Biomedical subjects

R Morgenstern

Publications and source records attributed to R Morgenstern.

At least 37 records · Page 2Linked to original sources

PubMed2000

The influence of pallidal deep brain stimulation on striatal dopaminergic metabolism in the rat.

Deep brain stimulation of the globus pallidus internus has been recently shown to alleviate parkinsonian symptoms and levodopa-induced dyskinesias. However, its exact mechanisms of action are unclear. Pallidal neurones are connected via various pathways to the dopaminergic nigrostriatal system. In the present study we investigated the hypothesis that deep brain stimulation of the entopeduncular nucleus (corresponds to the human internal pallidum) affects striatal dopaminergic metabolism in naive and 6-hydroxydopamine (6-OHDA) lesioned rats using microdialysis. Our results show that stimulation of the entopeduncular nucleus does not significantly affect striatal dopamine metabolism (of dopamine, 3, 4-dihdroxyphenylacetic acid and homovanillic acid) in naive and 6-OHDA-lesioned animals. They contrast with our previous observations that deep brain stimulation of the subthalamic nucleus increases striatal dopamine metabolism suggesting differential effects of these nuclei on striatal dopamine metabolism.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2000

Reactivity of cysteine-49 and its influence on the activation of microsomal glutathione transferase 1: evidence for subunit interaction.

Microsomal glutathione transferase 1 is a homotrimeric detoxication enzyme protecting against electrophiles. The enzyme can also react with electrophiles, and when modification occurs at a unique Cys49 the reaction often results in activation. Here we describe the characterization of the chemical properties of this sulfhydryl (kinetic pK(a) was 8.8 +/- 0.3 and 9.0 +/- 0.1 with two different reagents) and we conclude that the protein environment does not lower the pK(a). Upon a direct comparison of the reactivity of Cys49 and low molecular weight thiols [L-Cys and glutathione (GSH)], the protein sulfhydryl displayed a 10-fold lower reactivity. The reactivity was correlated to reagent concentration in a linear fashion with a polar reagent, whereas the reactivity toward a hydrophobic reagent displayed saturation behavior (at low concentrations). This finding indicates that Cys49 is situated in a hydrophobic binding pocket. In a series of related quinones, activation occurs with the more reactive and less sterically hindered compounds. Thus, activation can be used to detect reactive intermediates during the metabolism of foreign compounds but certain intermediates can (and will) escape undetected. The reactivities of the three cysteines in the homotrimer were shown not to differ dramatically as the reaction of the protein with 4, 4'-dithiodipyridine could be fitted to a single exponential. On the basis of this result, a probabilistic expression could be used to relate the overall degree of modification to fractional activation. When N-ethylmaleimide activation (determined by the 1-chloro-2, 4-dinitrobenzene assay) was plotted against modification (determined with 4,4'-dithiodipyridine), a nonlinear relation was obtained, clearly showing that subunits do not function independently. The contribution to activation by single-, double-, and triple-modified trimers, were 0 +/- 0.06, 0.74 +/- 0.09, and 0.97 +/- 0.06, respectively. The double-modified enzyme appears partly activated, but this conclusion is more uncertain due to the possibility of independent modification of the purified enzyme upon storage. It is, however, clear that the single-modified enzyme is not activated whereas the triple-modified enzyme is fully activated. These observations together with the fact that MGST1 homotrimers bind only one substrate molecule (GSH) strongly support the view that subunits must interact in a functional manner.

Animals↗

PubMed2000

The three-dimensional map of microsomal glutathione transferase 1 at 6 A resolution.

Microsomal glutathione transferase 1 (MGST1) is representative of a superfamily of membrane proteins where different members display distinct or overlapping physiological functions, including detoxication of reactive electrophiles (glutathione transferase), reduction of lipid hydroperoxides (glutathione peroxidase), and production of leukotrienes and prostaglandin E. It follows that members of this superfamily constitute important drug targets regarding asthma, inflammation and the febrile response. Here we propose that this superfamily consists of a new class of membrane proteins built on a common left-handed four-helix bundle motif within the membrane, as determined by electron crystallography of MGST1 at 6 A resolution. Based on the 3D map and biochemical data we discuss a model for the membrane topology. The 3D structure differs significantly from that of soluble glutathione transferases, which display overlapping substrate specificity with MGST1.

