Second Phase Of Inhibition Research Articles

Differences of the ipsilateral silent period in small hand muscles

The ipsilateral silent period (iSP) is thought to depend on activity transmitted by the corpus callosum but ipsilateral corticospinal pathways may also contribute. Because the presence of ipsilateral corticospinal pathways differs between small hand muscles, we compared the iSP in the first dorsal interosseous (FDI) and abductor pollicis brevis (APB) muscles. The iSP was elicited in 20 healthy subjects by focal transcranial magnetic stimulation of one primary motor cortex during maximal voluntary contraction of the ipsilateral target muscle. The iSP duration was significantly longer in the FDI than APB because of an irregularly occurring second phase of inhibition in the FDI that was absent in the APB. Although the first phase of inhibition is transmitted by the corpus callosum, we provide evidence that the second phase is mediated through ipsilateral corticospinal pathways. Therefore, for specific assessment of callosal conduction, the iSP should be measured in the APB rather than FDI.

MX, a by‐product of water chlorination, lacks in vivo genotoxicity in gpt delta mice but inhibits gap junctional intercellular communication in rat WB cells

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a by-product of water chlorination, is a potent bacterial mutagen and rat carcinogen. In the present study, the in vivo mutagenicity, cell proliferative activity, and carcinogenicity of MX were investigated in gpt delta mice. Groups of 5 male and female 7-week-old gpt delta C57BL/6J transgenic mice were given MX at doses of 0, 10, 30, or 100 ppm in their drinking water for 12 weeks, and then killed to assess in vivo mutagenicity using 6-thioguanine and Spi- selection, and cell proliferative activity using immunohistochemistry for proliferating cell nuclear antigen (PCNA). Further groups of 10 male and female gpt delta mice were given 0 or 100 ppm MX for 78 weeks, and a full necropsy with histopathological examination of all organs was conducted to detect neoplastic lesions. The 12-week MX treatment did not result in mutagenicity in the livers or lungs or cell proliferative activity in several organs of the mice, and the 78-week treatment did not cause carcinogenicity. Additional investigations were conducted to evaluate the potential of MX to inhibit gap junctional intercellular communication (GJIC) in rat liver epithelial cells (WB cells) by the scrape loading/dye transfer method. Inhibition of GJIC was detected within 2 hr with a noncytotoxic dose of MX (4 microg/ml), followed by partial restoration after 5 hr. A second phase of inhibition occurred after 10 hr and then the lowered level persisted for the 24 hr-incubation period. Dose-dependent inhibition was evident at both 2 hr and 24 hr, with much stronger effects at the former time. These findings indicate that MX is not mutagenic, mitogenic or carcinogenic in mice, and suggest that the compound exerts epigenetic actions leading to GJIC inhibition.

Kinetic Model of Biphasic Character of Catalyse Inhibition

A mathematical model of kinetics of fungal catalase inhibition with hydroxylamine in presence of substrate (hydrogen peroxide) has been developed. The scheme includes an intermediate formation and slow reversible native enzyme production. The model is based on differential equations of nonstationary kinetics of the enzyme action. The computer simulation was carried out using adaptive Runge-Kuta method.
 Good satisfactory is achieved if the kinetic constants calculated by modeling nonstationary state of hydrogen peroxide decomposition are used for calculations of inhibition of the catalases at the second phase of inhibition and for kinetics of the intermediate formation, that was determined with stop-flow spectrophotometer.

Convulxin Binding to Platelet Receptor GPVI: Competition with Collagen Related Peptides

