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Many previous works have demonstrated that acetylcholinesterase (AChE) was enantioselectively inhibited by chiral organophosphorus insecticides (OPs) and that a significant difference in reactivation existed for AChE inactivated by (1R)- versus (1S,3S)-stereoisomers of isomalathion. It had been known that α-naphthyl acetate esterase (ANAE), an enzyme which might play an essential role in the growth of plants and the defense of plants against environmental stress by regulating the concentration of hormones in plants, can be inhibited by OPs. However, it was unknown whether interaction of ANAE with chiral OPs was enantioselective. The present work investigated the inhibition kinetics and spontaneous reactivation of ANAE inactivated by enantiomers of malaoxon, isomalathion, and methamidophos. The order of inhibition potency is (R) > (S) for malaoxon, (1R,3R) > (1R,3S) > (1S,3R) > (1S,3S) for isomalathion, and (S) > (R) for methamidophos according to bimolecular rate constants of inhibition (ki), which is consistent with the order observed in the enantioselective inhibition of AChE by malaoxon, isomalathion, and methamidophos. The difference in spontaneous reactivation of AChE inactivated between (1R)- and (1S,3S)-isomers of isomalathion is conserved for ANAE. The observations indicated ANAE and AChE have similar selective inhibition kinetics and postinhibitory reactions in reaction with chiral OPs.

A malathion-resistant (RM) strain of Culex pipiens pallens Coq was obtained by successively selecting a field population with malathion in the laboratory. The synergistic effect of iprobenfos on malathion toxicity and alpha-naphthyl acetate (alpha-NA) esterase assay revealed that malathion resistance in the RM strain was associated with increased alpha-NA esterase activity and the synergism was mainly due to the inhibition by iprobenfos of this activity. There was no difference in alpha-NA esterase activity between the larvae and female adults in the susceptible (S) strain, but the activity in the adults was 13-fold higher than in the larvae of the RM strain. To understand the effect of the application of a mixture of iprobenfos and malathion on the evolution of malathion resistance, an artificial strain (Syn) was generated by mixing the RM and S strains with 0.1 frequency of the malathion-resistant individuals. The offspring of the Syn strain were divided into two sub-strains, Rm and Rm+ibp, which were successively treated with, respectively, malathion alone and malathion + iprobenfos (1:2) at LC70. In the mixture, the fungicide iprobenfos acted as a synergist of malathion. After treatment for 10 generations, the resistance level to malathion was 317.4-fold for the Rm sub-strain, whereas for the Rm+ibp sub-strain it was only 38.9-fold, compared with the Syn strain. Similar results were obtained by measurement of alpha-NA esterase activity from both larvae and female adults. The alpha-NA esterase activities in larvae and female adults at F10 generation were 2.6- and 10.9-fold from the Rm+ibp sub-strain and 5.7- and 98.5-fold from the Rm sub-strain, respectively, compared with the Syn strain. The above results suggested that iprobenfos, although it cannot completely stop or prevent the onset of malathion resistance, could dramatically delay its evolution., (Copyright 2005 Society of Chemical Industry.)

Clinical use of chlorpromazine (CPZ), an antipsychotic drug, is limited due to its hepatotoxicity. CPZ is found to inhibit in vitro intracellular carboxylesterases (CE), such as alpha-naphthyl acetate esterase, naphthol AS-D chloroacetate esterase, and alpha-naphthyl butyrate esterase in polymorphonuclear neutrophils, hepatocytes, and neuronal brain cells from mice. CPZ inhibits CE of all these cell types, whereby the degree of the inhibition depends on the incubation time and CPZ concentration. The polymorphonuclear neutrophils are most sensitive to CPZ. Comparable results were obtained with polymorphonuclear neutrophils from mice and humans. Since leukocytes are much more available than hepatocytes or neuronal cells in humans, we assume that CE in peripheral blood leukocytes (neutrophils and monocytes) can be used as markers for indication of pending liver damage by CPZ.