Amino Acid Motifs↗

PubMed2000

Comparison by microdialysis of striatal L-DOPA after its systemic administration in rats with probes implanted acutely or through a guide cannula.

Different methods of microdialysis probe implantation are utilized according to the purpose and needs of each particular study. However, very few experiments have systematically examined whether these different techniques have an impact on the obtained data. In the present study we examined the influence of two different microdialysis methods - acute probe implantation vs. insertion into a preimplanted guide cannula - on the striatal extracellular availability of systemically administered L-DOPA. Furthermore, we monitored the effects of L-DOPA administration on dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC). In rats that received a guide cannula 4 days prior to probe insertion and microdialysis, extracellular L-DOPA concentrations increased to concentrations that were about nine times higher than in rats with acute implantation of a microdialysis probe. Extracellular DOPAC concentrations were also higher in the chronic preparations but dopamine concentrations showed no differences between groups. Our results suggest that the observed differences may be due to inflammatory disruption of the BBB following chronic implantation of a guide cannula.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2000

Z oscillations in ion-induced fullerene fragmentation

Multiply charged ion-induced fragmentation and ionization of C60 is governed by electronic and nuclear stopping of the projectile as well as charge exchange. We have studied the collision dynamics as a function of the projectile atomic number Z ( 2</=Z</=18) for constant projectile velocity v at charge states q = 2,3. Strong oscillations of electronic-stopping-related quantities such as ionization and fragmentation are observed. From the properties of the oscillations, we obtain information on the effective electron density of the fullerene.

Journal Article↗

PubMed2000

Structural organization of the microsomal glutathione S-transferase gene (MGST1) on chromosome 12p13.1-13.2. Identification of the correct promoter region and demonstration of transcriptional regulation in response to oxidative stress.

The structure and regulation of the microsomal glutathione S-transferase gene (MGST1) are considerably more complex than originally perceived to be. The MGST1 gene has two alternative first exons and is located in the 12p13.1-13.2 region. Two other potential first exons were determined to be nonfunctional. The region between the functional first exons cannot direct transcription. Thus, one common promoter element directing transcription exists, and RNA splicing occurs such that only one of the first exons (containing only untranslated mRNA) is incorporated into each mRNA species with common downstream exons. MGST1 expression and regulation are therefore similar to those of other hepatic xenobiotic handling enzymes, which also produce mRNA species differing only in the 5'-untranslated regions to yield identical proteins. MGST1 was previously considered a "housekeeping" gene, as non-oxidant inducers had little effect on activity. However, the promoter region immediately upstream of the dominant first exon transcriptionally responds to oxidative stress. In this respect, MGST1 is similar to glutathione peroxidases that also transcriptionally respond to oxidative stress. The discovery that MGST1 utilizes alternative first exon splicing eliminates a problem with the first description of MGST1 cDNA in that it appeared that MGST1 expression was in violation of the ribosomal scanning model. The identification that the first exon originally noted is in fact a minor alternative first exon far downstream of the primary first exon eliminates this conundrum.

5' Untranslated Regions↗

PubMed2000

Human glutathione dependent prostaglandin E synthase: gene structure and regulation.

A P1 clone containing the gene for human glutathione dependent PGE synthase (PGES) was isolated and characterized. The gene is divided into three exons, spans 14.8 kb and was localized to chromosome 9q34. 3. In A549 cells, the protein and activity levels of PGES were increased by interleukin-1beta. This increase was prevented by phenobarbital. Reporter constructs containing the 5'-flanking region of exon 1, which exhibited strong promoter activity, responded accordingly, except that interleukin-1beta induced a transient increase followed by a decrease. As cyclooxygenase 2 expression has been reported to respond in a similar fashion, a transcriptional regulatory basis for the observed co-regulation with PGES is implied. The strong down-regulation by phenobarbital raises important issues concerning its mechanisms of action.