Convulxin (CVX), a potent platelet aggregating protein from the venom of the snake Crotalus durissus terrificus, is known to bind to the platelet collagen receptor, glycoprotein VI (GPVI). CVX binding to human platelets was investigated by flow cytometry, using fluorescein labeled convulxin (FITC-CVX). Scatchard analysis indicated high and low affinity binding sites with Kd values of 0.6 and 4 nM and Bmax values of 1200 and 2000 binding sites per platelet. FITC-CVX binding was inhibited by collagen related peptides (CRPs) comprising a repeated GPO sequence, namely GCO(GPO)10GCOGNH2 and GKO(GPO)10GKOGNH2, which also bind to receptor GPVI. These peptides (monomeric or cross-linked forms) gave a high affinity inhibition of 10–20% for concentrations between 10 ng/ml and 5 μg/ml, followed by a second phase of inhibition at concentrations greater than 5 μg/ml. It was shown also that the inhibition of FITC-CVX binding by CRPs was independent on the time of preincubation of platelets with CRPs, and the same percentage of inhibition was seen with various concentrations of convulxin. Confocal microscopy of the distribution of FITC-CVX binding sites on platelets showed an homogeneous distribution of FITC-CVX bound to GPVI, although some limited clustering may exist.

Inhibitory effect of pentachlorophenol on gap junctional intercellular communication in rat liver epithelial cells in vitro

To understand the initiating/promoting actions of pentachlorophenol (PCP), a non-mutagenic hepatocarcinogen, and its metabolite, tetrachlorohydroquinone (TCHQ), we investigated the effects of each chemical on gap junctional intercellular communication (GJIC) in rat liver epithelial cells (WB cells) by the scrape-loading and dye transfer method. After treatment with PCP, the GJIC was initially inhibited at 4 h but was restored in 6–8 h, followed by a second phase of inhibition between 16 and 24 h. Both the first and second inhibitions were concentration-dependent and were restored by 2–4 h after removal of PCP. The phosphorylation state of connexin 43 (CX43) and its localization on the plasma membrane were unchanged up to 24 h after treatment; however, this was accompanied by a decrease in the CX43 protein level. No inhibitory effect was apparent on the GJIC of cells treated with TCHQ. These results suggest that PCP may play a critical role of promoting activity via non-mutagenic mechanisms.

P3D123 - Inhibitory effect of pentachlorophenol on gap junctional intercellular communication in rat liver epithelial cells in vitro

To understand the initiating/promoting actions of pentachlorophenol (PCP), a non-mutagenic hepatocarcinogen, and its metabolite, tetrachlorohydroquinone (TCHQ), we investigated the effects of each chemical on gap junctional intercellular communication (GJIC) in rat liver epithelial cells (WB cells) by the scrape-loading and dye transfer method. After treatment with PCP, the GJIC was initially inhibited at 4 h but was restored in 6-8 h, followed by a second phase of inhibition between 16 and 24 h. Both the first and second inhibitions were concentration-dependent and were restored by 2-4 h after removal of PCP. The phosphorylation state of connexin 43 (CX43) and its localization on the plasma membrane were unchanged up to 24 h after treatment; however, this was accompanied by a decrease in the CX43 protein level. No inhibitory effect was apparent on the GJIC of cells treated with TCHQ. These results suggest that PCP may play a critical role of promoting activity via non-mutagenic mechanisms.

Electrophysiological investigation of thalamic neuronal mechanisms of motor disorders in parkinsonism: an influence of D 2ergic transmission blockade on excitation and inhibition of relay neurons in motor thalamic nuclei of cat

