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Biomedical subjects

R Morgenstern

Publications and source records attributed to R Morgenstern.

At least 73 records · Page 4Linked to original sources
PubMed1996

Foetal ventral mesencephalic cell suspension grafts to the 6-hydroxydopamine-lesioned rat reduce the rate of dopamine uptake in the contralateral striatum.

The present study employed fast cyclic voltammetry, at carbon-fibre microelectrodes, to monitor and compare the rate of dopamine uptake in the rat striatum contralateral to (a) the 6-hydroxydopamine (6-OHDA)-lesioned/grafted striatum and (b) the 6-OHDA-lesioned/ sham grafted striatum. Cell suspensions of foetal rat ventral mesencephalic tissue were grafted into the dopamine-depleted striatum of unilaterally 6-OHDA-lesioned rats. Six weeks after grafting, animals with functional, mature grafts were monitored for dopamine elimination in the contralateral striatum following electrical stimulation of the median forebrain bundle, before and after treatment with the dopamine uptake inhibitor GBR 12909. Compared to animals with sham grafts, amphetamine-amplified rotational behaviour was significantly reduced in animals with grafts of foetal ventral mesencephalic tissue. Fast cyclic voltammetric measurements followed by evaluation with the aid of a kinetic model revealed that in grafted animals, the rate of dopamine uptake via the high affinity uptake mechanism, following treatment with GBR 12909, was significantly reduced when compared to sham grafted animals.

Amphetamine↗
PubMed1996

Foetal nigral cell suspension grafts influence dopamine release in the non-grafted side in the 6-hydroxydopamine rat model of Parkinson's disease: in vivo voltammetric data.

The present study employed differential-pulse voltammetry to assess the influence of foetal ventral mesencephalic grafts on dopamine overflow in the contralateral caudate putamen of the 6-hydroxydopamine rat model of Parkinson's disease. The experimental design involved measurements of dopamine overflow in the grafted and contralateral striatum. Control measurements of dopamine overflow were performed in 6-hydroxydopamine-lesioned rats only and the caudate putamen of normal control rats. Cell suspensions of foetal rat ventral mesencephalic tissue were grafted into the dopamine-depleted caudate putamen of unilaterally 6-hydroxydopamine-lesioned rats. At 6 weeks, animals with functional, mature grafts (as assessed by amphetamine-amplified behavioural asymmetry), were pretreated with pargyline (75 mg/kg i.p.), and both striatal sides were monitored for dopamine overflow for 90 min following amphetamine sulphate administration (5 mg/kg i.p.). The time course of dopamine overflow inside the graft was similar to that in the contralateral caudate putamen of the same animal, the normal control animal and the contralateral caudate putamen of 6-hydroxydopamine-lesioned animals. However, in grafted animals the mean dopamine overflow detected in the contralateral caudate putamen was approximately 34% lower than the concentration of dopamine detected in the contralateral caudate putamen of 6-hydroxydopamine-lesioned control animals and approximately 39% lower than the concentration of dopamine detected in the caudate putamen of the normal control animal. There was no statistical difference in the concentration of amphetamine-induced dopamine overflow between the caudate putamen contralateral to the 6-hydroxydopamine lesion and the caudate putamen of the normal control animal. These data suggest that intrastriatal foetal ventral mesencephalic suspension grafts reduce amphetamine-induced dopamine release in the contralateral non-grafted caudate putamen.

Amphetamine↗
PubMed1995

Heterologous expression of rat liver microsomal glutathione transferase in simian COS cells and Escherichia coli.

The cDNA coding for rat liver microsomal glutathione transferase was subcloned into the mammalian expression vector pCMV-5 and the construct was transfected into, and transiently expressed in, simian COS cells. This resulted in high expression (0.7% of the microsomal protein). The activity towards 1-chloro-2,4-dinitrobenzene in microsomes was 15-30 nmol/min per mg, which increased upon N-ethylmaleimide treatment to 60-200 nmol/min per mg. Control and antisense-vector-treated cells displayed very low activity (3-6 nmol/min per mg). A DNA fragment coding for rat microsomal glutathione transferase was generated by PCR, cloned into the bacterial expression vector pSP19T7LT and transformed into Escherichia coli strain BL21 (DE3) (which contained the plasmid pLys SL). Isopropyl beta-D-thiogalactopyranoside (IPTG; 1 mM) induced the expression of significant amounts of enzymically active protein (4 mg/l of culture as measured by Western blots). The recombinant protein was purified and characterized and found to be indistinguishable from the rat liver enzyme with regard to enzymic activity, molecular mass and N-terminal amino acid sequence. Human liver cDNA was used to obtain the coding region of human microsomal glutathione transferase by PCR. This PCR product was cloned into pSP19T7LT, which, upon induction with IPTG, yielded significant amounts (9 mg/l of culture) of active enzyme in BL21 (DE3) cells. Thus, for the first time, it is now possible to express both human and rat microsomal glutathione transferase in an enzymically active form in Escherichia coli.