Base Sequence↗

PubMed2000

High frequency stimulation of the subthalamic nucleus influences striatal dopaminergic metabolism in the naive rat.

High frequency stimulation (HFS) of the subthalamic nucleus (STN) can partially alleviate motor symptoms in patients with Parkinson's disease (PD). However, the mechanism of action of HFS is incompletely understood. We investigated the effect of HFS (130 Hz) and low frequency stimulation (LFS, 20 Hz) of the STN on striatal dopaminergic transmission and metabolism using in vivo microdialysis in anaesthetized and freely moving rats. While LFS had no effect, HFS of the STN produced a delayed, stable and intensity-dependent increase of extracellular dopamine metabolites. Striatal extracellular levels of dopamine and 5-HIAA were not influenced by HFS or LFS in the present experimental paradigm. We conclude that HFS of the STN influences striatal dopaminergic metabolism in naive, nonlesioned rats.

3,4-Dihydroxyphenylacetic Acid↗

PubMed2000

Phenotype determination of a common Pro-Leu polymorphism in human glutathione peroxidase 1.

Oxidative stress has been implicated in human illness such as cardiovascular and neurodegenerative disease. The genetic mechanisms involved are only poorly understood. Here we describe the determination of the allelic frequency and phenotype of a common polymorphism in Se-dependent glutathione peroxidase 1 (GPX1) in Finnish/Swedish populations. A proline/leucine variant occurs at position 197 close to the C-terminus of the protein. The more common allele encoding the Pro variant is present at 59% in a Finnish/Swedish population (n = 66) and at 73% in a Swedish population (n = 315). The genotypes encoding Pro/Pro, Pro/Leu, and Leu/Leu are distributed according to the Hardy-Weinberg relationship. The Swedish population consisted of 101 stroke cases and 214 controls. No significant association between allele frequency and risk to suffer from stroke was evident. Erythrocyte GPX activity was determined in the Finnish/Swedish population and no significant differences were obtained between the genotypes. It can be concluded that the Pro/Leu genetic variation does not appear to compromise the defense against oxidative stress in red blood cells nor to be associated with stroke.

DNA↗

PubMed2000

Direct analysis of single-nucleotide polymorphism on double-stranded DNA by pyrosequencing.

Pyrosequencing, a new method for DNA sequencing, is gaining widespread use for many different types of DNA analysis. The method takes advantage of four coupled enzymes in a single tube assay to monitor DNA synthesis in real time using a luminometric detection system. Here, we demonstrate the use of pyrosequencing for direct analysis of single-nucleotide polymorphism on double-stranded PCR product. Pyrosequencing data on the human glutathione peroxidase gene (GPX1) from several individuals were analysed and three different allelic variants were determined and confirmed. The possibility of further simplifying the sequencing and template-preparation steps is discussed.

Alleles↗

PubMed2000

Membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG). A widespread protein superfamily.

The members of the MAPEG superfamily have been aligned and found to be distantly related, with a common pattern of hydropathy. Figure 2A shows the multiple sequence alignments of the human members and Figure 2B the corresponding superimposed hydropathy profiles. The alignment in Figure 2A demonstrates a total of six strictly conserved residues. The Arg-51 in LTC4 synthase has been suggested to function as proton donor for the opening of the LTA4 epoxide. This arginine is found in all but the FLAP sequences in accordance with the observation that FLAP has no known enzyme activity. Also the Tyr-93 in LTC4 synthase has been suggested to function as a base for the formation of the thiolate anion of glutathione. This tyrosine is not conserved in MGST1 or MGST1-L1. Table 1 summarizes some other properties of the individual human proteins. They are all of the same size, ranging from 147 to 161 amino acids. Only FLAP differs in that its isoelectric point is more neutral than that of the other, more basic proteins. The genes encoding these proteins all reside on different chromosomes (when known) (Table 1). In addition to the human proteins, MAPEG members have been identified in plants, fungi, and bacteria. It is clearly a challenge to elucidate their role in these different phyla in relation to their defined physiological functions in humans.