In acute experiments on cats anaesthetized with ketamine (25 mg/kg, i.m.) and immobilized with myorelaxine (2 mg/kg, i.v.) the activity of two groups of motor thalamic (nucleus ventralis anterior thalami-nucleus ventralis lateralis thalami) relay neurons was studied. The neurons ( n = 7) receiving afferents from deep cerebellar nuclei and projecting to the motor area 4γ were included in the first group, and those ( n = 12) receiving afferents from nucleus entopeduncularis and projecting to the supplementary motor area 6 were included in the second one. All changes in the background activity and reactions to cerebellothalamic or nucleus entopeduncularis stimulation developing under the influence of D 2 receptor antagonist haloperidol (1.5–1.7 mg/kg, i.v.) have been studied in the same cell. Under haloperidol influence both groups of neurons showed a reliable decrease of background activity and generation of high frequency discharges accompanied by a shift in the mode of interspike interval histograms. A regular decrease of probability and increase of response latencies after stimulation of afferent input were observed in neurons receiving afferents from the cerebellum. nucleus ventralis anterior thalami-nucleus ventralis lateralis thalami neurons with an inhibitory input from nucleus entopeduncularis, a shortening of inhibition from 17.5 ± 3.6to9.1 ± 1.8ms(P <0.05) under the haloperidol influence was evident. If the inhibition evoked by nucleus entopeduncularis stimulation consisted of two phases separated by a period of excitation ( n = 4), the duration of the second phase of inhibition after haloperidol injection regularly increased and the excitation separating the phases of inhibition became more prominent. Observation on the spontaneous activity and reactions of the same neuron for 2 h or more showed a gradual moderation of the changes evoked by haloperidol. On the basis of data obtained it is concluded that the blockade of D 2 receptors is followed by the increase of inhibitory processes in the relay neurons of motor thalamic nuclei. The suggestion is discussed that during the blockade of D 2 receptors afferent impulsation to the motor cortex is being restricted and its influence on segmental apparatus of the spinal cord decreases. These conditons are beneficial for the development of spasticity (rigidity). At the same time, hyperpolarization of the relay neurons promotes the development of oscillatory processes at least in part of them and creates conditions for forming of tremor generators.

Resiniferatoxin and piperine: Capsaicin-like stimulators of oxygen uptake in the perfused rat hindlimb

The naturally occurring capsaicin-like molecules, resiniferatoxin (RTX, Euphorbia spp.) and pipeline ( Piper nigrum), each stimulated oxygen uptake (V̇O 2) in association with increased vascular resistance in a concentration-dependent manner when infused into the perfused rat hindlimb. 5 μM glyceryl trinitrate (GTN, a nitrovasodilator) significantly blocked the oxygen and pressure responses to both RTX and piperine, indicating a close relationship between changes in V̇O 2 and the vasoconstriction. Concentrations greater than those required for maximal V̇O 2 resulted in an inhibition of V̇O 2, although perfusion pressure continued to increase. Time course studies showed that both RTX and piperine at high doses resulted in a tri-phasic response. An initial phase of transient V̇O 2 stimulation was followed by a second phase of inhibition. A third phase involving an often larger but transient stimulation of V̇O 2 followed removal of the agents and continued after the pressure returned to basal. The actions of RTX and piperine were similar to those of other active capsaicin-like molecules tested previously in this system, including capsaicinoids ( Capsicum spp.), gingerols ( Zingiber officinale), and shogaols ( Zingiber officinale). RTX was the most potent, and piperine the least potent of this series. Although receptor involvement has yet to be unequivocally established, the data are consistent with the presence of a functional capsaicin-like (vanilloid) receptor in the vasculature of the rat hindlimb that mediates vasoconstriction and oxygen uptake. These findings may have implications for the future development of thermogenic agents.

Evidence favouring presynaptic inhibition between antagonist muscle afferents in the human forearm.

1. A single motor-threshold electrical stimulus to the radial nerve in the spiral groove produced three phases of inhibition in the H reflex of relaxed wrist and finger flexor muscles. The first phase of inhibition occurred with a conditioning-test interval of -1 to 3 ms and represents disynaptic Ia reciprocal inhibition of flexor motoneurones by afferent input from extensor muscles. The second phase occurred with a conditioning-test interval of 5-40 ms. The characteristics of the second inhibitory phase have been investigated in the present study. 2. The threshold intensity of radial nerve stimulation required to produce the second phase of inhibition was 0.7 X motor threshold and was the same as that required to produce the first (disynaptic) phase of inhibition. 3. The time course of the effect of a radial nerve stimulus on the size of e.m.g. response to transcranial electrical stimulation of the motor cortex also was studied. The results were compared to those obtained with H reflex testing. The time course and depth of the first phase of inhibition were identical with both test inputs. The second phase of inhibition, however, was seen only with the H reflex test stimulus. When the corticospinal volley was timed to arrive at the cervical cord 30 ms after the radial nerve afferent volley, the flexor muscle response was facilitated rather than inhibited. 4. A single stimulus (2-3 X sensory threshold) to the superficial (cutaneous) branch of the radial nerve at the wrist failed to produce inhibition of the flexor H reflex. Instead, the H reflex was facilitated when the median nerve Ia volley (for the H reflex) was timed to arrive at the cervical cord between 30-40 ms after the radial nerve cutaneous afferent volley. In contrast to results using mixed nerve stimulation in the spiral groove, there was no difference between the effect of this cutaneous input on the cortically evoked e.m.g. response and the H reflex. 5. We conclude that the second phase of flexor H reflex inhibition is produced by activity in large-diameter (group I) extensor muscle afferents which acts on the H reflex pathway at a premotoneuronal site.(ABSTRACT TRUNCATED AT 400 WORDS)