Animals↗
PubMed1995

Fetal mesencephalic grafts decrease the rate of dopamine uptake in the non-lesioned striatum of unilaterally 6-OHDA lesioned rats: an in vivo voltammetric study.

In the present study the influence of intraventricular fetal mesencephalic grafts on the elimination rate of extracellular dopamine (DA) in the non-lesioned striatum of previously unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats was investigated. The elimination of DA was measured after electrical stimulation of the medial forebrain bundle in vivo before and after treatment with the high affinity uptake inhibitor GBR 12909 (20 mg/kg i.p.) using fast cyclic voltammetry (FCV). Rotational behavior induced by amphetamine (AMPH, 2 mg/kg i.p.) and tyrosine hydroxylase (TH) immunohistochemistry were used to prove the functional recovery and the ingrowth of the graft, respectively. After grafting, the number of rotations was decreased and TH-positive cells and fibers were found in the grafted striatum. Voltammetric measurements with the aid of a kinetic model revealed a smaller rate constant for the in vivo elimination of extracellular DA in the non-lesioned striatum of grafted rats compared to that of non-grafted controls. This effect was abolished after treatment with GBR 12909. Our results will be discussed in relation to the method used and according to recent investigations of the specific [3H]DA uptake into striatal synaptosomes in vitro. Based on these data we conclude that grafts, placed to the lesioned striatum, reduce the DA uptake rate in the non-lesioned striatum due to the reduction of the number of functional DA transporters.

Analysis of Variance↗
PubMed1995

Grafts modulate dopamine transporters of the non-lesioned striatum.

The effect of intraventricular fetal mesencephalic grafts placed in the previously 6-hydroxydopamine (6-OHDA) lesioned striatum on the kinetics of [3H]dopamine (DA) uptake into striatal synaptosomes prepared from the non-lesioned (contralateral) striatum was studied in rats. Using WIN 35,065 as specific [3H]DA uptake inhibitor, the equilibrium dissociation constant (Km) of [3H]DA uptake into synaptosomes of the non-lesioned (contralateral) striatum did not differ between grafted and nongrafted controls 7 months after grafting. However, the maximal rate of specific [3H]DA uptake (Vmax) was markedly decreased in the non-lesioned striatum of grafted animals. This result indicates that fetal mesencephalic grafts reduce the [3H]DNA uptake in the non-lesioned (contralateral) striatum by reducing the number of functional DA transporters.

Analysis of Variance↗
PubMed1995

The projection structure of microsomal glutathione transferase.

Through the use of electron crystallography, it has been possible to obtain high resolution structural information regarding a mammalian protein that spans the lipid bilayer. Two-dimensional crystals of the detoxification enzyme microsomal glutathione transferase were induced by slow detergent removal from a mixture containing low amounts of phospholipid. Images of specimens stabilized in tannin were collected using electron cryomicroscopy. The projection structure at 4 A shows tightly packed trimers of the protein. Each of them contains an inner core of six parallel alpha-helices delineating a central low density region. The helical bundle is partly surrounded by elongated domains.

Animals↗
PubMed1995

Ontogenesis of rat liver microsomal glutathione transferase.

The ontogenesis of rat liver microsomal glutathione transferase was investigated by activity measurements and immunochemical methods. The activity rises from a very low level (3% of adults) at day 8 pre-partum to adult levels at days 50-150. Increases are associated with the neonatal and late-suckling clusters. Interestingly the capacity to become activated by N-ethylmaleimide is much lower in females early and late in life (days 35-100 and 300-550). After the initial increases (from 10% of adult levels at day 8 pre-partum), protein levels determined immunochemically remain constant throughout life with no apparent sex differences. The developmental pattern of microsomal glutathione transferase resembles those of other drug-metabolizing enzymes indicating that the function of the enzyme is required in adult life.

Aging↗
PubMed1995

Microsomal glutathione transferase: lipid-derived substrates and lipid dependence.