Amino Acid Sequence↗

PubMed1999

Hypophysectomy and/or peroxisome proliferators strongly influence the levels of phase II xenobiotic metabolizing enzymes in rat testis.

The objectives of the present work were to determine the influence of hypophysectomy and/or peroxisome proliferators (PP) on certain xenobiotic-metabolizing enzyme activities, i.e. glutathione transferases (GST), glutathione peroxidase (GPX), phenol sulphotransferases (pSULT), phenol UDP-glucuronosyl transferases (pUGT), catalase, NADP(H) quinone oxidoreductase (QR) and epoxide hydrolases (EH) in the rat testes. Adult male rats, hypophysectomized and their sham-operated controls, were treated for 10 days with clofibrate (0.5%), perfluorooctanoic acid (0.05%, PFOA), acetylsalicylic acid (1%, ASA) and di(2-ethylhexyl)phthalate (2%, DEHP) in their diet. The results show that, in addition to both body and testis weight, hypophysectomy caused dramatic changes in most of the xenobiotic-metabolizing enzyme activities, which have been measured here. The most pronounced effects were seen in cytosolic QR (2.2-fold increase), pUGT (95% reduction), pSULT (75% reduction), mitochondrial catalase (75% reduction), microsomal EH (70% reduction) and microsomal GST (55% reduction). Treatment with PP, i.e. perfluorooctanoic acid (PFOA), clofibrate, acetyl salicylic acid (ASA) and di(2-ethylhexyl)phthalate (DEHP) showed varied effects on the xenobiotic-metabolizing enzyme activities, the highest effects (10-60% reduction) were seen in sham-operated animals. These effects were not so pronounced or were not seen in hypophysectomized rats except for the case of PFOA treatment, which caused increases of enzyme activities. The highest increases were seen with microsomal GST (70%), GPX (75%) and cytosolic EH (75%). It is concluded from these experiments that the regulation of several xenobiotic-metabolizing enzymes in the rat testis is affected by the pituitary and/or pituitary hormones and that different peroxisome proliferators have variable effects on the levels of these xenobiotic-metabolizing enzymes. The general trend of reduction in enzyme activities implies that the testis is less protected under conditions that can perturb hormonal status.

Animals↗

PubMed1999

Identification of human prostaglandin E synthase: a microsomal, glutathione-dependent, inducible enzyme, constituting a potential novel drug target.

Human prostaglandin (PG) E synthase (EC 5.3.99.3) is a member of a recently recognized protein superfamily consisting of membrane associated proteins involved in eicosanoid and glutathione metabolism (the MAPEG family). Previous designations of the protein are PIG12 and MGST1-L1. PGE synthase was expressed in Escherichia coli, and both cytosolic and membrane fractions were prepared. Western blot analysis specifically detected a 15- to 16-kDa protein in the membrane fraction. Both fractions were incubated with prostaglandin H2 in the presence or absence of reduced glutathione. The membrane but not the cytosolic fraction was found to possess high glutathione-dependent PGE synthase activity (0.25 micromol/min/mg). The human tissue distribution was analyzed by Northern blot analysis. High expression of PGE synthase mRNA was detected in A549 and HeLa cancer cell lines. Intermediate level of expression was demonstrated in placenta, prostate, testis, mammary gland, and bladder whereas low mRNA expression was observed in several other tissues. A549 cells have been used as a model system to study cyclooxygenase-2 induction by IL-1beta. If A549 cells were grown in the presence of IL-1beta, a significant induction of the PGE synthase was observed by Western blot analysis. Also, Western blot analysis specifically detected a 16-kDa protein in sheep seminal vesicles. In summary, we have identified a human membrane bound PGE synthase. The enzyme activity is glutathione-dependent, and the protein expression is induced by the proinflammatory cytokine IL-1beta. PGE synthase is a potential novel target for drug development.

Amino Acid Sequence↗

PubMed1999

The projection structure of the membrane protein microsomal glutathione transferase at 3 A resolution as determined from two-dimensional hexagonal crystals.

