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PubMed1996

Metrifonate induces cholinesterase inhibition exclusively via slow release of dichlorvos.

Metrifonate, a long-acting cholinesterase (ChE) inhibitor with very low toxicity in warm-blooded animals, inhibits rat brain and serum cholinesterase (ChE) in vitro through its hydrolytic degradation product, dichlorvos. This conclusion is based on the finding that metrifonate-induced ChE inhibition showed the same pH dependence as its reported dehydrochlorination to dichlorvos. The ChE inhibition induced by dichlorvos was not pH dependent. It was mediated by a competitive drug interaction with the catalytic site of the enzyme, which led to irreversible inhibition within several minutes of incubation. After this time, addition of further substrate to the inhibited enzyme was not able to promote drug dissociation and hence enzyme reactivation. Similar characteristics of inhibition, i.e. interaction with the substrate binding site and time-dependent switch to non-competitive inhibition were observed with the reference compound, physostigmine. However, the physostigmine-induced inhibition of ChE could be readily reversed by further substrate addition. Another reference compound, tetrahydroaminoacridine (THA), also induced a reversible inhibition of rat brain and serum cholinesterase, but with a mechanism of action different from that of both dichlorvos and physostigmine in that enzyme inhibition occurred rapidly upon drug addition at an allosteric site on the enzyme surface. It is suggested that the unique slow release plus the slow inhibition of ChE by dichlorvos is responsible for the lower toxicity of metrifonate compared to that of directly acting ChE inhibitors.

Animals↗
PubMed1994

False-positive human immunodeficiency virus screening test related to rabies vaccination.

We report a false-positive result on an enzyme immunoassay screening test for antibodies to human immunodeficiency virus in a 32-year-old nonpregnant woman who belonged to none of the usual risk groups. Because of the patient's employment in an animal care facility, she had received a series of three vaccinations for rabies, and 16 days after the last vaccination, she had donated a unit of blood. It was during routine screening on a sample drawn at the time of donation that the repeatedly reactive enzyme immunoassay screening test occurred. The results of a Western blot were indeterminant. A polymerase chain reaction assay was negative for proviral DNA.

Adult↗
PubMed1993

Dissection of the human acetylcholinesterase active center determinants of substrate specificity. Identification of residues constituting the anionic site, the hydrophobic site, and the acyl pocket.

Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.

Acetylcholinesterase↗
PubMed1981

[Enzymatic conversion of polymers. Nature of apparent product inhibition in the course of enzymatic degradation of polymer substrates].

The general regularities for the kinetics of enzymatic conversion of polymers were studied. It was proposed that the formal kinetic analysis of progress kinetic curves for enzymatic degradation of polymers inevitably results in the apparent effects of product inhibition, even in the case when the products are not virtually bound to the enzyme and there is no real product inhibition at all. An experimental verification of this hypothesis was performed, using maltodextrins hydrolysis by Aspergillus niger glucoamylase as an example. It was shown that a progressive decrease of the enzyme reactivity with respect to a polymer substrate in the course of its degradation is kinetically equivalent to a progressive decrease of the velocity of an enzymatic reaction due to the product inhibition. These two cases cannot practically be distinguished by conventional methods of analysis of progress kinetic curves.

Aspergillus niger↗
PubMed1993

Identification of insulin-stimulated protein kinase-1 as the rabbit equivalent of rskmo-2. Identification of two threonines phosphorylated during activation by mitogen-activated protein kinase.

An improved procedure has been developed for the isolation of insulin-stimulated protein kinase-1 (ISPK-1), an S6 kinase-II homologue, by which 0.5 mg highly purified enzyme can be obtained within four days. The sequences of tryptic peptides from ISPK-1 (100 residues) revealed 100% identity with the predicted protein product of rskmo-2, a cDNA clone isolated from a mouse F2 cell line library [Alcorta, D. A., Crews, C. M., Sweet, L. J., Bankston, L., Jones, S. W. and Erikson, R. L. (1989) Mol. Cell. Biol. 9, 3850-3859], demonstrating that rskmo-2 encodes an S6 kinase-II. Two isoforms of mitogen-activated protein (MAP) kinase (p42mapk and p44mapk) were the only ISPK-1-reactivating enzymes detected after Mono Q chromatography of extracts prepared from rabbit skeletal muscle or phaeochromocytoma 12 cells stimulated by nerve or epidermal growth factors. One of the residues on ISPK-1 phosphorylated by p42mapk was a threonine located nine residues N-terminal to the conserved Ala-Pro-Glu motif in the C-terminal protein kinase domain, an analogous location to phosphorylation sites essential for the activity of cAMP-dependent protein kinase, MAP kinase and p34cdc2. A further threonine located five residues N-terminal to the same Ala-Pro-Glu motif was also phosphorylated, probably via autophosphorylation catalysed by ISPK-1 itself.

