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C J Cao

Publications and source records attributed to C J Cao.

9 recordsLinked to original sources
PubMed1999

Cytotoxicity of organophosphate anticholinesterases.

Organophosphate (OP) anticholinesterases were found to modulate metabolic activities of human neuroblastoma cells and hepatocytes, which was detectable by the Cytosensor microphysiometer. The nerve gas ethyl-S-2-diisopropylaminoethyl methylphosphorothiolate (VX), at 10 microM, produced significant reduction in cell metabolism within 2 min, as measured by changes in the acidification rate of the medium. The reduction was dose- and time-dependent and irreversible after 4 h of exposure. Two alkaline degradation products of VX produced no cytotoxicity. Exposure for 24 h to 3 microM VX caused 36% and 94% irreversible loss of metabolism in hepatocytes and neuroblastoma cells, respectively. The insecticides parathion and chlorpyrifos stimulated hepatocyte metabolism but inhibited neuroblastoma cells. Their oxons were more active. Exposure of neuroblastoma cells for 4 h to VX, parathion, paraoxon, diisopropylfluorophosphate or chlorpyrifos gave an LC50 of 65, 775, 640, 340, or 672 microM, respectively, whereas 24 h gave an LC50 of 0.7, 3.7, 2.5, 29, and 31 microM, respectively. Preincubation of hepatocytes with phenobarbital enhanced their response to parathion and VX due to metabolic bioactivation. Atropine partially blocked the effects of VX and paraoxon on both cell types, which suggests the involvement of a muscarinic receptor as the target for cytotoxicity. There was no correlation between OP in vivo neurotoxicity and in vitro cytotoxicity. It is suggested that the former results from their cholinesterase inhibition, while the latter results from action on different targets and requires much higher concentrations.

Acetylcholinesterase↗
PubMed1998

Toxicity of sea nettle toxin to human hepatocytes and the protective effects of phosphorylating and alkylating agents.

The sea nettle jellyfish toxin (SNTX), which contains several polypeptides, was highly toxic to human hepatocytes. The Cytosensor microphysiometer was used continuously to monitor cell media acidification rate as an index of cellular metabolic activity. Cells exposed to > 1 microg SNTX protein/ml media exhibited a transient increase in metabolic activity, followed by a sharp decrease and cell death within minutes. The kinetics of the transient increase and subsequent decline increased with higher concentrations of SNTX. The biphasic and time-dependent response of hepatocytes to SNTX suggests that more than one mechanism may be involved in the toxicity of its different polypeptides. SNTX-induced cytotoxicity of hepatocytes was reduced by the presence of high titer antibodies against a heterologous jellyfish. Phenobarbital-induced cells became more vulnerable to SNTX, suggesting that some toxin component(s) require(s) bioactivation. Short-term exposure (1-2 h) to 10 microg/ml of the calcium ionophore calcimycin, or the non-selective monovalent cation ionophore gramicidin, had no effect on metabolic activity. However, 165 microg/ml gramicidin or 53 microg/ml calcimycin produced slight transient activation followed by steady decline in metabolic activity, while 20 h exposure to either ionophore produced total cell death. Exposure to even a 10-fold lower concentration of either ionophore killed 88% and 75%, respectively. This contrasts with the toxicity of SNTX which is detectable in minutes with as little as 3 microg/ml. Since pre-exposure to the organophosphate anticholinesterases VX and paraoxon, or the chemotherapeutic alkylating agents cyclophosphamide and mechlorethamine reduced the cytotoxic effects of SNTX, it suggests that phosphorylation or alkylation of cell protein(s) interferes with SNTX toxicity.

Alkylating Agents↗
PubMed1991

Uncoupling of Ca2+ transport from ATP hydrolysis activity of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase.

