Monoamine oxidases (MAO-A and MAO-B) are enzymes that play a key role in the degradation of endog... more Monoamine oxidases (MAO-A and MAO-B) are enzymes that play a key role in the degradation of endogenous and dietary monoamines. A full-length cDNA of the B-type of MAO, isolated from a human liver cDNA library, was cloned into a prokaryotic expression vector (pET11c).Escherichia coliwhich was transfected with the recombinant plasmid expressed an insoluble protein product with the expected molecular weight
Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in se... more Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in several human conditions such as congestive heart failure, diabetes mellitus, and inXammation. The reactive aldehydes and hydrogen peroxide produced by SSAO have been suggested to contribute to the progression of vascular complications associated with these conditions. In addition, SSAO activity has been shown to be involved in the leukocyte extravasation process at sites of inXammation. To facilitate characterization and development of speciWc and selective inhibitors of SSAO, we have developed a method for production of recombinant human SSAO. The extracellular region (residues 29-763) of human SSAO was expressed in HEK293 cells in fusion with a mutated Schistosoma japonicum glutathione Stransferase (GST) and secreted to the culture medium. The mutGST-SSAO fusion protein was puriWed in a single step by glutathione-aYnity chromatography followed by site-speciWc cleavage using a GST-3C protease fusion protein to remove the mutGST fusion partner. A second glutathione-aYnity chromatography step was then used to capture both the mutGST fusion partner and the GST-3C protease, resulting in milligram quantities of pure, enzymatically active, and soluble recombinant human SSAO.
Pfl�gers Archiv European Journal of Physiology, 2001
The intrarenal natriuretic hormone dopamine (DA) is metabolized by catechol-O-methyltransferase (... more The intrarenal natriuretic hormone dopamine (DA) is metabolized by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO). We have previously shown that inhibition of COMT by entacapone results in a potent D1-like receptor-mediated natriuretic response. The present study was performed using anaesthetized rats to compare the importance of MAO and COMT in DA-mediated natriuresis by use of the MAO inhibitor phenelzine. Urinary sodium and DA excretion remained unchanged after MAO inhibition, while excretion of the main metabolite dihydroxyphenylacetic acid (DOPAC) decreased by 55%. The response was unaltered if 5-hydroxytryptamine receptors (5-HT1A) were blocked during MAO inhibition. We also investigated the specific renal activities of MAO and COMT in rat renal cortex during DA-influenced natriuresis. Specific COMT activity in the renal cortex was reduced by 13% after isotonic sodium loading (5% of body mass) whereas renal MAO-A and MAO-B activities remained unaltered. Furthermore, preliminary data obtained from spontaneously hypertensive rats, whose basal urinary DA excretion is higher than that of normotensive Wistar-Kyoto rats, show a tendency for renal COMT activity to be lower. It is concluded that MAOinhibition by phenelzine does not alter sodium excretion. Furthermore, specific renal cortical COMT activity is reduced during partly D1-like receptor-mediated natriuresis, whereas MAO activity remains unchanged. The results suggest that MAO is less important than COMT in regulating DA-mediated natriuresis in the rat kidney.