Catalytic activities of lingual lipase were investigated by enzyme histochemistry in post-mortem tongues from male rats. Sections of fresh-frozen or formalin-calcium fixed tissue were incubated with naphthol-AS-nonanoate and alpha-naphthyl acetate substrate mixtures. The effects of pH level, sodium taurocholate activator and E600 inhibitor were also examined. The use of cryostat sections of tissues fixed in formalin-calcium and of nonanoate substrate within the range of pH 4.4-6.4, were optimal for localizing maximum reaction product, captured by Fast Blue BB, in acini and demilunes of the posterior deep and superficial lingual glands respectively. The reaction product corresponded with the distribution of secretory granules and failed to develop when taurocholate was omitted from the incubation medium. Similarly localized E600-resistant reaction product occurred with the acetate substrate and hexazotized New Fuchsin at pH 7.4, in the absence of taurocholate. Lipase and conventional esterase activities appear to be superimposed in posterior lingual glands of rat. The ability of their acini and demilunes to hydrolyse nonanoate substrate at an acidic pH optimum, when activated by sodium taurocholate, seems attributable to lipase destined for secretion into saliva--hence convenient for routine histochemical identification of the enzyme.

Wallerian degeneration of a peripheral nerve is mainly characterized by axon and myelin degradation and is paralleled by a massive invasion of peripheral macrophages into the nerve. These cells enter the nerve attracted by a cascade of chemokines and cytokines but require proteolytic and enzymatic factors which enables them to cross the blood-nerve barrier. Here we investigated whether alpha-naphthyl (alpha-NA) esterases -- which have been shown to be exclusively expressed in human monocytes -- play a role during Wallerian degeneration. These enzymes were blocked by the specific inhibitor bis(4-nitrophenyl)-phosphate (BNPP) in an established in vitro model of Wallerian degeneration. Sciatic nerve segments of mice were co-cultured with peritoneal macrophages and BNPP was added to the cultures in various concentrations and at different timepoints. The macrophage numbers and myelin density in the nerve segments and the myelin load of macrophages were evaluated. After BNPP treatment the macrophage number within the nerve was significantly diminished and the myelin load within the macrophages was decreased, resulting in elevated levels of preserved myelin within the nerves. These experiments clearly showed a double effect of the alphaNA esterase inhibitor BNPP on macrophages. First, it suggests a role for alphaNA esterases on the migratory potential of macrophages since their invasion into the nerves was diminished. Second, the reduced myelin uptake is due to the inhibition of phagocytic capacity of these cells by BNPP. The therapeutical use of this inhibitor for treatment of autoimmune diseases such as multiple sclerosis or Guillain-Barré syndrome remains to be investigated.

Three alpha-naphthyl acetate hydrolyzing esterase isozymes were purified from microsomes prepared from Reticulitermes flavipes workers. The two step process involved sequential preparative IEF followed by continuous elution preparative electrophoresis on a 5% non-denaturing polyacrylamide gel. The first IEF run resulted in 5.4-fold purification with a yield of 46.1%. Subsequent IEF further purified the esterases 14.3-fold and 12% yield. Preparative electrophoresis of the pooled IEF fractions produced three major peaks of alpha-naphthyl acetate hydrolyzing activity. The esterases were correspondingly designated microsomal esterase (ME) 1, ME 2, and ME 3 based on increasing molecular retention on a native PAGE gel. ME 1, ME 2, and ME 3 were acidic proteins with pI values of 4.61, 4.70, and 4.77, respectively. Molecular mass as determined by gel filtration chromatography of ME 1, ME 2, and ME 3 was 69, 64, and 62 kDa, respectively. SDS-PAGE gels produced a single band for each of the isozymes with a molecular mass of 63 kDa indicating that the esterases were monomers. Specific activities of ME 1, ME 2, and ME 3 increased with increasing pH and the enzymes were active over a broad temperature range (25-55 degrees C). The three purified isozymes were inhibited at low concentration by paraoxon (10(-10) M), chlorpyrifos (10(-6) M), DEF (10(-6) M), and PMSF (10(-6) M) indicating that they were "B" type serine esterases. Conversely, inhibition was not observed at 10(-4) M eserine, PHMB, or CaCl(2), further supporting the conclusion that the microsomal esterases were of the "B" type. None of the isozymes was inhibited by 10(-4) M imidacloprid, fipronil, or PBO. Quantitatively, ME 1, ME 2 and ME 3 metabolized t-permethrin at 21.8, 21.0, and 38.8 nmol/h/mg protein, representing a purification factor of 333-, 318-, and 591-fold over microsomes, respectively. The three isozymes produced the same type and number of t-permethrin metabolites.

