liver histopathology, and plasma enzyme levels consistent with control were found in the group receiving OTP, BSO, and AAP. Treatment with BSO only had no influence on hepatotoxicity parameters. These results indicate that the increased GSH levels in the OTP-treated hamster are coincidental to the sparing effect of OTP and are not central to the protection scheme in AAP-induced hepatotoxicity.

Acetaminophen↗
PubMed2005

Role of caspases in acetaminophen-induced liver injury.

The mode of cell death after acetaminophen (AAP) overdose is controversially discussed. A recent study reported a protective effect of the pancaspase inhibitor Z-VAD-fmk against AAP toxicity in vivo but the mechanism of protection remained unclear. Therefore, the objective of this investigation was to assess if Z-VAD-fmk or the low doses of dimethyl sulfoxide (DMSO) used as solvent were responsible for the protection. Treatment with 10 mg/kg Z-VAD-fmk or diluted DMSO (0.25 ml/kg) for 15 min before but not 2.5 h after AAP prevented the oxidant stress (hepatic glutathione disulfide content; nitrotyrosine staining), DNA fragmentation (anti-histone ELISA, TUNEL assay) and liver injury (plasma ALT activities) at 6 h after administration of 300 mg/kg AAP. Even a lower dose (0.1 ml/kg) of DMSO was partially effective. DMSO pretreatment also attenuated the initial decline in hepatic glutathione levels. On the other hand, 10 microM Z-VAD-fmk was unable to prevent AAP-induced cell death in primary cultured mouse hepatocytes. We conclude that Z-VAD-fmk does not protect against AAP-induced liver injury and, therefore, caspases are not involved in the mechanism of AAP-induced liver injury. In contrast, the protection in vivo is caused by the diluted DMSO, which is used to solubilize the inhibitor Z-VAD-fmk. The results emphasize that even very low doses of DMSO, which are generally necessary to dissolve water-insoluble inhibitors, can have a profound impact on the toxicity of drugs and chemicals when metabolic activation is a critical aspect of the mechanism of cell injury.

Acetaminophen↗
PubMed2001

Differential effects of acetaminophen pretreatment on hepatic aflatoxin B(1)-DNA binding, cellular proliferation, and aflatoxin B1-induced hepatic foci in rats and hamsters.

Effects of acetaminophen (AAP) pretreatment on hepatic aflatoxin B1 (AFB(1))-DNA binding, cellular proliferation, and AFB(1)-induced glutathione S-transferase placental form (GST-P) positive hepatocytes and foci have been examined in young male rats and hamsters. Intraperitoneal (i.p.) dosing of 600mg AAP 3h before AFB(1) i.p. injection showed three-fold more AFB(1)-DNA binding in hamsters and 40% less binding in rats. Cell proliferation analyzed by bromodeoxyuridine incorporation was not significant (0.4-0.6%) 24-96h after AAP (600mg) treatment of rats; however, proliferation was stimulated and was maximum (11%) in hamsters at 72h after AAP treatment. Dosing of rats with AFB(1) alone at 0.5 or 2.5mg level gave an appreciable number of GST-P positive minifoci (two to nine cells) with a few foci larger than 100 microm; pretreatment with AAP (300 or 600mg) 48h before 0.5 or 2.5mg AFB(1) had no effect on the number and focal area of foci. In hamsters, 1 or 2mg AFB(1) alone yielded GST-P positive hepatocytes without any minifoci. Pretreatment with AAP (600mg) 48 or 72h before 1 or 2mg AFB(1) produced increases in both GST-P positive hepatocytes and minifoci. Thus, marked changes are observed after AAP pretreatment in hamsters compared to rats.

Acetaminophen↗
PubMed2002

Low and high affinity amino acid H+-cotransporters for cellular import of neutral and charged amino acids.