A correlative study of the binding of dexamethasone in hypothalamic blocks in vitro with its ability to inhibit the release of bioactive corticotrophin-releasing factor

Rat hypothalamic blocks incubated in vitro were used to study the characteristics of binding of [ 3H]dexamethasone and other steroids to cytosolic binding sites. Cytosols prepared following incubation of the tissue with [ 3H]dexamethasone for 2 h contained specifically bound steroid in amounts that depended upon the concentration of potassium (but not sodium) ions in the extracting buffer. There was an increase in bound [ 3H]dexamethasone extracted as the potassium ion concentration increased up to 0.1 M, but not beyond. Dexamethasone, when added to hypothalami in vitro caused a biphasic inhibition of bioactive corticotrophin-releasing factor (CRF) release, and the extent of the second phase of inhibition was dose-related. 11-Epicortisol, when added in a 100-fold molar excess over dexamethasone was able to prevent the second phase of inhibition caused by the latter steroid, as in the binding studies it was able to cause a 50% reduction in the binding of [ 3H]dexamethasone. In the functional studies it was shown that 11-epicortisol was able to “rescue” the tissue from dexamethasone-mediated delayed inhibition of CRF secretion if added to the blocks 30 min (but not later) after the agonistic steroid.

Biphasic effect on platelet aggregation by phospholipase a purified from Vipera russellii snake venom

A basic phospholipase A was isolated from Vipera russellii snake venom. It induced a biphasic effect on washed rabbit platelets suspended in Tyrode's solution. The first phase was a reversible aggregation which was dependent on stirring and extracellular calcium. The second phase was an inhibitory effect on platelet aggregation, occurring 5 min after the addition of the venom phospholipase A without stirring or after a recovery from the reversible aggregation. The aggregating phase could be inhibited by indomethacin, tetracaine, papaverine, creatine phosphate/creatine phosphokinase, mepacrine, verapamil, sodium nitroprusside, prostaglandin E 1 or bovine serum albumin. The venom phospholipase A released free fatty acids from synthetic phosphatidylcholine and intact platelets. p- Bromophenacyl bromide-modified venom phospholipase A lost its phospholipase A enzymatic and platelet-aggregating activities, but protected platelets from the aggregation induced by the native enzyme. The second phase of the venom phospholipase A action showed a different degree of inhibition on platelet aggregation induced by some activators in following order: arachidonic acid > collagen > thrombin > ionophore A23187 . The longer the incubation time or the higher the concentration of the venom phospholipase A, the more pronounced was the inhibitory effect. The venom phospholipase A did not affect the thrombin-induced release reaction which was caused by intracellular Ca 2+ mobilization in the presence of EDTA, but inhibited collagen-induced release reaction which was caused by Ca 2+ influx from extracellular medium. The inhibitory effect of the venom phospholipase A and also lysophosphatidylcholine or arachidonic acid could be antagonized or reversed by bovine serum albumin. It was concluded that the first stimulatory phase of the venom phospholipase A action might be due to arachidonate liberation from platelet membrane. The second phase of inhibition of platelet aggregation and the release of ATP might be due to the inhibitory action of the split products produced by this venom phospholipase A.