Rat liver microsomal glutathione transferase was found to display glutathione peroxidase activity toward a variety of oxidized lipids. 1-Linoleoyl-2-palmitoyl phosphatidylcholine hydroperoxide, 2-linoleoyl-1-palmitoyl phosphatidylcholine hydroperoxide, 2-linoleoyl-1-palmitoyl phosphatidylethanolamine hydroperoxide, and cholesteryl linoleate hydroperoxide all served as substrates (0.02, 0.04, 0.02, and 0.02 mumol/min mg, respectively). The phospholipid hydroperoxide glutathione peroxidase activity of the enzyme was found not to require detergent and increased when liposomes containing peroxidized phospholipid were fused with liposomes containing microsomal glutathione transferase. Methyl linoleate ozonide serves as a very efficient substrate for the microsomal glutathione transferase. The unactivated and N-ethylmaleimide-activated enzyme displayed specific activities of 0.74 and 5.9 mumol/min mg, respectively. Upon examination of a series of 4-hydroxyalk-2-enals it was found that the catalytic efficiency of the enzyme increases from the 4-hydroxyhept-2-enal up to the 4-hydroxytetradec-2-enal. The specific activities with the various 4-hydroxyalk-2-enals tested varied between 0.28 and 0.95 mumol/min mg. The phospholipid dependence of the microsomal glutathione transferase was examined in proteoliposomes formed by cholate dialysis. Phosphatidyl choline, phosphatidyl serine, phosphatidyl ethanolamine, and rat liver microsomal phospholipids could all be used successfully to reconstitute the enzyme. In conclusion, microsomal glutathione transferase can detoxify a number of lipid peroxidation products as well as a fatty acid ozonide. The results imply a protective role for the enzyme under conditions of oxidative stress.

Animals↗
PubMed1995

Graft-induced reduction in [3H]dopamine uptake into the non-lesioned striatum.

The effect of intraventricular fetal mesencephalic grafts placed in the 6-hydroxydopamine (6-OHDA) lesioned striatum on [3H]dopamine (DA) uptake into synaptosomes was studied. Apomorphine (APO)- and amphetamine (AMPH)-induced rotational behaviour was continuously reduced by the grafts over 7 months. After this time, a non-significant increase in [3H]DA uptake into synaptosomes of the lesioned striatum and an approximately 40% decrease in [3H]DA uptake into synaptosomes of the non-lesioned striatum were found. Grafts placed in the non-lesioned striatum did not significantly change both rotational behaviour or [3H]DA uptake. The results show that unilateral fetal mesencephalic grafts producing behavioural improvement in the unilateral 6-OHDA lesioned rat induce changes in dopaminergic structures in the non-lesioned striatum.

Amphetamine↗
PubMed1995

Kinetic studies on rat liver microsomal glutathione transferase: consequences of activation.

Rat liver microsomal glutathione transferase is activated by sulfhydryl reagents and proteolysis. This property varies, however, depending on the combination, concentration and reactivity of the substrates. Thus, a multi-dimensional diagram can be envisioned in which the parameters affecting enzyme activity and activation are visualized. In principle activation could stem from an alteration in enzyme mechanism, transition-state complementarity, product release rate or pH-rate behaviour. These studies appear to rule out these possibilities and an alternate hypothesis is suggested based on the following experiments: (i) alternate substrate diagnosis of the kinetic mechanism of microsomal glutathione transferase indicates a random sequential mechanism. Non-activated and activated enzyme follow the same mechanism by these criteria. (ii) The microsomal glutathione transferase stabilizes a Meisenheimer complex between 1,3,5-trinitrobenzene and glutathione. The formation constants were similar for the unactivated and activated enzyme ((15 +/- 1).10(3) and (14 +/- 1).10(3) M-1, respectively, at pH 8). Inasmuch as the Meisenheimer complex resembles the transition state there is no evidence for an increased stabilization upon activation. (iii) The catalytic rate constant kcat does not vary with the viscosity in the assay medium. Thus, product release is not rate limiting for the unactivated and activated microsomal glutathione transferase (with saturating 1-chloro-2,4-dinitrobenzene and varying GSH). (iv) The pH dependence of the Kf-values for Meisenheimer complex formation exhibited pKa values close to 6 for both the activated and unactivated microsomal glutathione transferase. The pH profile of kcat (with saturating 1-chloro-2,4-dinitrobenzene and variable GSH concentrations) showed apparent pKa values of 5.7 +/- 0.5 and 6.3 +/- 0.4 for the unactivated and activated enzyme, respectively, indicative of a very similar requirement for deprotonation of the enzyme-GSH-1-chloro-2,4-dinitrobenzene complex. (v) Examination of the kinetic parameters (obtained with GSH as the variable substrate against increasingly reactive electrophilic substrates) in Hammett plots shows that the activation mechanism entails a more efficient utilization of GSH. It is suggested that a higher rate of formation of the glutathione thiolate anion occurs in the activated enzyme.

