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PubMed1985

Interaction of alpha-crystallin with lens plasma membranes. Affinity for MP26.

The binding of the major water-soluble lens protein alpha-crystallin to the lens plasma membrane has been investigated by reassociating purified alpha-crystallin with alpha-crystallin-depleted membranes and with phospholipid vesicles in which the lens membrane protein MP26 had been reconstituted. alpha-Crystallin reassociates at high affinity (Kd = 13 X 10(-8)M) with alkali-washed lens plasma membranes but not with lens plasma membranes treated with guanidine/HCl, nor with phospholipid vesicles or erythrocyte membranes. Binding to lens plasma membranes is dependent on salt, temperature and pH and occurs in a saturable manner. Reconstitution of MP26 into phospholipid vesicles and subsequent analysis of alpha-crystallin binding suggests the involvement of this transmembrane protein. Binding ist not influenced by pretreatment of membranes with proteases, suggesting that the 4-kDa cytoplasmic fragment of MP26 is not necessary for alpha-crystallin binding. Labeling experiments using (trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine as a probe for intrinsic membrane proteins further showed that alpha-crystallin contains hydrophobic regions on its surface which might enable this protein to make contact with the lipid bilayer. Newly synthesized alpha-crystallin, in lens culture, is not associated with the plasma membrane, suggesting that the assembly of alpha-crystallin aggregates does not take place in a membrane-bound mode.

Animals↗
PubMed1986

Labeling of individual amino acid residues in the membrane-embedded F0 part of the F1 F0 ATP synthase from Neurospora crassa. Influence of oligomycin and dicyclohexylcarbodiimide.

Three F0 subunits and the F1 subunit beta of the ATP synthase from Neurospora crassa were labeled with the lipophilic photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). In the proteolipid subunit which was the most heavily labeled polypeptide labeling was confined to five residues at the NH2-terminus and five residues at the C-terminus of the protein. Labeling occurred at similar positions compared with the homologous protein (subunit c) in the ATP synthase from Escherichia coli, indicating a similar structure of the proteolipid subunits in their respective organisms. The inhibitors oligomycin and dicyclohexylcarbodiimide did not change the pattern of accessible surface residues in the proteolipid, suggesting that neither inhibitor induces gross conformational changes. However, in the presence of oligomycin, the extent of labeling in some residues was reduced. Apparently, these residues provide part of the binding site for the inhibitor. After reaction with dicyclohexylcarbodiimide an additional labeled amino acid was found at position 65 corresponding to the invariant carbodiimide-binding glutamic acid. These results and previous observations indicate that the carboxyl side chain of Glu-65 is located at the protein-lipid interphase. The idea is discussed that proton translocation occurs at the interphase between different types if F0 subunits. Dicyclohexylcarbodiimide or oligomycin might disturb this essential interaction between the F0 subunits.

Amino Acid Sequence↗
PubMed1988

F0 part of the ATP synthase from Escherichia coli. Influence of subunits a, and b, on the structure of subunit c.

Four different sets of proteoliposomes were prepared from F0, subunit c, a complex of subunits a and c (ac complex) and an ac complex supplemented with subunit b. Only liposomes containing intact F0 or all subunits of F0 were active in proton translocation and F1 binding [Schneider, E. and Altendorf, K. (1985) EMBO J. 4, 515-518]. The conformation of subunit c in the different preparations was analyzed by labelling the proteoliposomes with the hydrophobic photoactivatable reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). Subsequent isolation and Edman degradation of this polypeptide revealed distinct radioactive labelling patterns over the entire amino acid sequence. In the F0 complex and in the ac complex subunit c retains a labelling pattern which is related to that found in TID-labelled membrane vesicles of Escherichia coli [Hoppe et al. (1984) Biochemistry 23, 5610-5616]. In the absence of subunit a, considerably more and different amino acid residues of subunit c are modified. The labelling data are discussed in relation to structural aspects of F0 and functional properties of proteoliposomes reconstituted with F0 or individual subunits.

Azirines↗
PubMed1987

Effects of intrahippocampal injections of the cholinergic neurotoxin AF64A on presynaptic cholinergic markers and on passive avoidance response in the rat.

1. The effects of the intrahippocampal injection of ethylcholine mustard aziridinium ion (AF64A) on the following cholinergic markers were examined. 2. Bilateral injection of 10 nmol of AF64A into the dorsal hippocampus caused a loss of choline acetyltransferase (CAT) activity in the dorsal hippocampus, ventral hippocampus, cortex and striatum. 3. This treatment caused significant decreases in ACh contents in the dorsal hippocampus, ventral hippocampus and cortex, but not in the striatum. 4. The step-down latencies during the test trials of both 10 and 50 nmol AF64A-treated groups were significantly shorter than those of vehicle-treated groups. 5. None of the control animals stepped down to the large box where they had previously received a foot shock, but 40% of the 10 nmol and 60% of the 50 nmol AF64A-treated animals stepped down to the large box within 600 s. 6. These results indicate that AF64A treatment produces biochemical and functional deficits in the cholinergic neurons in the CNS.