Mode of neuromuscular blocking action of ceruleotoxin

Ceruleotoxin is an acidic toxin protein from a Bungarus venom which has recently been clarified as B. fasciatus venom. The toxin at a low concentration (10 −6g/ml) abolished the twitch response of the indirectly stimulated biventer cervicis muscle of the chick, without affecting the response to acetylcholine or carbamylcholine. At a higher concentration (10 -5g/ml), the toxin, in addition to inhibition of the twitch response, caused contracture in the chick biventer cervicis muscle and reduced the response to acetylcholine or carbamylcholine in both the innervated chick cervical and denervated rat diaphragm muscles. Electrophysiological studies on the rat phrenic nerve - diaphragm preparation showed that ceruleotoxin at a low concentration caused an initial inhibition followed by recovery of the quantal content of endplate potentials and then a second phase of inhibition leading to complete failure. At a higher concentration, the toxin gradually reduced the resting membrane potential of the diaphragm muscle to a level lower than 50 mV within 2 hr. In addition, enzyme assay showed that the toxin possessed phospholipase A activity comparable to that of other basic phospholipases A 2 of snake venom origin. It is concluded that ceruleotoxin is a phospholipase A 2 with presynaptic and myotropic actions on vertebrate nerve - muscle system similar to those of notexin from Notechis scutatus scutatus venom.

In vivo regulation of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase: immunotitration of the enzyme after short-term mevalonate or cholesterol feeding.

In recent studies using either a single dose of mevalonolactone administered by intragastric tube or a single meal containing 2% cholesterol, it was demonstrated that rat liver hydroxymethylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] (HMG-CoA reductase) the major regulatory enzyme in cholesterol biosynthesis, is subject to two phases of inhibition. The first phase of inhibition is explained by in vivo phosphorylation of the enzyme; however, the nature of the second phase of inhibition remained obscure. The present study tested two possible explanations for this second phase of inhibition--increased enzyme turnover leading to a decreased concentration of HMG-CoA reductase molecules, and further inactivation of existing enzyme molecules. The results with the technique of immunotitration of HMG-CoA reductase show that, in short-term studies conducted up to 2 hr after the administration of a single dose of mevalonolactone or up to 6 hr after a single meal of rat chow containing 2% cholesterol, the in vivo regulation of rat liver HMG-CoA reductase during the first half of the dark period does not occur by increased enzyme turnover but, instead, existing enzyme is further inactivated.

Site of action of caudoxin, a neurotoxic phospholipase A 2 from the horned puff adder ( Bitis caudalis) venom

Caudoxin, a single-chain phospholipase A 2 isolated from the venom of Bitis caudalis is a toxic polypeptide with a formula weight of 13,332 dalton. The ld 50 in mice (i.p.) was 0.18 (0.15–0.22) mg/kg. In the chick biventer cervicis muscle preparation the toxin (1–10 μg per ml) caused complete neuromuscular blockade without affecting the response of the muscle to acetylcholine. In the mouse phrenic nerve-diaphragm preparation, the toxin abolished the indirectly elicited contraction without inhibiting that evoked directly. When this preparation was bathed in a low calcium (0.6 mM) medium, the toxin induced a triphasic change in the indirectly evoked contractions: an immediate initial inhibition followed by augmentation and then a second phase of inhibition leading to irreversible neuromuscular blockade. Electrophysiological studies in the same preparation showed a similar triphasic change in the quantal content of endplate potentials. The frequency of miniature endplate potentials first increased and then decreased, while the resting membrane potential was not significantly decreased by the toxin. Histological study showed that the toxin caused local myonecrosis only at a higher dose (2 mg/kg mouse). It is concluded that caudoxin produced a neuromuscular block by acting selectively on a presynaptic site. However, the site of binding appears to be different from that of β-bungarotoxin since combination of the toxin with β-bungarotoxin caused potentiation of its neuromuscular blocking action rather than addition.

The in vivo regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase. Phosphorylation of the enzyme as an early regulatory response following cholesterol feeding.