In reconstituted rabbit skeletal muscle (Ca2+ + Mg2+)-ATPase proteoliposomes, Ca(2+)-uptake is decreased by more than 90% with T2 cleavage (Arg-198). However, no difference in the ATP dependence of hydrolysis activity is seen between SR and trypsin-treated SR. A large decrease in E-P formation and hydrolysis activity of the enzyme appear only at T3 cleavage, which represents the cleavage of A1 fragment to A1a + A1b forms. The disappearance of hydrolysis activity due to digestion is prior to the disappearance of E-P formation. No significant difference is found in the passive Ca2+ efflux between control SR and tryptically digested SR in the absence of Mg2+ + ruthenium red or in the presence of ATP. However, the passive Ca2+ efflux rate for tryptically digested SR is much larger than control SR in the presence of Mg2+ + ruthenium red. These results show that the Ca2+ channel cannot be closed after trypsin digestion of SR membranes by the presence of the Ca2+ channel inhibitors, Mg2+ and ruthenium red. In the reconstituted proteoliposomes, the Ca2+ efflux rates are the same regardless of digestion (T2); also, efflux is not affected by the presence or absence of Mg2+ + ruthenium red. These results indicate that T2 cleavage causes 'uncoupling' of the 'Ca(2+)-pump' from ATP hydrolytic activity. A theoretical model is developed in order to fit the extent of tryptic digestion of the A fragment of the (Ca2+ + Mg2+)-ATPase polypeptide with the loss of Ca(2+)-transport. Fits of the theoretical equations to the data are consistent with that Ca(2+)-transport system appears to require a dimer of the polypeptide (Ca2+ + Mg2+)-ATPase.

Adenosine Triphosphate↗
PubMed1991

Acetylcholinesterase fiber-optic biosensor for detection of anticholinesterases.

An optical sensor for anticholinesterases (AntiChEs) was constructed by immobilizing fluorescein isothiocyanate (FITC)-tagged eel electric organ acetylcholinesterase (AChE) on quartz fibers and monitoring enzyme activity. The pH-dependent fluorescent signal generated by FITC-AChE, present in the evanescent zone on the fiber surface, was quenched by the protons produced during acetylcholine (ACh) hydrolysis. Analysis of the fluorescence response showed Michaelis-Menten kinetics with a Kapp value of 420 microM for ACh hydrolysis. The reversible inhibitor edrophonium (0.1 mM) inhibited AChE and consequently reduced fluorescence quenching. The biosensor response immediately recovered upon its removal. The carbamate neostigmine (0.1 mM) also inhibited the biosensor response but recovery was much slower. In the presence of ACh, the organophosphate (OP) diisopropylfluorophosphate (DFP) at 0.1 mM did not interfere with the ACh-dependent fluorescent signal quenching, but preexposure of the biosensor to DFP in absence of ACh inhibited totally and irreversibly the biosensor response. However, the DFP-treated AChE biosensor recovered fully after a 10-min perfusion with pralidoxime (2-PAM). Echothiophate, a quaternary ammonium OP, inhibited the ACh-induced fluorescence quenching in the presence of ACh and the phosphorylated biosensor was reactivated with 2-PAM. These effects reflected the mechanism of action of the inhibitors with AChE and the inhibition constants obtained were comparable to those from colorimetric methods. The biosensor detected concentrations of the carbamate insecticides bendiocarb and methomyl and the OPs echothiophate and paraoxon in the nanomolar to micromolar range. Malathion, parathion, and dicrotophos were not detected even at millimolar concentrations; however, longer exposure or prior modification of these compounds (i.e., to malaoxon, paraoxon) may increase the biosensor detection limits. This AChE biosensor is fast, sensitive, reusable, and relatively easy to operate. Since the instrument is portable and can be self-contained, it shows potential adaptability to field use.

Acetylcholinesterase↗
PubMed1990

Tertiary structure and energy coupling in Ca2(+)-pump system.