Our study probed the effects of the beta-2 adrenergic agonist, formoterol and the macrolide antib... more Our study probed the effects of the beta-2 adrenergic agonist, formoterol and the macrolide antibiotic, roxithromycin, on muscle wasting in a well-characterized animal model of cancer cachexia. Female Wistar rats were inoculated with Yoshida AH130 ascites hepatoma (AH) cells to induce rapid and severe cachexia as demonstrated by wet weight determinations of the hearts, gastrocnemius muscles and carcasses. The control animals received saline (vehicle) inoculations. The AH-inoculated rats were treated once daily for four days by i.p. injection with a vehicle control, 1 mg/kg formoterol, 5 and 50 mg/kg roxithromycin or 1 mg/kg formoterol plus 5, 25, 40 and 50 mg/kg roxithromycin. The saline-inoculated animals were treated by i.p. injection with vehicle control, 1 mg/kg formoterol, 5 and 40 mg/kg roxithromycin. As a result, formoterol alone reduced the loss of muscle mass in the AH-inoculated rats by approximately one-half, consistent with literature reports. Roxithromycin alone at 5 mg/kg did not affect muscle mass in the AH-inoculated rats. Roxithromycin given alone at 50 mg/kg reduced the loss of muscle mass in AH-inoculated animals by approximately one-half. With respect to the antagonizing muscle loss, formoterol combined with either 5 or 25 mg/kg roxithromycin did not reach statistical significance versus formoterol alone, while formoterol plus either 40 or 50 mg/kg roxithromycin enhanced protection against muscle loss versus formoterol alone. The gastrocnemius weights in the AH-inoculated rats treated with formoterol combined with 40 mg/kg roxithromycin were not significantly different from the muscle weights in the saline-inoculated controls. To sum up, formoterol and roxithromycin apparently exert anti-cachectic effects in an additive fashion and may offer the potential for combination therapy in cachexia.
Many studies show that monoamine oxidase B in blood cells is a biological marker for personality ... more Many studies show that monoamine oxidase B in blood cells is a biological marker for personality characteristics such as sensation seeking. The mechanism underlying this association is so far not explored. In the present study we have performed electrophoretic mobility-shift assays to investigate the pattern of protein binding to a 150 bp fragment of the proximal 5'-flanking region of the human monoamine oxidase B gene. We compared the pattern using nuclear extracts from human brain and lymphocytes. Interestingly, a correlation was observed between monoamine oxidase B enzyme activity in blood cells (platelets) and the binding pattern of two uncharacterized transcription factors. These data are well in line with the long-standing notion that interindividual differences in platelet monoamine oxidase may represent differences in expression of the enzyme rather than genotypic variation.
Monoamine oxidase type A (MAOA) has been implicated to be part of mechanisms underlying human tem... more Monoamine oxidase type A (MAOA) has been implicated to be part of mechanisms underlying human temperament and psychiatric disorders. We hypothesised that a functional polymorphism in the 5' untranslated region of the MAOA gene is associated with specific personality traits. In 371 healthy Caucasians, we estimated personality traits by the use of the Karolinska Scales of Personality (KSP), Scandinavian Universities Scales of Personality, Health-Relevant 5-Factor Personality inventory, Temperament and Character Inventory and the revised NEO Personality Inventory. In the same subjects, we analysed the genotype of a polymorphic region consisting of a variable number of a 30-bp repeat sequence located approximately 1.2 kb upstream of the MAOA gene. After correction for multiple testing, no statistically significant differences between MAOA genotype and personality were observed in men (n = 206) nor in women (n = 165). We conclude that the structure of this MAOA promoter region does not have a large impact on the expression of personality characteristics in the present Swedish population.
To find genes that underlie disease susceptibilities, genome-wide single nucleotide polymorphisms... more To find genes that underlie disease susceptibilities, genome-wide single nucleotide polymorphisms (SNPs) have been analyzed using high-throughput matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). As a proof-of-concept for this approach, gene regions have been identified that were previously associated by others with certain diseases or traits. On the same technology platform, accurate and absolute transcriptional profiling can be performed and applied to allele expression analysis. Here, we provide a brief review of the technology and its applications to disease gene discovery.