Amino Acid Sequence↗
PubMed1991

The potency of inducers of NAD(P)H:(quinone-acceptor) oxidoreductase parallels their efficiency as substrates for glutathione transferases. Structural and electronic correlations.

Induction of glutathione transferases (EC. 2.5.1.18), NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2; quinone reductase) and other detoxification enzymes is a major mechanism for protecting cells against the toxicities of electrophiles, including many carcinogens. Although inducers of these two enzymes belong to many different chemical classes, they nevertheless contain (or acquire by metabolism) electrophilic centres that appear to be essential for inclusive activity, and many inducers are Michael reaction acceptors [Talalay, De Long & Prochaska (1988) Proc. Natl. Acad. Sci. U.S.A., 85, 8261-8265]. The inducers therefore share structural and electronic features with glutathione transferase substrates. To define these features more precisely, we examined the inductive potencies (by measuring quinone reductase in murine hepatoma cells) of two types of glutathione transferase substrates: a series of 1-chloro-2-nitrobenzenes bearing para-oriented electron-donating or -withdrawing substituents and a wide variety of other commonly used and structurally unrelated glutathione transferase substrates. We conclude that virtually all glutathione transferase substrates are inducers, and their potencies in the nitrobenzene series correlate linearly with the Hammett sigma or sigma- values of the aromatic substituents, precisely as previously reported for their efficiencies as glutathione transferase substrates. More detailed information on the electronic requirements for inductive activity was obtained with a series of methyl trans-cinnamates bearing electron-withdrawing or -donating substituents on the aromatic ring, and in which the electronic densities at the olefinic and adjacent carbon atoms were measured by 13C n.m.r. Electron-withdrawing meta-substituents markedly enhance inductive potency in parallel with their increased non-enzymic reactivity with GSH. Thus, methyl 3-bromo-, 3-nitro- and 3-chloro-cinnamates are 21, 14 and 8 times more potent inducers than the parent methyl cinnamate. This finding permits the design of more potent inducers, which are important for elucidation of the molecular mechanisms of induction.

Animals↗
PubMed1976

Active-site determinations on forms of mammalian brain and eel acetylcholinesterase.

Three forms of brain acetylcholinesterase were purified from bovine caudate-nucleus tissue and determined by calibrated gel filtration to have mol.wts. of approx. 120 000 (C), 230 000 (B) and 330 000 (A). [3H]Di-isopropyl phosphorofluoridate (isopropyl moiety labelled) was purified from commercial preparations and its concentration estimated by an enzyme-titration procedure. Brain acetylcholinesterase preparations and enzyme from eel electric tissue were allowed to react with [3H]di-isopropyl phosphorofluridate in phosphate buffer until enzyme activity was inhibited by 98%. Excess of [3H]di-isopropyl phosphorofluoridate that had not reacted was separated from the labelled enzyme protein by gel filtration, or by vacuum filtration or by extensive dialysis. The specificity of active-site labelling was confirmed by use of the enzyme reactivator, pyridine 2-aldoxime. The forms of brain acetylcholinesterase were calculted to contain approximately two (C) four (B) and six (A) active sites per molecule respectively. Acetylcholinesterase (mol.wt. 250 000) from electric-eel tissue was estimated to contain two active sites per molecule. Gradient-gel electrophoresis was used to confirm the estimation of molecular weights of brain acetylcholinesterase forms made by gel filtration. Under the conditions of electrophoresis acetylcholinesterase form A was stable, but form B was converted into a species of approx. 120 000 mol. wt. Similarly, form C of the brain enzyme was converted into a 60 000-mol.wt. form during electrophoresis. These results are in general accord with the suggestion that the multiple forms of brain acetylcholinesterase may be related to the aggregation of a single low-molecular-weight species.