Europium luminescence from europium bound to sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase indicates that there are two high affinity calcium binding sites. Furthermore, the two calcium ions at the binding sites are highly coordinated by the protein as the number of H2O molecules surrounding the Ca2+ ions are 3 and 0.5. In the presence of ATP, calcium ions are occluded even further down to 2 and zero H2O molecules, respectively. The Ca2+ - Ca2+ intersite distance is estimated to be 8-9 A and the average distance from the Ca2+ sites to CrATP is about 18 A. Digestion of the (Ca2+ + Mg2+)-ATPase at the T2 site (Arg 198) causes uncoupling of Ca2(+)-transport from ATPase activity while calcium occlusion due to E1-P formation remains unchanged. Further tryptic digestion beyond T2 and in the presence of ATP diminishes Ca2+ occlusion to zero while 50% of the ATPase hydrolytic activity remains. Tryptic digestion beyond T2 and in the absence of ATP diminishes ATPase hydrolytic activity to 50% of normal while Ca2+ occlusion remains intact. These data are consistent with a mechanism in which the functional enzyme must be in the dimeric form for occlusion and calcium uptake to occur, but each monomer can hydrolyze ATP.

Adenosine Triphosphate↗
PubMed1990

ATP-regulated neuronal catecholamine uptake: a new mechanism.

Uptake of the catecholamines (CA), dopamine (DA) and norepinephrine (NE) into synaptosomes prepared from rat and bovine brains was potentiated by ATP (from 0.1 to 5.0 mM) in a dose-dependent manner. Other nucleotides, particularly the nonhydrolyzable ATP analogs beta,gamma-imidoadenosine-5'-triphosphate (AMP-PNP) and beta,gamma-methyladenosine-5'-triphosphate (AMP-PCP) also potentiated [3H]DA and [3H]NE uptake. Several endogenous 5'-nucleotide triphosphates (e.g. GTP, UTP and CTP) potentiated [3H]CA uptake, but were less effective than ATP. Among the ATP metabolites, only ADP potentiated uptake whereas AMP and adenosine did not. [3H]Dopamine uptake measured in Krebs bicarbonate buffer had a Km of 2.1 microM and a Vmax of 163.9 pmol/mg prot./min. In presence of ATP, [3H]DA uptake had much higher affinity (Km = 0.56 microM) and larger capacity (Vmax = 333 pmol/mg prot./min) than uptake in absence of added ATP. Furthermore, [3H]DA uptake in presence of ATP had faster rate of uptake, and was independent of temperature while in absence of added ATP it was temperature-dependent. This ATP-dependent [3H]DA uptake was retained by synaptosomal ghosts that were obtained after lysing the striatal synaptosomes and removing their contents of synaptic vesicles and mitochondria. It is proposed that, in addition to the carrier-mediated (neuronal) uptake of CA, there is neuronal uptake that is regulated by ATP and inhibited by cocaine, which may be more relevant for terminating the synaptic action of CA because of its faster rate of uptake and larger capacity.

Adenosine Triphosphate↗
PubMed1989

Putative cocaine receptor in striatum is a glycoprotein with active thiol function.

Dopamine transporters of bovine and rat striata were identified by their specific [3H]cocaine binding and cocaine-sensitive [3H]dopamine [( 3H]DA) uptake. Both binding and uptake functions of bovine striatal transporters were potentiated by lectins. Concanavalin A (Con A) increased the velocity but did not change the affinity of the transporter for DA; however, it increased its affinity for cocaine without changing the number of binding sites. This suggests that the DA transporter is a glycoprotein and that Con A action on it produces conformational changes. Inorganic and organic mercury reagents inhibited both [3H]DA uptake and [3H]cocaine binding, though they were all more potent inhibitors of the former. n-Ethylmaleimide inhibited [3H]DA uptake totally but [3H]cocaine binding only partially. Also, n-pyrene maleimide had differential effects on uptake and binding, inhibiting uptake and potentiating binding. [3H]DA uptake was not affected by mercaptoethanol up to 100 mM, whereas [3H]cocaine binding was inhibited by concentrations above 10 mM. On the other hand, both uptake and binding were fairly sensitive to dimercaprol (less than 1 mM). The effects of all these sulfhydryl reagents suggest that the DA transporter has one or more thiol group(s) important for both binding and uptake activities. The Ellman reagent and dithiopyridine were effective inhibitors of uptake and binding only at fairly high concentration (greater than 10 mM). Loss of activity after treatment with the dithio reagents may be a result of reduction of a disulfide bond, which may affect the transporter conformation.

4-Chloromercuribenzenesulfonate↗