The formation of two-dimensional crystals of the membrane-bound enzyme microsomal glutathione transferase is sensitive to fractional changes in the lipid-to-protein ratio. Variation of this parameter results in crystal polymorphism. The projection structure of a p6 crystal form of the enzyme has been determined by the use of electron crystallography. The unit cell at 3 A resolution is comprised of two trimers. The hexagonal p6 and the orthorhombic p21212 crystal types have common elements in the packing arrangement which imply dominant crystal contacts. An overall structural similarity between the protein molecules in the two crystal forms is suggested by the projection maps. Furthermore, a comparison of the p6 and p21212 projection maps identifies additional corresponding protein densities which could not be assigned to the microsomal glutathione transferase trimer previously. Surprisingly, an ambiguity of the rotational orientation was found for trimers interspersed at certain positions within the crystal lattice.

Animals↗

PubMed1999

Leukotriene C4 is a tight-binding inhibitor of microsomal glutathione transferase-1. Effects of leukotriene pathway modifiers.

Microsomal glutathione transferase-1 (MGST-1) is an abundant protein that catalyzes the conjugation of electrophilic compounds with glutathione, as well as the reduction of lipid hydroperoxides. Here we report that leukotriene C4 is a potent inhibitor of MGST-1. Leukotriene C4 was found to be a tight-binding inhibitor, with a Ki of 5.4 nM for the unactivated enzyme, and 9.2 nM for the N-ethylmaleimide activated enzyme. This is the first tight-binding inhibitor characterized for this enzyme. Leukotriene C4 was competitive with respect to glutathione and non-competitive toward the second substrate, CDNB. Analysis of stoichiometry supports binding of one molecule of inhibitor per homotrimer. Leukotrienes A4, D4, and E4 were much weaker inhibitors of the purified enzyme (by at least 3 orders of magnitude). Leukotriene C4 analogues, which have been developed as antagonists of leukotriene receptors, were found to display varying degrees of inhibition of MGST-1. In particular, the cysteinyl-leukotriene analogues SKF 104,353, ONO-1078, and BAYu9773 were strong inhibitors (IC50 values: 0.13, 3. 7, and 7.6 microM, respectively). In view of the partial structural similarity between MGST-1, leukotriene C4 synthase, and 5-lipoxygenase activating protein (FLAP), it was of interest that leukotriene C4 synthesis inhibitors (which antagonize FLAP) also displayed significant inhibition (e.g. IC50 for BAYx1005 was 58 microM). In contrast, selective 5-lipoxygenase inhibitors such as zileuton only marginally inhibited activity at high concentrations (500 microM). Our discovery that leukotriene C4 and drugs developed based on its structure are potent inhibitors of MGST-1 raises the possibility that MGST-1 influences the cellular processing of leukotrienes. These findings may also have implications for the effects and side-effects of drugs developed to manipulate leukotrienes.

Animals↗

PubMed1999

Low yield of polymorphisms from EST blast searching: analysis of genes related to oxidative stress and verification of the P197L polymorphism in GPX1.

To determine new polymorphisms in the antioxidant enzymes superoxide dismutase, glutathione peroxidases, catalase, and microsomal glutathione transferase 1, a search of the human expressed sequence tags (EST) database was performed (with BLAST 2.0). When any mutation, indicated by the BLAST search, gave rise to a nonconservative amino acid change we performed polymerase chain reaction (PCR) restriction analysis and/or sequence analysis of genomic DNA from human subjects in order to verify these potential polymorphisms. Of nine indicated polymorphisms from the EST analysis found in four different antioxidant enzymes, we could verify one, an amino acid substitution Pro-Leu at amino acid position 197 (P197L), in the glutathione peroxidase 1 gene. The corresponding allele frequencies were approximately 70/30%. In addition, a silent mutation (1167T/C) in the catalase gene indicated by the BLAST search could also be verified. Six to nine individuals were analyzed per indicated polymorphism, so that only common polymorphisms would be found. The indicated mutations not verified by direct analysis thus cannot be excluded as allelic variation in the human population. These results show that the EST database can be used to search for polymorphisms in genes with high abundance in the human EST database. In addition to the EST analysis, PCR/single-strand conformation polymorphism (SSCP) was employed for the analysis of the microsomal glutathione transferase 1 gene. No polymorphism in the coding sequence could be detected in the gene by either method. The high degree of conservation of the microsomal glutathione transferase 1 gene indicates an important physiological function for this enzyme.