Amino Acid Sequence↗
PubMed1995

Protein-protein interaction affinity chromatography of leukotriene C4 synthase.

A novel affinity chromatography purification for human leukotriene C4 synthase is described. It is based on a specific interaction between leukotriene C4 synthase and microsomal glutathione S-transferase which occurs in the presence of magnesium ion. Microsomal glutathione S-transferase was immobilized on NHS-activated Sepharose 4B and used as an affinity matrix. Microsomes from 12-O-tetradecanoyl phorbol 13-acetate-treated human erythroleukemia cells were solubilized with taurocholic acid and applied on the affinity matrix at 0.1 M Mg2+ concentration. After washing with a buffer containing Mg2+, the enzyme was eluted with a glutathione-containing buffer lacking Mg2+. This facile one-step procedure gave a 166-fold purification of leukotriene C4 synthase with a yield of 44%. Analyses of proteins specifically adsorbed to the affinity matrix revealed components with apparent molecular weights of 18, 37, 48, and 60 kDa.

Animals↗
PubMed1994

3-O-methyl-DOPA is not involved in the development of behavioral supersensitivity after repeated L-dopa administration in 6-OHDA lesioned rats.

The underlying cause of long-term complications of L-DOPA therapy in Parkinson's disease is largely unknown. Recently, centrally and peripherally acting catechol-O-methyltransferase (COMT) inhibitors became available. These drugs are capable of inhibiting the generation of 3-O-methyl-DOPA (3-OMD), a major metabolite of L-dopa developing considerable plasma levels during L-dopa therapy. The use of these drugs offers the opportunity to study the involvement of 3-OMD in the development of behavioral supersensitivity following repeated doses of L-dopa over 11 days in rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopaminergic system. Repeated daily administration of L-dopa/Carbidopa produced continuous increase of contralateral rotations to both L-dopa/Carbidopa and to challenge doses of apomorphine. This increase was not influenced by peripherally and peripherally plus centrally acting COMT inhibitors, OR-462 and OR-486, respectively, administered simultaneously with L-dopa/Carbidopa. Both COMT inhibitors suppressed the L-dopa induced increase of 3-OMD plasma levels, OR-486 being more effective than OR-462. This indicates that 3-OMD is not involved in the development of behavioral supersensitivity following repeated L-dopa treatment in rats with unilateral 6-OHDA lesion of the nigrostriatal system.

Animals↗
PubMed1994

Fetal mesencephalic grafts influence the dopamine release in the non-lesioned striatum of 6-OHDA-lesioned rats: a behavioral and in vivo voltammetric study.

This study was performed to investigate the effect of intraventricular fetal mesencephalic grafts on the non-lesioned nigro-striatal system of rats with unilateral lesions of dopaminergic neurones in the medial forebrain bundle and to analyze the involvement of the non-lesioned side in functional recovery due to grafts in this model. Grafts placed to the lesioned striatum produced a continuous decrease of apomorphine (APO, 0.25 mg/kg intraperitoneally (i.p.)) and amphetamine (AMPH, 5 mg/kg, i.p.) induced rotations over 7 months after grafting. Using differential pulse voltammetry (DPV) in anaesthetized and pargyline (75 mg/kg, i.p.) pretreated rats we found that 8 months after grafting in the lesioned striatum AMPH (5 mg/kg, i.p.) produced a higher increase of the dopamine (DA) signal (i.e., it became measurable), whereas in the non-lesioned striatum the same treatment produced a smaller increase of the DA signal, both compared to that in sham-grafted controls. After grafting onto the non-lesioned striatum, only a slight decrease of APO-induced rotations was observed, whereas AMPH-induced rotations were increased. In these animals AMPH did not produce a measurable DA signal in the lesioned striatum. The DA signal in the non-lesioned striatum was slightly higher than that of non-grafted controls. These results show clearly that unilateral fetal mesencephalic grafts producing behavioural recovery in the unilateral 6-OHDA model of the rat produce changes at dopaminergic mechanisms in the non-lesioned striatum.

Amphetamine↗