The catastrophic epidemic of subacute myelo-optic neuropathy (SMON) affected Japan around 1970 wi... more The catastrophic epidemic of subacute myelo-optic neuropathy (SMON) affected Japan around 1970 with thousands of victims. The cause was attributed to high doses of locally acting oxyquinolines. It has been speculated that oxyquinoline derivatives of the clioquinol type can disturb the retention of vitamin B12 through chelation of Co2+. In the present paper, possible effects of clioquinol on the uptake and tissue distribution of [57Co]-cyanocobalamin have been studied in mice. In vivo experiments showed markedly decreased accumulation of radiolabelled vitamin B12 in the kidney and skin in animals that were pre-treated with clioquinol. The chloroform:water partition coefficients for [57Co]-cyanocobalamin in the presence or absence of clioquinol were also determined. No statistically significant alterations in the partition coefficient for [57Co]-cyanocobalamin in the presence of clioquinol was evident, indicating that clioquinol does not bind cobalt. In addition, transmethylation reactions in the CNS in mice treated with clioquinol were studied. Specific activities of methionine adenosyltransferase, and S-adenosylhomocysteine levels were not affected. In contrast, clioquinol treatment caused a significant increase in the levels of S-adenosylmethionine in the brain. The data of the present study show that clioquinol treatment can affect the accumulation of vitamin B12 in the kidney and the skin but not in the brain. These results do not support the hypothesis that clioquinol causes its damage to the nervous system by a direct chemical interaction with vitamin B12.
Monoamine oxidases (MAO) play a critical role in the degradation of endogenous and exogenous amin... more Monoamine oxidases (MAO) play a critical role in the degradation of endogenous and exogenous amines throughout the body. There are two distinct MAO isoforms, MAO-A and MAO-B, which both are encoded in genes on the X chromosome. Alterations in MAO-B activity have previously been connected with several neurological disorders. Platelet MAO (trbc-MAO) is exclusively of the B-type and the catalytic activity of this enzyme is under strong, yet unknown, genetic control. Specific trbc-MAO activity has been reported to be increased in certain neurodegenerative diseases and to correlate with personality traits such as sensation seeking and impulsiveness. In the present study, we investigated if trbc-MAO activity is associated with genotype at a variable region (A/G dimorphism) in intron 13 of the human gene encoding MAO-B. The MAOB intron 13 allele status and levels of trbc-MAO were determined for 55 Caucasian non-smoking males.
Plasma activity of the enzyme semicarbazide-sensitive amine oxidase (SSAO) is high in diabetes. P... more Plasma activity of the enzyme semicarbazide-sensitive amine oxidase (SSAO) is high in diabetes. Production of angiotoxic substances (an aldehyde, hydrogen peroxide, and ammonia) in vessel walls is catalysed by SSAO, suggesting a role for SSAO in the development of complications of diabetes. The objective of the present study was to follow up plasma SSAO activity (measured radiometrically), HbA(1c) (using ion exchange chromatography), and retinopathy (by fundus photography) after 2.8 years, in 34 patients with Type 2 diabetes. We also measured urinary levels of an SSAO substrate, methylamine, by fluorometric high-performance liquid chromatography (HPLC). As at baseline, plasma SSAO activity was now higher in subjects with retinopathy (mean 19.5) than in subjects without retinopathy (mean 16.0), 95% confidence interval (CI) for difference 0.6-6.3 nmol benzylamine ml(-1) plasma h(-1). SSAO activity had not changed significantly since baseline, mean difference -1.65 and 95% CI for difference -3.76 to 0.46 nmol benzylamine ml(-1) plasma h(-1). Mean HbA(1c) level remained higher for patients with retinopathy (now 7.9%) compared to those without retinopathy (6.1%), 95% CI for difference 0.6-3.0%. Comparing baseline and the present study, retinopathy was nonproliferative; level had worsened for five and improved for two patients. Urinary methylamine/creatinine ratio was lower in the group of patients with retinopathy (mean 0.99) than in those without retinopathy (mean 1.78), 95% CI for difference 0.1-1.5 microg mg(-1). The results of the present study are compatible with a role for SSAO in the development of diabetic retinopathy.