Acetylcholinesterase↗
PubMed1995

Kinetic analysis of enzymic activities: prediction of multiple forms of 17 beta-hydroxysteroid dehydrogenase.

An overview of the application of kinetic methods to the delineation of 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) heterogeneity in mammalian tissues is presented. Early studies of 17 beta-HSD activity in animal liver and kidney subcellular fractions were suggestive of multiple forms of the enzyme. Subsequently, detailed characterization of activity in cytosol and subcellular membrane fractions of human placenta, with particular emphasis on inhibition kinetics, yielded evidence of two kinetically-differing forms of 17 beta-HSD in that organ. Gene cloning and transfection experiments have confirmed the identity of these two proteins as products of separate genes. 17 beta-HSD type 1 is a cytosolic enzyme highly specific for C18 steroids such as 17 beta-estradiol (E2) and estrone (E1). 17 beta-HSD type 2 is a membrane bound enzyme reactive with testosterone (T) and androstenedione (A), as well as E2 and E1. Useful parameters for the detection of multiple forms of 17 beta-HSD appear to be the E2/T activity ratio, NAD/NADP activity ratios, steroid inhibitor specificity and inhibition patterns over a wide range of putative inhibitor concentrations. Evaluation of these parameters for microsomes from samples of human breast tissue suggests the presence of 17 beta-HSD type 2. The 17 beta-HSD enzymology of human testis microsomes appears to differ from placenta. Analysis of human ovary indicates granulosa cells are particularly enriched in the type 1 enzyme with type 2-like activity in stroma/theca. Mouse ovary appears to contain forms of 17 beta-HSD which differ from 17 beta-HSD type 1 and type 2 in their kinetic properties.

17-Hydroxysteroid Dehydrogenases↗
PubMed2005

Analysis of covalently bound polyketide intermediates on 6-deoxyerythronolide B synthase by tandem proteolysis-mass spectrometry.

Polyketide natural products are biosynthesized via successive chain-elongation events mediated by elaborate protein assemblies. Facile detection of protein-bound intermediates in these systems will increase our understanding of enzyme reactivity and selectivity. We have developed a tandem proteolysis/mass spectrometric method for monitoring substrate loading and elongation in 6-deoxyerythronolide B synthase (DEBS), responsible for production of the macrolide precursor to erythromycin. Information regarding ketosynthase loading and polyketide unit elongation is readily acquired without need for complex protein or small molecule labels. A panel of structurally related substrates is evaluated through competition experiments and kinetic assays using LC-MS to resolve closely related species. Strong stereochemical effects are observed for ketosynthase substrate specificity. Semiquantitative kinetic analyses allow the resolution of the effects of structural and stereochemical changes on the individual ketosynthase-catalyzed steps of acyl-enzyme formation and polyketide chain extension.

Acyl Carrier Protein↗
PubMed2000

Transplantation of tissue grafts into the anterior eye chamber: a method to study intrinsic neurons.

Intrinsic nerves play a crucial role in the regulation of body functions. It is therefore of paramount importance to be able to study the biology of intrinsic nerves in tissue grafts. The anterior eye chamber of rats has long been used to study different biological mechanisms, growth and differentiation of a variety of tissues, because of the transparency of the cornea, which also allows macroscopic changes to be seen. Despite its extended use, a detailed, easy to follow description of the technique of tissue and cell transplantation into the anterior eye chamber has not been presented. In this study, pancreatic tissue fragments were transplanted into the anterior eye chamber of rats alone or with brain tissue fragments to examine the survival and viability of intrinsic nerves in these tissue fragments, which have been detached from their original extrinsic nerves. The pancreatic transplants contained intact 5-HT and AChE-positive intrinsic neurons. The brain tissue grafts contained many AChE-enzyme reactive cells. The method is simple and can be used to study the morphology or physiology of intrinsic neurons in any tissue fragment. The grafts are easily vascularised and reinnervated because of the rich blood and nerve supply of the iris which forms the bed of the anterior eye chamber. The graft will also survive with ease because the anterior eye chamber is an immunologically privileged site. In conclusion, the intrinsic nerves of pancreatic and brain tissue fragments can survive after several weeks of transplantation into the anterior eye chamber of rats. In addition to this, these intrinsic nerves have the ability to produce and or store neurotransmitters and their enzymes.