Acetylcholine↗
PubMed1987

Neurochemical effects following AF64A injection into the basal nuclei of rats.

AF64A, a specific cholinergic neurotoxin, was injected into the basal nuclei of rats. The injected sites were the bilateral nucleus basalis of Meynert (NBM) and the medial septal nucleus (MSN), well known to be the nuclei of origin of the two major cholinergic pathways. The remote effects of injection were estimated by the regional choline acetyltransferase (CAT) activity in the frontal cortex, striatum and hippocampus. The injection of AF64A (1 nmol in 1 microliter) produced a reduction in the CAT activity in each projected site: NBM lesions in the frontal cortex and MSN lesions in the hippocampus after one and three weeks. Twelve weeks after the injection, the reduced CAT activity had returned to normal levels. This neurochemical effect shows plasticity and recovery with time. The injections of small amounts of AF64A (0.2 and 0.1 nmol in 1 microliter) produced no chemical changes after one week.

Acetylcholinesterase↗
PubMed1983

Selective presynaptic cholinergic neurotoxicity following intrahippocampal AF64A injection in rats.

Compound AF64A, ethylcholine mustard aziridinium ion (0.4-8 nmol) was stereotaxically administered into rat dorsal hippocampus, and neurochemical changes were determined 5 days later. AF64A treatment, over an almost 10-fold dose range, resulted in a significant (up to 70%) decline in choline acetyltransferase activity. In the same tissue samples, Na+-dependent choline transport activity was also lowered, with most decreases ranging between 10 and 50% of controls; however, there was no significant correlation (r = 0.39) between these two parameters. Acetylcholinesterase activity was not affected by AF64A treatment when assayed by either histochemical or enzymatic methods. AF64A reduced acetylcholine levels by 43%, but did not alter norepinephrine content or serotonin uptake. These results demonstrate that AF64A can induce a specific, long-term reduction of cholinergic presynaptic biochemical markers in rat hippocampus. Thus, AF64A can serve as a useful new tool to study the cholinergic system and as an important agent to help develop animal models representing disorders of central cholinergic hypofunction.

Acetylcholine↗
PubMed1984

Effect of ethyl choline mustard on choline dehydrogenase and other enzymes of choline metabolism.

The effect of ethyl choline mustard (ECMA), and effective irreversible inhibitor of choline transport, was investigated on the enzymes of choline metabolism. ECMA at concentrations of 50 microM hardly affected choline acetyltransferase and caused only a 20% inhibition of choline kinase at a concentration of 1 mM. However, the mustard was an extremely effective inhibitor of choline dehydrogenase, producing 50% inhibition at concentrations of 6 microM. The inhibition was prevented by incubation in the presence of choline or by prior reaction of the mustard with thiosulphate. Separation of the components of the ECMA solution on TLC suggested that only the compound with an aziridine ring was an effective inhibitor of choline dehydrogenase. The inhibition was resistant to the washing out of excess unreacted mustard. The rate constant of inhibition was 395 M-1 X S-1. By the use of [3H]ECMA a single polypeptide in the enzyme preparation having a MW of 67,000 was labelled. The labelling was thiosulphate-sensitive and prevented by incubation with choline. It is concluded that ECMA is an irreversible inhibitor of choline dehydrogenase. It is at least as effective an inhibitor of choline dehydrogenase as of the choline transport system, although it does not appreciably inhibit choline acetyltransferase or choline kinase in the micromolar range.

Alcohol Oxidoreductases↗
PubMed1985

AF64A: an active site directed irreversible inhibitor of choline acetyltransferase.

Ethylcholine mustard aziridinium ion (AF64A, MEChMAz) has been proposed as a cholinergic neuron-specific neurotoxin. We report that in further studies on its mechanism of action incubation of the cholinergic neuroblastoma X glioma cell line, NG-108-15, with 100 microM AF64A resulted in a rapid decrease in cellular choline acetyltransferase (ChAT) activity which preceded cytotoxicity. Thus, a 60-85% decrease in ChAT activity was measured within 5 h of AF64A exposure, whereas cell lysis (measured as the release of the cytosolic enzyme lactate dehydrogenase into the medium) did not become apparent until 18 h of AF64A exposure. This led us to examine the effects of AF64A on partially purified ChAT. We report a concentration- and time-dependent inhibition of partially purified ChAT by AF64A that could not be reversed by dialysis but could be prevented by coincubation of the enzyme and AF64A with choline but not with acetyl-coenzyme A. We present kinetic evidence that choline and AF64A compete for the same site on the enzyme. In addition, thiosulfate, which inactivates the aziridinium ion, eliminated AF64A's capacity to inhibit the enzyme. AF64A also irreversibly inhibited partially purified choline kinase and acetylcholinesterase but not lactate dehydrogenase, alcohol dehydrogenase, carboxypeptidase A, or chymotrypsinogen, enzymes that do not use choline as a substrate or product. Thus, the data suggest that AF64A acts as an irreversible active site directed inhibitor of ChAT and possibly other enzymes recognizing choline.