In the present report we describe the astrocytic localization and content of monoamine oxidase-B ... more In the present report we describe the astrocytic localization and content of monoamine oxidase-B (MAO-B) by means of a 3H-L-deprenyl emulsion autoradiography in primary cultures of rat astrocytes, in cryosectioned astrocytoma surgical specimen, and in cryosections of human spinal cords from patients dying in amyotrophic lateral sclerosis (ALS) and controls. The occurrence of MAO-B enzyme protein depends on the degree of cellular differentiation as demonstrated by studies on astrocytes in primary cultures analyzed at two different stages of maturation. Highly differentiated cells exhibited high relative enzyme concentration whereas glioblasts lacked or showed very low contents of MAO-B enzyme. This was further substantiated by studies performed on human astrocytoma tissue using 3H-L-deprenyl emulsion autoradiography in combination with immunohistochemical detection of glial fibrillary acidic protein (GFAP). Regional increases of MAO-B concentration were found in ALS lumbar sections with quantitative 3H-L-deprenyl autoradiography. On the basis of results obtained from double staining for GFAP and MAO-B, the increase in MAO-B seemed to be due to an increased number of astrocytes as well as an increased content of MAO-B in reactive species of astrocytes. A cell culture model has been used that produces cells with morphology and GFAP-content similar to reactive cells. These astrocytes exhibited high relative content of the MAO-B enzyme protein. In the light of the presented data, taking into account the finding that a subpopulation of reactive cells contained low levels of MAO-B, a heterogeneity among reactive astrocytes was observed.
In the present study, levels ofS-adenosylmethionine (SAM) andS-adenosylhomocysteine (SAH) in whol... more In the present study, levels ofS-adenosylmethionine (SAM) andS-adenosylhomocysteine (SAH) in whole blood as well asl-methionineS-adenosyltransferase (MAT) activity in erythrocytes were assayed in a series of 20 patients with Parkinson's disease and 12 healthy control subjects. A significant difference was found with regard to SAM levels between patients and controls, with the detected levels being 383.1 ± 41.5 nMfor the parkinsonian
To measure plasma semicarbazide-sensitive amine oxidase (SSAO) activities and detect retinopathy ... more To measure plasma semicarbazide-sensitive amine oxidase (SSAO) activities and detect retinopathy in Type 2 diabetes mellitus (DM). Cross-sectional, population-based study of 65 diabetes patients (61 diagnosed from the age of 30 years) with or without retinopathy as determined by fundus photography in primary care. HbA1c was analysed by ion exchange chromatography on a Mono S for HbA1c column. SSAO activities were assayed radiometrically and formaldehyde-albumin adducts by ELISA in plasma samples from patients and 136 healthy controls. Subjects with diabetes had higher plasma SSAO activity, measured as nmol benzylamine x mlplasma(-11) x h(-1)(mean 20.6), than controls (mean 14.3), P<0.0001; 95% confidence interval (CI) for difference 4.9-7.7. SSAO activity was higher in patients with retinopathy (mean 23.2) than in those without (mean 18.9), P=0.012; 95% CI for difference 1.0-7.5, and related to the HbA1c value. No statistically significant relationship between diabetes duration and SSAO activity was found. With HbA1c values and insulin treatment entered into a multiple logistic regression model, SSAO activity no longer predicted retinopathy, P increasing from 0.025 to 0.17. SSAO activity and the presence of any retinopathy were unrelated to titres of antibodies against formaldehyde-treated human serum albumin. SSAO activity, earlier found to be elevated in Type 1 DM, is also elevated in Type 2 DM. The SSAO family of enzymes may be involved in the development of diabetic retinopathy, possibly by catalysing the formation of toxic metabolites. A potent and specific inhibitor of human SSAO might help prevent retinopathy in Type 1 and Type 2 DM.