Amides and acidic amino acids represent the major long distance transport forms of organic nitrogen. Six amino acid permeases (AAPs) from Arabidopsis mediating transport of a wide spectrum of amino acids were isolated. AAPs are distantly related to plasma membrane amino acid transport systems N and A and to vesicular transporters such as VGAT from mammals. A detailed comparison of the properties by electrophysiology after heterologous expression in Xenopus oocytes shows that, although capable of recognizing and transporting a wide spectrum of amino acids, individual AAPs differ with respect to specificity. Apparent substrate affinities are influenced by structure and net charge and vary by three orders of magnitude. AAPs mediate cotransport of neutral amino acids with one proton. Uncharged forms of acidic and basic amino acids are cotransported with one proton. Since all AAPs are differentially expressed, different tissues may be supplied with a different spectrum of amino acids. AAP3 and AAP5 are the only transporters mediating efficient transport of the basic amino acids. In vivo competition shows that the capability to transport basic amino acids in planta might be overruled by excess amides and acidic amino acids in the apoplasm. With the exception of AAP6, AAPs do not recognize aspartate; only AAP6 has an affinity for aspartate in the physiologically relevant range. This property is due to an overall higher affinity of AAP6 for neutral and acidic amino acids. Thus AAP6 may serve a different role either in cooperating with the lower affinity systems to acquire amino acids in the low concentration range, as a system responsible for aspartate transport or as an uptake system from the xylem. In agreement, a yeast mutant deficient in acidic amino acid uptake at low aspartate concentrations was complemented only by AAP6. Taken together, the AAPs transport neutral, acidic and cationic amino acids, including the major transport forms, i.e. glutamine, asparagine and glutamate. Increasing proton concentrations strongly activate transport of amino acids. Thus the actual apoplasmic concentration of amino acids and the pH will determine what is transported in vivo, i.e. major amino acids such as glutamine, asparagine, and glutamate will be mobilized preferentially.

Algorithms↗
PubMed2000

Evolutionarily conserved features of the arginine attenuator peptide provide the necessary requirements for its function in translational regulation.

Neurospora crassa arg-2 mRNA contains an evolutionarily conserved upstream open reading frame (uORF) encoding the Arg attenuator peptide (AAP) that confers negative translational regulation in response to Arg. We examined the regulatory role of the AAP and the RNA encoding it using an N. crassa cell-free translation system. AAPs encoded by uORFs in four fungal mRNAs each conferred negative regulation in response to Arg by causing ribosome stalling at the uORF termination codon. Deleting the AAP non-conserved N terminus did not impair regulation, but deletions extending into the conserved region eliminated it. Introducing many silent mutations into a functional AAP coding region did not eliminate regulation, but a single additional nucleotide change altering the conserved AAP sequence abolished regulation. Therefore, the conserved peptide sequence, but not the mRNA sequence, appeared responsible for regulation. AAP extension at its C terminus resulted in Arg-mediated ribosomal stalling during translational elongation within the extended region and during termination. Comparison of Arg-mediated stalling at a rare or common codon revealed more stalling at the rare codon. These data indicate that the highly evolutionarily conserved peptide core functions within the ribosome to cause stalling; translational events at a potential stall site can influence the extent of stalling there.

Amino Acid Sequence↗
PubMed2004

The catalytic role of glutamate 151 in the leucine aminopeptidase from Aeromonas proteolytica.

Glutamate 151 has been proposed to act as the general acid/base during the peptide hydrolysis reaction catalyzed by the co-catalytic metallohydrolase from Aeromonas proteolytica (AAP). However, to date, no direct evidence has been reported for the role of Glu-151 during catalytic turnover by AAP. In order to elucidate the catalytic role of Glu-151, altered AAP enzymes have been prepared in which Glu-151 has been substituted with a glutamine, an alanine, and an aspartate. The Michaelis constant (K(m)) does not change upon substitution to aspartate or glutamine, but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.05%), glutamine (0.004%), and alanine (0%). Examination of the pH dependence of the kinetic constants k(cat) and K(m) revealed a change in the pK(a) of a group that ionizes at pH 4.8 in recombinant leucine aminopeptidase (rAAP) to 4.2 for E151D-AAP. The remaining pK(a) values at 5.2, 7.5, and 9.9 do not change. Proton inventory studies indicate that one proton is transferred in the rate-limiting step of the reaction at pH 10.50 for both rAAP and E151D-AAP, but at pH 6.50 two protons and general solvation effects are responsible for the observed effects in the reaction catalyzed by rAAP and E151D-AAP, respectively. Based on these data, Glu-151 is intrinsically involved in the peptide hydrolysis reaction catalyzed by AAP and can be assigned the role of a general acid and base.

Aeromonas↗
PubMed2001

The development and preliminary validation of a new measure of adult attachment: the adult attachment projective.