Acetylcholinesterase↗
PubMed1998

Combined effect of organophosphorus hydrolase and oxime on the reactivation rate of diethylphosphoryl-acetylcholinesterase conjugates.

Reactivation of inhibited acetylcholinesterase (AChE) is essential for rapid recovery after organophosphate (OP) poisoning. However, following administration of an oxime reactivator, such as pralidoxime mesylate (P2S), in patients poisoned with certain diethylphosphorothioate pesticides, no reactivation is observed, presumably due to reinhibition by circulating anti-cholinesterase OPs. Pretreatment alone with organophosphorus hydrolases (OPH) that are capable of rapidly hydrolyzing OPs was demonstrated, in animals, to confer significant protection against OP toxicity. One strategy to augment the potentially therapeutic scope of OPHs is a combined post-exposure treatment consisting of a drug(s) commonly used against OP toxicity and a suitable hydrolase. In this study, we examined the in vitro ability of OPH from Pseudomonas sp. (OPHps) to prevent reinhibition of P2S-reactivated AChE by excess OPs. The kinetic parameters of the reactivation of a series of diethylphosphoryl-AChE (DEP--AChE) conjugates, obtained by the use of various diethylphosphates, were determined and compared with the rates of reactivation in the presence of OPHps, with and without the OP inhibitors in the reactivation medium. Extrapolation of the in vitro results to in vivo conditions suggests that an OPHps concentration as low as 1 microgram/mL blood would result in a 100-fold decrease in the concentration of circulating anti-AChE pesticides within less than one blood-circulation time, thereby minimizing reinhibition of the reactivated enzyme. Thus, for DEP-based pesticides, the combination of P2S-OPH treatment can significantly improve clinical recovery after OP intoxication. In addition, it is shown here for the first time that an OPH can effectively hydrolyze quaternary ammonium-containing OPs. This indicates that hydrolysis of phosphorylated oximes, toxic side products of oxime treatment, may also be accelerated by OPHs.

Acetylcholinesterase↗
PubMed1995

Identification of factor-XIIIa-reactive glutaminyl residues in the propolypeptide of bovine von Willebrand factor.

von Willebrand factor is a large multimeric plasma protein which plays important roles in platelet aggregation, blood coagulation and probably also in the adhesion of endothelial cells. A 100-kDa propeptide, called the propolypeptide of von Willebrand factor (pp-vWF), is generated during biosynthesis. We found that pp-vWF served as a substrate for transglutaminases including human factor XIIIa and guinea pig liver transglutaminase [Usui, T., Takagi, J. & Saito, Y. (1993) J. Biol. Chem. 268, 12311-12316]. As such, it could form cross-linked copolymers with the extracellular matrix protein, laminin, making it all the more likely that pp-vWF plays a role in cell adhesion phenomena [Takagi, J., Sudo, Y., Saito, T. & Saito, Y. (1994) Eur. J. Biochem. 222, 861-867]. In this work, we identified the Gln residues in pp-vWF specifically reacting with blood coagulation factor XIIIa as amine acceptors. The fluorescent amine, dansylcadaverine, was employed for labeling the enzyme-reactive sites of the protein. Following partial proteolysis, fragments containing the labeled Gln residues were isolated by passage through an anti-dansyl affinity chromatographic column. Amino acid sequence analyses of the fragments revealed that, out of about 40 Gln residues in pp-vWF, only four could be modified in the factor-XIIIa-catalyzed reaction.

Amino Acid Sequence↗
PubMed1982

1-Bromopinacolone, an active site-directed covalent inhibitor for acetylcholinesterase.