DNA Primers↗

PubMed1999

A genetic polymorphism in connexin 37 as a prognostic marker for atherosclerotic plaque development.

BACKGROUND AND OBJECTIVES: Atherosclerosis is a multifactorial disease, in part characterized by chronic inflammatory changes in the vessel wall and loss of normal physical and biochemical interactions between endothelial cells and smooth muscle cells. Previous studies [Hu J., Cotgreave IA. J Clin Invest; 99: 1-5] have provided molecular links between inflammation and myoendothelial communication via gap junctions, suggesting that these structures may be important in the development of the atherosclerotic vessel phenotype. In order to strengthen this premise, the aim of the present work was to probe for structural polymorphisms in connexin 37, a gap junctional protein uniquely expressed in endothelial cells, and to assess for potential genotypic segregation in individuals displaying atherosclerotic plaque. METHODS AND RESULTS: Computer-based comparisons of Expressed Sequence Tags (ESTs) predicted a polymorphism in the human gap junctional protein connexin 37 (cx37). The C1019-T mutation results in a proline to serine shift at codon 319 (cx37*1-cx37*2). A Restriction Fragment Length Polymorphism (RFLP) assay, involving the insertion of a novel Drd I cleavage site in the proline variant revealed a statistically significant over-representation of the cx37*1 allele in association with atherosclerotic plaque-bearing individuals (Odds-ratio for the homozygote = 2.38, Chi2 = 7.693, P = 0.006), in comparison to individuals lacking plaque, irrespective of a history of hypertension. CONCLUSIONS: These data suggest that the C1019-T polymorphism in cx37 may provide 'single gene marker', which could be useful in assessing atherosclerotic plaque development, particularly in cardiovascular risk groups such as those with borderline hypertension.

Adult↗

PubMed1999

Distribution of microsomal glutathione transferase 1 in mammalian tissues. A predominant alternate first exon in human tissues.

An extensive Northern blot analysis of microsomal glutathione transferase 1 in human and rat tissues was performed. When normalized against the glyceraldehyde-3-phosphate dehydrogenase or actin expression it was evident that the predominant expression occurs in liver and pancreas. An ontogenetic, as well as a functional, basis for the high levels in these two organs is possible. The relative expression levels in man ranged from: liver and pancreas (100%), to kidney, prostate, colon (30-40%), heart, brain, lung, testis, ovary, small intestine (10-20%), placenta, skeletal muscle, spleen, thymus and peripheral blood leucocytes (1-10%). Liver-enriched expression was detected in human fetal tissues with lung and kidney displaying lower levels (10-20%). No transcripts could be detected in fetal brain or heart. When comparing the expression levels between rat and man it is apparent that human extrahepatic mRNA levels are much higher relative to liver. Rat microsomal glutathione transferase mRNA expression ranges from 0.2 to 10% that of liver, with adrenal, uterus, ovary and stomach displaying the highest levels of the organs tested. Based on these observations, and the fact that the enzyme is encoded by a highly conserved single-copy gene, it is suggested that microsomal glutathione transferase 1 performs essential functions vital to most mammalian cell types. We suggest that protection against oxidative stress constitutes one such function. Human expressed sequence tag (EST) characterization yielded four alternate mRNA transcripts with different 5'-ends (four alternate noncoding exons 1). The predominant exon (based on the observed EST frequency) revealed a tissue distribution similar to that obtained using the reading frame as probe. Thus, it appears that one exon preferentially gives rise to mature mRNA in the human tissues examined. This exon is different from the one reported in the original cDNA characterized.

Animals↗