In this paper, we describe the development of and our preliminary work to empirically validate the Adult Attachment Projective (AAP), a new adult attachment classification system that is based on the analysis of individuals' responses to a set of seven attachment-related drawings. The AAP classification system uses evaluations of three dimensions (Discourse, Content and Defensive Processing) to designate four major adult classification groups: Secure, Dismissing, Preoccupied, and Unresolved. Preliminary validation of the AAP is based on 75 participants drawn from three separate samples. The results indicate strong interjudge reliability and convergent agreement between the AAP and Adult Attachment Interview (AAI) classifications. The AAP, thus, appears to be a promising developmental measure for assessing the representation of attachment in adults. Contributions of the AAP to attachment theory and research are discussed. The similarities and differences between AAP stories and other child and adult representational measures are also discussed.

Adult↗
PubMed1984

Modifying effects of butylated hydroxyanisole, ethoxyquin and acetaminophen on induction of neoplastic lesions in rat liver and kidney initiated by N-ethyl-N-hydroxyethylnitrosamine.

Studies were made on the effects of butylated hydroxyanisole (BHA), ethoxyquin (EQ) and acetaminophen (AAP) on the induction of neoplastic lesions in the liver and kidney of rats initiated by N-ethyl-N-hydroxyethylnitrosamine (EHEN). The number and area of histochemical gamma-glutamyltranspeptidase-positive (gamma-GT+) foci per unit area of liver section in rats given BHA, EQ or AAP were significantly less than in rats given EHEN alone. Similarly, the number of hyperplastic nodules (HN) in groups given BHA or AAP and their area in groups given BHA, EQ or AAP were significantly less than in control groups. Induction of hepatocellular carcinoma (HCC) was also clearly inhibited by these three chemicals. No liver lesions were found in any animals given BHA, EQ or AAP orally without EHEN. In contrast, the incidence and quantitative values of preneoplastic lesions and renal cell adenoma were significantly increased in groups given BHA, EQ or AAP. The results clearly demonstrated that BHA, EQ and AAP inhibited the development of gamma-GT+ foci, HN and HCC, whereas they enhanced the appearance of preneoplastic and neoplastic lesions in the kidney.

Acetaminophen↗
PubMed1983

Study of alanine aminopeptidase excretion as a test of gentamicin nephrotoxicity.

We studied the urinary excretion of the proximal tubular enzyme alanine aminopeptidase (AAP) by 38 patients who received gentamicin and by 45 similar control patients. AAP excretion by the control patients was highly variable. The pattern of AAP excretion distinguished patients with pre-renal and renal causes for increases in serum creatinine (SCr) but this can be done more simply with the urine to serum creatinine ratio. Peak AAP excretion did not reflect renal damage in oliguric patients and di not identify gentamicin patients with a renal cause for increases in SCr. The level of AAP excretion in the first three days of gentamicin treatment did identify the three patients with renal causes for an increase in SCr and was highest in a patient with symptomatic hypomagnesaemia due to impairment of proximal tubular function. We concluded that study of AAP excretion in normal volunteers receiving three-day courses of aminoglycosides is a valid screening test for nephrotoxicity but that AAP excretion is too non-specific to use in clinical practice.

Aminopeptidases↗
PubMed1997

Alanyl aminopeptidase from human seminal plasma: purification, characterization, and immunohistochemical localization in the male genital tract.

Alanyl aminopeptidase (AAP) was purified to homogeneity from human seminal plasma. The calculated molecular weight of the purified enzyme was approximately 137,000+/-5,000 from light scattering, 140,000 (main) and 137,000 (minor) from non-denatured PAGE and 153,000 from SDS-PAGE in the absence or presence of 2-mercaptoethanol (2-ME). These findings suggest that the enzyme is monomeric in form in human seminal plasma. The enzyme hydrolyzed several amino acid 4-methyl-coumaryl-7-amide (MCA) substrates. The order of Kcat/Km values of AAP at optimal pH (pH 7.5) was Ala- > Lys-Ala- > or = Met- > Leu- > Phe- > Arg- > or = Arg-Arg- > Lys- > Gly-MCAs. AAP was potently inhibited by bestatin, leuhistin, actinonin, amastatin, and 1,10-phenanthroline. These findings suggest that AAP is an aminopeptidase. We determined that the amino acid sequence of the first 22 residues of the enzyme was Ser1-Thr-Thr-Pro-Ser5-Ala-Ser-Ala-Thr-Thr10-Asn-Pro-Al a-Ser-Ala15-Thr-Thr-Leu-Asp-Gln20-Ser-Lys-. This sequence was completely coincident with that downstream of the transmembrane site of human intestinal alanyl aminopeptidase N (CD13). We also isolated cDNA encoding AAP from human prostate cDNA library, sequenced its structure, and confirmed human seminal plasma AAP to be identical with alanyl aminopeptidase N. We postulated that native human seminal plasma alanyl aminopeptidase is released into the seminal plasma after the specific site is cleaved by elastase or an elastase-like enzyme. The enzyme level in human seminal plasma determined by single radial immunodiffusion was 5.2+/-3.2 mg/100 ml (mean+/-SD, n=20) in individuals 20-47 years of age. AAP was immunohistochemically stained in the luminal site-cell membrane of epithelial cells in the prostatic gland and ductuli efferentes of the testis.