1-Bromopinacolone, BrPin, acts initially as a reversible competitive inhibitor for acetylcholinesterase, KI = 0.18 mM in hydrolysis of acetylcholine. Unlike bromoacetone, with time it acts as an irreversible covalent inhibitor. BrPin has a hydrolytic half-life of 30 h at the pH of incubation, 7.8. The enzyme-BrPin complex is 50% inactivated in 2 h. First order kinetics are observed; the rate constant is proportional to the concentration of complex. Retardation by cationic inhibitors of the inactivation is consistent with inactivation occurring as a result of binding of BrPin to the active site. Efficiency of irreversible inhibition by BrPin is essentially the same for hydrolysis of cationic and uncharged substrates, acetylcholine, 3,3-dimethylbutyl acetate, phenyl acetate, n-butyl acetate, and indophenyl acetate. In contrast, a cationic alkylating agent, N,N-dimethyl-2-phenylaziridinium ion, DPA, acts noncompetitively; it inactivates completely toward cationic, and partially toward uncharged substrates, and does so slightly more rapidly than BrPin, but less than would be commensurate with its greater intrinsic reactivity. Enzyme first treated with DPA is inactivated by BrPin toward hydrolysis of 3,3-dimethylbutyl acetate. It is proposed that BrPin, and not DPA, binds and reacts in, and may be a useful labeling agent for, the active site.

Acetylcholinesterase↗
PubMed1991

Lipoylation of the E2 components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli.

The number of functional lipoyl groups in the dihydrolipoyl acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex from Escherichia coli has been re-assessed by means of a combination of protein-chemical and mass-spectrometric techniques. (1) After the complex had been treated with N-ethyl[2,3-14C]maleimide in the presence of pyruvate, the lipoyl domains were excised from the complex, treated with NaBH4 and re-exposed to N-ethyl[2,3-14C]maleimide. All the chemically reactive lipoyl groups in the native complex were found to be catalytically active. (2) Proteolytic digests of the separated lipoyl domains were examined for the presence of the lipoylation-site peptide, GDKASME, with and without the lipoyl group in N6-linkage to the lysine residue. Only the lipoylated form of the peptide was detected, suggesting that all three lipoyl domains are fully substituted at this site. (3) The behaviour of each lipoyl domain was examined on ion-exchange chromatography in response to alkylation with 4-vinylpyridine after either chemical reduction of the lipoyl group with dithiothreitol or reductive acetylation by the pyruvate dehydrogenase complex in the presence of pyruvate. All three domains exhibited a quantitative shift in retention time, confirming that each domain was fully substituted by an enzymically reactive lipoyl group. (4) When subjected to electrospray mass spectrometry, each domain gave a mass consistent with a fully lipoylated domain, and no aberrant substitution of the target lysine residue was detected. The same result was obtained for the lipoyl domain from the E. coli 2-oxoglutarate dehydrogenase complex. (5) Previous widespread attempts to assess the number of functional lipoyl groups in the pyruvate dehydrogenase multienzyme complex, which have led to the view that a maximum of two lipoyl groups per E2 chain may be involved in the catalytic mechanism, are in error.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1998

Loss of myocardial capillary endothelial-cell alkaline phosphatase (ALP) activity in primary endothelial cell culture.

Primary cultures of rat myocardial capillary endothelial cells were established and characterized. A range of typical endothelial cell-specific markers were retained in vitro. Cell kinetic studies in confluent endothelial-cell cultures in vitro revealed a roughly 50-fold increase in the proportion of cells in s-phase, indicating a very considerable shortening of cell turnover time, compared to in vivo conditions. Alkaline phosphatase enzyme activity and encoding mRNA are strongly expressed in myocardial capillary endothelial cells in vivo, but were not detectable in vitro. This was true in cell cultures from two strains of rat, which revealed significantly different enzyme expression levels in vivo. In co-cultures of pericytes and endothelial cells, positive ALP enzyme reaction was detected in pericytes, which in vivo show only very weak enzyme reactivity. Treatment of cell cultures with </=10 M retinoic acid had no effect in pure endothelial cell cultures, but did increase ALP expression of pericytes in co-cultures. The observation of a loss of endothelial ALP expression in vitro supports other in vitro as well as our own in vivo observations, indicating a negative correlation of ALP expression and proliferative activity of endothelial cells.

Alkaline Phosphatase↗
PubMed1985

Twentyfold increase in alkaline phosphatase activity by sequential reversible activation of the enzyme followed by coupling with a copolymer of ethylene and maleic anhydride.