This study examines the distribution of alpha-naphthyl acetate esterase enzymes in reduced enamel epithelium, i.e., post secretory ameloblasts (PSA) and external cells of reduced enamel epithelium (ERE) of continuously growing incisor and molar tooth germs and in the covering oral epithelium (OE). Jaws of guinea pig embryos, 25-50 days of gestation age, were pretreated, frozen, serially cut, and incubated with alpha-naphthyl acetate as substrate and hexazotized pararosaniline as capture agent for demonstration of enzyme activity. In addition, sections were preincubated with various inhibitors of enzyme activity. A strong enzyme reaction, essentially unaffected by pretreatment procedures, was demonstrated in all cells of PSA and ERE, and in suprabasal cells of OE. Preliminary characterization of the alpha-naphthyl acetate esterase enzymes by means of inhibitors suggests a prevailing presence of B-esterase enzymes in both oral and dental epithelia. PSA were selectively stained when 2 x 10(-3) M arsanilic acid was used as inhibitor, ERE were selectively inhibited by 10(-5) M eserine, and cells of OE were selectively stained when 2 x 10(-3) M HgCl2 was used as inhibitor. We therefore conclude that the alpha-naphthyl acetate esterase enzymes in combination with inhibitors may serve as histochemical markers for discrimination of dental and oral epithelium in the guinea pig prior to eruption of teeth.

Using image-analyzing equipment, we measured the effect of quinidine on the activity of naphthol AS-D chloroacetate esterase, alpha-naphthyl butyrate esterase, alpha-naphthyl acetate esterase, and acid phosphatase in individual cells of a human Hep G2 hepatoma cell line. The impact of the drug on the morphology of the cells was also observed. Depending on the concentration of quinidine applied, various changes occurred, the most extreme being cell death. However, at some drug concentrations that did not appear to affect visible cell structures, the activity of the esterases was decreased. This lessened enzyme activity did not seem to be related to the enzymes leaking from the cells, because the activity of acid phosphatase was unaffected. Inhibition of the esterase activity was related to the interval of exposure to quinidine in the perfusing medium and to the concentration of the drug. We consider the system described here to be a potential replacement for experiments with animals in the study of hepatotoxicity.

Normal human leucocytes and platelets contain esterases which hydrolyse alpha-naphthyl acetate (alpha NA). Purified preparations from these cells were investigated by isoelectric focusing and subsequent polyacrylamide gradient gel electrophoresis at pH 9.0. Extractable alpha NA esterases were separated according to isoelectric point (pI) and molecular weight (MW). Monocytes, lymphocytes, granulocytes and platelets contain a unique pattern of alpha NA esterases, most of which can be inhibited by diisopropyl fluorophosphate (DFP; 0.1 mM). Their activity, however, is not affected by eserine (0.1 mM) or p-hydroxymercuribenzoate (1 mM). No protease activity of these enzymes was detected; it is likely that the majority constitute carboxylesterases (EC 3.1.1.1). Monocytes contain five alpha NA esterases which are additionally inhibited by bis(4-nitrophenyl)-phosphate (0.1 mM) and sodium fluoride (40 mM). PIs are in the range 5.7-6.2 and MWs are 145 000, 155 000, 250 000, 290 000 and 340 000. These enzymes are specific for monocytes. Platelets are characterized by a group of alpha NA esterases having pIs between 6.5 and 8.0, these corresponding to MWs ranging from 15 000 to 400 000.

Following characterization of myeloid nonspecific esterases by isoelectric focusing (IEF), two main groups of alpha-naphthyl acetate (ANAE) esterase isoenzymes were defined and fractionated from cytoplasmic extracts by chromato focusing techniques according to differences in their isoelectric points (pI). The first of these ANAE enzyme groups was common to leukocytes of both granulocytic and monocytic lineage, while the other, which characteristically comprised a group of isoenzymes within the pI range 5.5-6.1, was specifically associated with monocytic differentiation. The properties of the two purified ANAE enzyme fractions were compared by inhibition (heat and sodium fluoride) and further electrophoretic studies, and the results discussed in relation to the cytochemical characterization of these enzymes as markers of specific myeloid cell differentiation.