Adult↗
PubMed2002

Mode of cell death after acetaminophen overdose in mice: apoptosis or oncotic necrosis?

Acetaminophen (AAP) overdose can cause severe liver injury and liver failure in experimental animals and humans. Recently, several authors proposed that apoptosis might be a major mechanism of cell death after AAP treatment. To address this controversial issue, we evaluated a detailed time course of liver injury after AAP (300 mg/kg) in fasted C3Heb/FeJ mice. Apoptotic hepatocytes were quantified in H&E-stained liver sections using morphologic criteria (cell shrinkage, chromatin condensation and margination, and apoptotic bodies). The number of apoptotic hepatocytes remained at baseline (0.2 +/- 0.1 cells/10 high-power fields [HPF]) up to 2 h after AAP administration. However, between 3 and 24 h, apoptotic cell death increased significantly, e.g., 6.3 +/- 0.8 cells/10 HPF at 6 h. Despite the increase in the number of hepatocytes meeting the morphological criteria of apoptosis, this cell fraction remained well below 1% of all parenchymal cells. No evidence for caspase-3 processing or increase in enzyme activity was detected at any time. These results were compared to the overall percent of necrotic cells in liver sections. Confluent areas of centrilobular necrosis were estimated to involve 40-60% of all hepatocytes between 3 and 24 h after AAP administration. These numbers correlated with the increase in plasma alanine aminotransferase activities, which reached a peak level of 5900 +/- 1350 U/l at 24 h. A similar result was obtained with higher doses of AAP and with the use of fed animals. Thus, oncotic necrosis and not apoptosis is the principal mechanism of liver-cell death after AAP overdose in vivo.

Acetaminophen↗
PubMed2004

Acetaminophen-induced oxidant stress and cell injury in cultured mouse hepatocytes: protection by N-acetyl cysteine.

The increase in cellular and mitochondrial glutathione disulfide (GSSG) levels and the GSSG:GSH ratio after acetaminophen (AAP) overdose suggest the involvement of an oxidant stress in the pathophysiology. However, the initial severe depletion of hepatocellular glutathione makes quantitative assessment of the oxidant stress difficult. Therefore, we tested the hypothesis that oxidant stress precedes the onset of cell injury in a cell culture model using 2',7'-dichlorofluorescein (DCF) fluorescence as a marker for intracellular oxidant stress. Cultured primary murine hepatocytes were exposed to 5 mM AAP. DCF fluorescence, XTT reduction, lactate dehydrogenase (LDH) release, and trypan blue uptake were determined from 0 to 12 h. After glutathione depletion at 3 h, DCF fluorescence increased by 16-fold and was maintained at that level up to 12 h. At 1.5 h after AAP, a significant decrease of the cellular XTT reduction capacity was observed, which continued to decline until 9 h. Cell necrosis (LDH release, trypan blue uptake) was detectable in 20% of cells at 6 h, with a significant further increase at later time points. Pretreatment with 20 mM N-acetylcysteine (NAC) 1 h before AAP enhanced cellular glutathione content, prevented or attenuated the AAP-induced decrease of GSH levels and XTT reduction capacity, respectively, and reduced the loss of cell viability. Additionally, treatment with NAC 2 h after AAP exposure prevented further deterioration of XTT reduction at 3 h and later, and attenuated cell necrosis. Thus, AAP-induced oxidant stress precedes cell necrosis and, in cultured hepatocytes, the oxidant stress is involved in the propagation of cell injury.

Acetaminophen↗
PubMed2005

Aerobic anoxygenic photosynthesis genes and operons in uncultured bacteria in the Delaware River.