Alkaline phosphatase, APase, (EC 3.1.31) from calf intestine, after shifting the equilibrium by effector molecules towards the dimeric form of the enzyme, was coupled (ratio 1:2, protein: copolymer) to a copolymer of ethylene and maleic anhydride, EMA. The water-soluble APase-EMA was separated from APase and the unbound EMA by DEAE-cellulose ion exchange chromatography. The specific activity of the APase-EMA, compared to APase, increased 26-fold at pH 7.1 and 10-fold at pH 8.6. The pH optimum of APase-EMA was shifted down from pH 9.5 (native APase) to 8.6. This change could be interpreted in terms of polyelectrolyte theory. APase-EMA retained 50-70% of its optimum activity in the pH range 7-8, while APase retained only 5-15% of its optimum activity within the same pH range. Its isoelectric point, pI, was 4.2 (APase 6.0) and it migrated on polyacrylamide gel electrophoresis in a single band, anodic movement twice as fast as APase. Parallel with the kinetic measurements, the reactive-enzyme sedimentation method was used to measure S20,w values. S20,w values obtained for APase-EMA, activated APase, and APase dialyzed against water were 6.56S, 6.46S, and 5.17S, respectively. Molecular weights, Mr, were determined by equilibrium sedimentation: the values obtained were 180,000, 160,000, and 84,500. Mr values of APase-EMA and APase (native) estimated by Sepharose-4B gel filtrations were essentially the same. The above-mentioned values remained unchanged for APase-EMA after intensive dialysis against water, whereas for the activated APase, separation from the effector molecules caused the equilibrium to shift back to the monomeric, very slightly active enzyme with concomitant changes of S20,w to 5.15 and Mr to 82,000.

Alkaline Phosphatase↗
PubMed1982

Pharmacokinetic characterization of extracorporeal therapy.

The efficiency of extracorporeal therapy with immobilized enzymes (reactive devices) and hemoperfusion cartridges (nonreactive devices) is evaluated in terms of a new pharmacokinetic parameter, the bioefficacy. A detailed analysis is provided in the former case, and it is shown how the bioefficacy can be calculated from a combined pharmacokinetic and reactor analysis. The theory is presented for evaluating bioefficacy in terms of linear pharmacokinetic models that view the reactor or other extracorporeal appliance as an artificial organ. Examples are drawn from recent work on methotrexate rescue with immobilized carboxypeptidase G1 and from literature data on the use of hemoperfusion to treat drug overdose to illustrate the calculations.

Enzymes, Immobilized↗
PubMed1997

Regulation of macrophage gene expression following invasion by Mycobacterium tuberculosis.

INTRODUCTION: Mycobacteria are intracellular pathogens which survive and grow in host macrophages. M. tuberculosis bacilli enter the macrophage via binding to several distinct cell surface molecules. Following phagocytosis, sustained intracellular bacterial growth depends on the ability to avoid destruction by macrophage-mediated host defences such as lysosomal enzymes, reactive oxygen and the reactive nitrogen intermediates. We used differential display reverse transcription polymerase chain reaction (DD RT-PCR) to identify host genes which are regulated during infection and hence which might be involved in the host-parasite cross talk. RESULTS: Live M. tuberculosis (strain H37Rv) was used to infect Balb/c peritoneal murine macrophages. mRNA from infected and uninfected macrophages was isolated at different time intervals after phagocytosis and subjected to DD RT-PCR. Oligo dT12NV and random 10mer primers were used for PCR amplification of cDNA. Macrophage genes which appeared to be differently regulated during infection were subjected to further reamplification by PCR in order to clone and sequence them. The differential expression of the selected bands was further analysed by an RNA protection assay and a Northern blot. RESULTS: Several differentially regulated bands were identified. One band, of 158 bp, was down regulated after infection. Sequencing of this band revealed a high level of homology (95% identity) to mouse cytochrome c oxidase subunit VIIc. The downregulation was specific for live virulent Mtb, while live BCG, heat killed Mtb and latex beads-mediated phagocytosis did not affect the transcriptional level of this enzyme. CONCLUSIONS: The cytochrome oxidase enzyme complex of the inner mytochondrial membrane catalyzes the reaction between ferrocytochrome c and oxygen. The reaction is the terminal event in the electron transport scheme. Downregulation of cytochrome c oxidase subunit VIIc could interfere with: (1) the host apoptotic programme; or (2) the host respiratory burst.

Animals↗