Cell culture and siRNA-mediated gene silencing MDA-MB-231 and MDA-MB-435 breast cancer cell lines... more Cell culture and siRNA-mediated gene silencing MDA-MB-231 and MDA-MB-435 breast cancer cell lines were obtained from the Developmental Therapeutics Program, NCI/NIH (Frederick, MD) and were cultured in DMEM and RPMI media, respectively, supplemented with 10% fetal calf serum (FCS, Omega Scientific, Tarzana, CA). MCF-7, T47D, NCI-H460, A375 and PC3 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured according to ATCC recommendations. Normal human dermal fibroblasts (NHDF) were obtained from BioWhittaker (Walkersville, MD) and cultured according to the manufacturer's instructions. Small-inhibitory RNA duplexes were designed according to the guidelines of Elbashir et al. (23) and were synthesized by Dharmacon Research Inc. (Lafayette, CO). Oligonucleotide siRNA sequences for human ICAM1 were designed as follows: siICAM1-1, 5 0 -AAACAACCGGAAGGU-GUAUGA; siICAM1-2, 5 0 -AAGCCAACCAAUGUGCUAUUC; siICAM1-3, 5 0 -AAGAUCACCAUGGAGCCAAUU; siICAM1-4, 5 0 -AACUGUCACUCG-AGAUCUUGA. A control siRNA siGL2 (5 0 -AACGUACGCGGAAUACUU-CGA), which is non-homologous to any human sequence, was obtained from Dharmacon. Additional control siRNA were designed to target human ICAM5
The human neuroblastoma cell line SH-SY5Y was used to study the regulation of methionine adenosyl... more The human neuroblastoma cell line SH-SY5Y was used to study the regulation of methionine adenosyltransferase (MAT II; E.C.2.5.1.6.) catalytic activity and transcript levels in cells of neuronal origin. The cells were exposed for 24 hr to a medium containing different concentrations of methionine (MAT substrate) as well as medium deficient of methionine. Furthermore, cells were treated with hydroxycobalamin, SAM, and the competitive MAT inhibitor cycloleucine. The MAT catalytic activity was inversely correlated to methionine concentrations, e.g. MAT Vmax increased 2-fold in cells grown in methionine-deficient medium as compared with cells cultured under standard conditions. Interestingly, MAT Km also increased from 9.04 +/- 0.44 to 12.08 +/- 0.83 in the methionine-deficient medium. Hydroxycobalamin caused an increase in activity at 40 microM while a decrease was observed at higher concentrations (100, 200, and 400 microM). Cycloleucine caused a significant inhibition of MAT catalytic activity, i.e. the inhibition was approximately 50% in the presence of 4 mM cycloleucine. The relevance of these results for the understanding of observations on MAT catalytic activity in brains of patients with Alzheimer's disease is discussed.
Biological markers for acquisition and extinction of fear conditioning were studied in 40 individ... more Biological markers for acquisition and extinction of fear conditioning were studied in 40 individuals selected for displaying either good or poor acquisition of fear conditioning. as estimated by the skin conductance response. Participants with a short serotonin transporter (5-HTT) promoter allele or low monoamine oxidase activity in platelets (trbc-MAO) displayed better acquisition than those with only long alleles or high trbc-MAO, whereas participants with a long dopamine D4 receptor (D4DR) exon III allele showed delayed extinction compared with those with only short alleles. The findings, that D4DR exon III and 5-HTT promoter genotypes and trbc-MAO activity are related to human fear conditioning, a basic form of associative learning, are consistent with animal studies suggesting a genetic contribution to fear conditioning. The authors suggest that in humans these genetic mechanisms are partly dopaminergic and serotonergic in origin.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2003
Semicarbazide-sensitive amine oxidases (SSAO) are widely distributed enzymes, with as yet not ful... more Semicarbazide-sensitive amine oxidases (SSAO) are widely distributed enzymes, with as yet not fully elucidated functions and roles, present in many tissues but also circulating in plasma. The enzyme also functions as an adhesion molecule, the vascular adhesion protein-1. In healthy humans, plasma SSAO activity is constant from birth until 16 years of age, when it drops to lower values, gradually increasing again at advanced ages. When measuring SSAO activity, care should be taken to ensure proper preparation and storage conditions, and it should be realized that quite a few drugs unintentionally are good inhibitors, and sometimes even substrates, of SSAO. Under normal conditions SSAO activity is constant and inter-individual variation is small. In various pathophysiological conditions plasma SSAO activities are increased, most notably in diabetes mellitus (both type I and type II), in congestive heart failure and in cirrhotic liver inflammation. In patients with other vascular and inflammatory diseases plasma SSAO is normal, while it is low in children with congenital lung diseases. Interpretation of these changes is speculative, since source and regulation of plasma SSAO are as yet unknown. However, in two situations where the disease-causing process was ended (transplantation, delivery), plasma SSAO returned to normal. Many questions remain to be answered.