We have demonstrated the feasibility of a system based on image processing to measure enzyme activity inside morphologically classified individual cells. When used in this system, quinidine inhibited naphthol AS-D chloroacetate esterase in polymorphonuclear neutrophils and alpha-naphthyl acetate esterase and alpha-naphthyl butyrate esterase in monocytes. These effects were dependent on the duration of exposure and on drug concentration. Dose-effect relationships were established and the concentration at which 50% inhibition (ID-50) occurred was used as a reference point to compare the toxicity of different compounds. Chloroquine, primaquine, and quinine (which, like quinidine, possess a quinoline ring) also affected the esterases, but had no effect on several other enzymes. Neostigmine inhibited the esterases we studied but only at a very high concentration. Various chemicals inhibited the enzymes within the cells, as has been demonstrated for the enzymes in plasma.

On continuation of study by KURZ and GOSLAR (1974) concerning the inhibitory effects to non specific esterases on liver and kidney, the behaviour of alpha-naphthyl-acetate-esterases on hypothalamus, neurohypophysis, adrenal and parotid glands of rats had been examined against aldehydes, organic solution-mediators and anorganic salts. It had shown a different sensitiveness, the esterases of tanycytes, nerve cells and pituicytes acted more sensitive than those of the adrenal cortex and parotid gland. The differences seen on the solution-mediators were less; on metal salts from different groups of periodical system which applied, the most intense blocking effect was found in the nerve cells. The esterases of tanycytes and the immediately extensively reacting pituicytes, as well as the adrenal cortex esterases reacted essentially less sensitive, the gland cells of parotid even almost not at all. The arguments of this behaviour are discussed and pointed out with different complexity of alpha-naphthylacetate splitting enzymes on individual organs.

Human monocytes contain a series of alpha-naphthyl acetate (alpha NA) esterases which are not present in other blood cells and which can be specifically inhibited by bis(4-nitrophenyl)-phosphate (BNPP). This inhibitor is non-toxic at the concentration used and thus enabled studies on the possible significance of this enzyme towards various monocyte functions. BNPP has no noticeable influence on adhesion and spreading of monocytes on glass surfaces, nor does it inhibit the phagocytosis of IgG-coated latex particles. BNPP does, however, diminish the spontaneous cytotoxicity of freshly isolated monocytes towards the erythroleukaemic cell line K562. In the single cell assay in agarose, BNPP treatment of monocytes leads to a decrease in the number of lytic and non-lytic effector-target cell conjugates. In contrast, the antibody-dependent cell-mediated cytotoxicity (ADCC) of monocytes as well as the natural cytotoxicity of lymphocytes towards K562 cells are not influenced by BNPP. The present investigations show that monocyte specific alpha NA esterases are involved in the spontaneous cytotoxicity of monocytes toward tumour cells.

Serial changes of immunosuppressive activity of serum and cell-free skin bleb fluid that can suppress the activity of acid a-naphthyl acetate esterase (ANAE) of lymphocytes and phagocytosis of macrophages were detected by immunoregulatory tests in vitro in 50 lung cancer and 42 esophageal cancer patients. In comparing these tests with those of 53 cases of noncancerous thoracic lesion and 69 normal adults, the immunosuppressive activity of serum and skin bleb fluid from cancer patients is significantly higher than that of noncancerous thoracic lesions and normal individuals (P less than .01). The activity is related to the stages of cancer, the size of primary tumor, the presence of lymph node metastasis, and tumor resect-ability, but not to histological classification, sex, and age. The immunosuppressive activity of serum and skin bleb fluid decreased gradually after the removal of the tumor and was eliminated on the 30th postoperative day. These results suggest that serum and skin bleb fluid from cancer patients may contain immunosuppressive factors that can suppress the immune responsive functions of lymphocytes and macrophages in vitro in a manner similar to that seen in vivo. Therefore, complete surgical removal of cancer is likely the most effective immunotherapy.