Animals↗
PubMed1987

Reversible and irreversible inhibition of high-affinity choline transport caused by ethylcholine aziridinium ion.

The effect of ethylcholine aziridinium ion (AF64A) on choline transport in hippocampal, striatal, and cerebrocortical synaptosomes was studied. Synaptosomes prepared from these three brain regions were equally sensitive to AF64A. Low concentrations of AF64A produced a reversible inhibition (IC50 values = 1.35-2.25 microM), whereas higher concentrations produced an irreversible inhibition (IC50 values = 25-30 microM), which started as competitive. The irreversible component of the inhibition was independent of extracellular Na+ concentration, a finding suggesting that the choline transporter is alkylated at its outward position. The kinetics of the inhibition were rapid and similar in the three brain regions examined. The high-affinity choline transport was more sensitive to the toxin than the low-affinity choline transport. Based on these results, we propose a kinetic model that explains the reversible and the irreversible inhibitions induced by AF64A. The possible relationships between the concentrations that in vitro produce reversible and irreversible inhibition and those that in vivo produce selective and nonselective cholinergic hypofunction are discussed.

Animals↗
PubMed1987

Specificity of ethylcholine mustard aziridinium as an irreversible inhibitor of choline transport in cholinergic and noncholinergic tissue.

The sensitivity of choline transport to inhibition by ethylcholine mustard aziridinium (ECMA) was studied in several tissues. Choline transport was found to be inhibited irreversibly by ECMA in guinea pig and rat synaptosomes but not inhibited in erythrocytes or kidney slices. If this finding can be extended to other tissues ECMA sensitivity may provide a simple criterion for identifying the choline carrier associated with cholinergic tissue.

Animals↗
PubMed1989

Clonidine prevents transient loss of noradrenaline in response to cholinergic hypofunction induced by ethylcholine aziridinium (AF64A).

Intracerebroventricular injection of ethylcholine aziridinium (AF64A) (2 nmol/ventricle) induced a considerable decrease in the level of acetylcholine (ACh) in hippocampus (from 21.14 +/- 0.84 to 10.04 +/- 0.59 pmol/mg of tissue; p less than 0.001) 4 days after application. The reduction of cholinergic function was accompanied by a decrease in the level of noradrenaline (NA) (from 1.96 +/- 0.08 to 1.41 +/- 0.06 pmol/mg of tissue; p less than 0.001). Two days after administration of AF64A (1 or 2 nmol/ventricle), the dose-dependent decrease in NA level was associated with an increase in the level of its major metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), resulting in a considerable increase in the MHPG/NA molar ratio (from 0.84 +/- 0.06 to 1.62 +/- 0.17; p less than 0.002). Chronic treatment of AF64A-injected rats with clonidine (0.02-0.2 mg/kg, i.p., every 8-12 h) had no significant effect on the loss of ACh content, whereas the decrease in NA content in hippocampus was completely prevented. Clonidine induced aggressive behavior in the AF64A-treated rats, in contrast to sedation in vehicle-injected rats. The response to clonidine under these experimental conditions and the increased MHPG/NA molar ratio in response to AF64A suggest that the transient loss of NA content following AF64A administration results from increased NA release. The increased noradrenergic activity in hippocampus may be linked to the reduction of tonic inhibitory cholinergic input. These results are discussed in relation to possible implications for senile dementia of the Alzheimer type.

Acetylcholine↗
PubMed1985

Dermatitis from aziridine hardener in printing ink.

13 of 51 workers developed dermatitis of the hands and face after handling a water-based ink containing a polyfunctional aziridine hardening agent. Improper work practices resulted in skin contamination with the ink and its ingredients. The aziridine hardener contained trimethylolpropane triacrylate (TMPTA). The incidence of dermatitis was highest among the ink mixers who handled the undiluted aziridine (6 of 8 workers affected), was lower among printers who handled ink containing 2 to 4% aziridine (7 of 22 workers affected), and was absent in workers who did not handle ink. The mean latency from first contact with the ink to the development of the rash was 3.2 months among the ink mixers, and 6.2 months among the printers. The present findings demonstrate the risk of handling aziridine hardeners when protective clothing is not properly used and when work practices result in direct skin contact. Further research should be performed to discern whether aziridine compounds themselves, free of TMPTA, can cause dermatitis.

Acrylates↗
PubMed1989

Treatment of multiple myeloma with carboquone-prednisolone.

18 previously untreated patients with multiple myeloma were entered in a clinical study of a combination chemotherapy, carboquone (CQ) and prednisolone (P) (CQ-P). Response was observed in 44% of patients treated with CQ-P judged by the criteria for response to chemotherapy. The median survival time was 33 months. Serious toxic reactions seldom occurred. The results suggest that the combination of CQ-P has efficacy in the treatment of multiple myeloma and could have a role among primary chemotherapeutic regimens for the disease. Further clinical trials should be conducted to establish a new drug combination in which CQ is included.

Aged↗