Although substantial evidence supports the conclusion that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the major regulatory enzyme in cholesterol biosynthesis, the molecular events involved in the in vivo regulation of this enzyme have remained obscure. In order to study this problem, rats received a single meal consisting of either rat chow or rat chow containing 2% cholesterol. The rats were killed 60 or 120 min after the beginning of feeding, and liver microsomes were prepared by ultracentrifugation. Two phases of inhibition of microsomal HMG-CoA reductase were observed. The first phase of inhibition, observed 60 min after the beginning of cholesterol feeding, was completely reversed by preincubation of the microsomes with purified phosphoprotein phosphatase. The second phase of inhibition, observed 120 min after the beginning of cholesterol feeding, was not reversed by phosphoprotein phosphatase. These results are consistent with the conclusion that phosphorylation of HMG-CoA reductase is the first step in a series of in vivo regulatory events which produce inactivation and ultimately degradation of the enzyme.

In vivo regulation of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase: enzyme phosphorylation as an early regulatory response after intragastric administration of mevalonolactone.

Although substantial evidence supports the conclusion that 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34] is the major regulatory enzyme in cholesterol biosynthesis, the molecular events involved in the in vivo regulation of this enzyme have remained obscure. To study this problem, rats were given a single 100-mg dose of mevalonolactone by intragastric tube. The rats were sacrificed 20 or 60 min later, and liver microsomes were prepared by ultracentrifugation. Two phases of inhibition of microsomal HMG-CoA reductase were observed. The first phase of inhibition, observed 20 min after mevalonolactone administration, was completely reversed by preincubation of the microsomes with purified phosphoprotein phosphatase. The second phase of inhibition, observed 60 min after mevalonolactone administration, was not reversed by phosphoprotein phosphatase. The reactivation of liver microsomal HMG-CoA reductase by phosphoprotein phosphatase was blocked by potassium fluoride or by phosphoprotein phosphatase inhibitor. Results obtained by immunotitration also showed that microsomal HMG-CoA reductase obtained from animals killed 20 min after mevalonolactone administration was significantly activated by phosphoprotein phosphatase treatment of the microsomes. These findings demonstrate that phosphorylation of rat liver HMG-CoA reductase is an early in vivo regulatory response after intragastric administration of mevalonolactone.

Studies on the mechanisms of the rapid modulation of 3-hydroxy-3-methylglutaryl coenzyme a reductase in intact liver by mevalonolactone and 25-hydroxycholesterol

A rapid, biphasic inhibition of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase (mevalonate:NADP + oxidoreductase (CoA-acylating), EC 1.1.1.34) was induced by intragastric administration of R,S-mevalonolactone. The initial phase had a t 1 2 of 5.3 min. 30 min after drug administration the inhibition could be reversed in vitro by cytosol or a partially purified cytosolic activator. The reactivation was prevented by 50mM NaF. Thus the initial inhibition appeared to be the result of reversible inactivation possibly by phosphorylation of the enzyme. Consistent with this was the finding that the net reductase activator (phosphatase) activity present in cytosol was decreased 64% in these animals. The rapid reversible inhibition could not be reproduced in vitro by incubating microsomes or postmitochondrial supernatants with mevalonate suggesting the intact cell was necessary for expression of the effect. The second phase of inhibition due to mevalonate administration had a t 1 2 of 1.3 h and was not reversible. It was attributed to inhibition of synthesis of reductase probably as the result of sterol accumulation in the cell. Perfusion of 25-hydroxycholesterol through livers isolated from animals at the circadian peak of cholesterol biosynthesis resulted in a rapid, 75–80% inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase. This inhibition was not reversed by incubation with cytosol or partially purified activator. Further, there was no apparent change in net activator levels in cytosol from the livers perfused with 25-hydroxycholesterol. This suggests the effect of this sterol on reductase does not involve reversible phosphorylation-dephosphorylation. On the basis of this study it is postulated that there are at least two mechanisms by which 3-hydroxy-3-methylglutaryl coenzyme A reductase activity can be rapidly suppressed in the intact liver. One is reversible and appears to be the result of alteration in the reductase kinase-phosphatase system. The second is irreversible and may be due to acceleration of the normal degradation system.