Photosynthesis genes and operons of aerobic anoxygenic photosynthetic (AAP) bacteria have been examined in a variety of marine habitats, but genomic information about freshwater AAP bacteria is lacking. The goal of this study was to examine photosynthesis genes of AAP bacteria in the Delaware River. In a fosmid library, we found two clones bearing photosynthesis gene clusters with unique gene content and organization. Both clones contained 37 open reading frames, with most of those genes encoding known AAP bacterial proteins. The genes in one fosmid were most closely related to those of AAP bacteria in the Rhodobacter genus. The genes of the other clone were related to those of freshwater beta-proteobacteria. Both clones contained the acsF gene, which is required for aerobic bacteriochlorophyll synthesis, suggesting that these bacteria are not anaerobes. The beta-proteobacterial fosmid has the puf operon B-A-L-M-C and is the first example of an uncultured bacterium with this operon structure. The alpha-3-proteobacterial fosmid has a rare gene order (Q-B-A-L-M-X), previously observed only in the Rhodobacter genus. Phylogenetic analyses of photosynthesis genes revealed a possible freshwater cluster of AAP beta-proteobacteria. The data from both Delaware River clones suggest there are groups of freshwater or estuarine AAP bacteria distinct from those found in marine environments.

Aerobiosis↗
PubMed2005

Glycogen synthase kinase-3 mediates acetaminophen-induced apoptosis in human hepatoma cells.

The mild analgesic drug acetaminophen (AAP) induces severe hepatic injury when taken at excessive doses. Recent evidence shows that the initial form of damage is through apoptosis, but this fails to go to completion and degenerates into necrosis. The aim of this study was to elucidate the mechanism through which AAP induces apoptosis using human HuH7 hepatoma cells as an in vitro model system to investigate the initial phase of AAP-induced hepatic injury. AAP-induced apoptosis in HuH7 cells as evidenced by chromatin condensation was preceded by the translocation of Bax to mitochondria and the cytoplasmic release of the proapoptotic factors cytochrome c and Smac/DIABLO. A concomitant loss of mitochondrial membrane potential occurred. Activation of the mitochondrial pathway of apoptosis led to the activation of execution caspases-3 and -7. AAP-induced apoptosis and cell death was blocked by inhibitors of caspases but not by inhibitors of calpains, cathepsins, and serine proteases. Apoptosis was unaffected by inhibitors of the mitochondrial permeability transition pore and by inhibitors of Jun NH(2)-terminal kinases, p38 mitogen-activated protein kinase, or mitogen-activated protein kinase kinase 1/2. However, pharmacological inhibition of glycogen synthase kinase-3 (GSK-3) delayed and decreased the extent of AAP-induced apoptosis. In comparison, endoplasmic reticulum stress-induced but not prooxidant-induced apoptosis of HuH7 cells was sensitive to GSK-3 inhibition. It is concluded that AAP-induced apoptosis involves the mitochondrial pathway of apoptosis that is mediated by GSK-3 and most likely initiated through an endoplasmic reticulum stress response.

Acetaminophen↗
PubMed2005

Peroxynitrite-induced mitochondrial and endonuclease-mediated nuclear DNA damage in acetaminophen hepatotoxicity.

Intracellular sources of peroxynitrite formation and potential targets for this powerful oxidant and nitrating agent have not been identified after acetaminophen (AAP) overdose. Therefore, we tested the hypothesis that peroxynitrite generated in mitochondria may be responsible for mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage. C3Heb/FeJ mice were treated with 300 mg/kg AAP and monitored for up to 12 h. Loss of mtDNA (assayed by slot blot hybridization) and substantial nDNA fragmentation (evaluated by anti-histone enzyme-linked immunosorbent assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and agarose gel electrophoresis) were observed as early as 3 h after AAP overdose. Analysis of nitrotyrosine protein adducts in subcellular fractions established that peroxynitrite was generated predominantly in mitochondria beginning at 1 h after AAP injection. Delayed treatment with a bolus dose of glutathione (GSH) accelerated the recovery of mitochondrial glutathione, which then effectively scavenged peroxynitrite. However, mtDNA loss was only partially prevented. Despite the absence of nitrotyrosine adducts in the nucleus after AAP overdose, nDNA damage was almost completely eliminated with GSH administration. A direct comparison of nDNA damage after AAP overdose with nDNA fragmentation during tumor necrosis factor receptor-mediated apoptosis showed similar DNA ladders on agarose gels but quantitatively different results in three other assays. We conclude that peroxynitrite may be partially responsible for mtDNA loss but is not directly involved in nDNA damage. In contrast, nDNA fragmentation after AAP overdose is not caused by caspase-activated DNase but most likely by other intracellular DNase(s), whose activation is dependent on the mitochondrial oxidant stress and peroxynitrite formation.

Acetaminophen↗