Effects of dithiothreitol on dopa oxidase activity from potato tubers

A filtrate, prepared from potato tuber by grinding in an isotonic medium, has been separated into a particulate and a ‘soluble’ fraction by ultracentrifugation. Following dialysis and lyophilization, both fractions catalysed the oxidation of l-DOPA, with approximately 30% of the l-DOPA: oxygen-oxidoreductase (EC 1.14.18.1; DOPA oxidase) activity being associated with the particulate fraction. When dithiothreitol (DTT, 10 −2M was included in the grinding medium, much lower yields of DOPA oxidase were obtained and 80% appeared to be associated with the particulate fraction. DTT proved to be a powerful inhibitor of DOPA oxidase. With concentrations of DTT causing only partial inhibition, the kinetics of the inhibited rate of dopachrome formation from l-DOPA were complex. When oxygen consumption was measured inhibition was not transient. The degree of inhibition was inversely related to the DOPA oxidase activity, indicating interaction of a product of this activity with DTT. Direct determination of -SH groups in DTT using 5,5′-dithiobis(2-nitrobenzoic acid (DTNB) showed that they were all oxidised during the initial phase of inhibition of dopachrome formation. It is concluded that the first phase of inhibition involves oxidation of DTT by an intermediate between l-DOPA and dopachrome. The second phase of inhibition also appeared to require -SH groups initially, since trans-4,5-dihydroxy-1,2-dithiane (oxidized DTT) caused very little inhibition at all.

Effect of stimulation of prefrontal cortex and amygdala on diencephalic neurons

Extracellular unit recordings were obtained from the nucleus medialis dorsalis (MD) and adjacent thalamic nuclei and from the hypothalamus of anesthetized cats. Electrical stimulation of the prefrontal cortex produces suppression of unit activity and rebound excitation in MD. In addition antidromic and short latency excitation was found which was followed by prolonged suppression of firing. This suppression was also followed by a strong postinihibitory activation of unit firing which was, in some cases, again followed by a second phase of inhibition. Amygdala stimulation produced similar results except that antidromic responses were found only in the intralaminar nuclei, and short-latency transynaptic excitation occurred somewhat later. The results are in consonance with the hypothesis that the same interneuronal mechanism is engaged by both afferent sources to MD. In the hypothalamus, fewer units responded to cortical and amygdalar stimulation and alternating sequences of excitation-inhibition-excitation were not found.

Time-dependent Inhibition of Aspartate Aminotransferase Isozymes by dl-Glyceraldehyde 3-Phosphate

Abstract dl-Glyceraldehyde 3-phosphate in low concentrations has been found to be a time-dependent inhibitor of aspartate aminotransferase (EC 2.6.1.1) isozymes. The cationic isozyme is more susceptible than the anionic isozyme to this inhibition. The l-isomer of glyceraldehyde 3-phosphate was as effective as the d-isomer for each isozyme. Study of various glycolytic intermediates indicated that the conjoint presence of the free aldehyde group and the phosphoryl residue of glyceraldehyde 3-phosphate was necessary for inhibition. The extent of inhibition was dependent on the duration of preliminary incubation, the concentration of glyceraldehyde-3-P, and the pH of the preliminary incubation mixture, but was independent of the concentration of enzyme. Half-maximum inhibition of each isozyme was obtained between pH 6.5 and 6.7. Maximum inhibition of the anionic isozyme was obtained at pH values of 8.4 and above, while the cationic isozyme was optimally inhibited at pH 7.4. The presence of α-ketoglutarate in the preliminary incubation mixture decreased inhibition while aspartate accentuated it. After 30 min of preliminary incubation with glyceraldehyde-3-P, dialysis of the anionic isozyme against buffer, aspartate, α-ketoglutarate, or pyridoxal 5-phosphate resulted in a substantial release of inhibition. In the case of the cationic isozyme, substantial release of inhibition was obtained only by dialysis against α-ketoglutarate. The addition of α-ketoglutarate to either the anionic or cationic isozyme-inhibitor preliminary incubation mixtures released inhibition, but only in the case of the anionic isozyme was the extent of release dependent on the duration of preliminary incubation. Prolonged incubation of this isozyme with glyceraldehyde-3-P resulted in the appearance of a second phase of inhibition. The mechanism of the inhibition of the isozymes by glyceraldehyde-3-P is discussed.