Coprrghl 0 Muirksguard 1994 hCTA NEUROI.OGICh OKAZAWA H. MURATA M, WATANABE M, KAMEI M. KANAZAWA ... more Coprrghl 0 Muirksguard 1994 hCTA NEUROI.OGICh OKAZAWA H. MURATA M, WATANABE M, KAMEI M. KANAZAWA I. Dopaminergic stimulation up-regulates the iri vivv expression of brain-derived neurotrophic factor (BDNF) in the striatum. FEBS Lett, 1992: 313: 138-142.
Monoamine oxidases (MAO-A and MAO-B) are enzymes that play a key role in the degradation of endog... more Monoamine oxidases (MAO-A and MAO-B) are enzymes that play a key role in the degradation of endogenous and dietary monoamines. A full-length cDNA of the B-type of MAO, isolated from a human liver cDNA library, was cloned into a prokaryotic expression vector (pET11c).Escherichia coliwhich was transfected with the recombinant plasmid expressed an insoluble protein product with the expected molecular weight
Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in se... more Elevated levels of semicarbazide-sensitive amine oxidase (SSAO) activity have been observed in several human conditions such as congestive heart failure, diabetes mellitus, and inXammation. The reactive aldehydes and hydrogen peroxide produced by SSAO have been suggested to contribute to the progression of vascular complications associated with these conditions. In addition, SSAO activity has been shown to be involved in the leukocyte extravasation process at sites of inXammation. To facilitate characterization and development of speciWc and selective inhibitors of SSAO, we have developed a method for production of recombinant human SSAO. The extracellular region (residues 29-763) of human SSAO was expressed in HEK293 cells in fusion with a mutated Schistosoma japonicum glutathione Stransferase (GST) and secreted to the culture medium. The mutGST-SSAO fusion protein was puriWed in a single step by glutathione-aYnity chromatography followed by site-speciWc cleavage using a GST-3C protease fusion protein to remove the mutGST fusion partner. A second glutathione-aYnity chromatography step was then used to capture both the mutGST fusion partner and the GST-3C protease, resulting in milligram quantities of pure, enzymatically active, and soluble recombinant human SSAO.
Pfl�gers Archiv European Journal of Physiology, 2001
The intrarenal natriuretic hormone dopamine (DA) is metabolized by catechol-O-methyltransferase (... more The intrarenal natriuretic hormone dopamine (DA) is metabolized by catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO). We have previously shown that inhibition of COMT by entacapone results in a potent D1-like receptor-mediated natriuretic response. The present study was performed using anaesthetized rats to compare the importance of MAO and COMT in DA-mediated natriuresis by use of the MAO inhibitor phenelzine. Urinary sodium and DA excretion remained unchanged after MAO inhibition, while excretion of the main metabolite dihydroxyphenylacetic acid (DOPAC) decreased by 55%. The response was unaltered if 5-hydroxytryptamine receptors (5-HT1A) were blocked during MAO inhibition. We also investigated the specific renal activities of MAO and COMT in rat renal cortex during DA-influenced natriuresis. Specific COMT activity in the renal cortex was reduced by 13% after isotonic sodium loading (5% of body mass) whereas renal MAO-A and MAO-B activities remained unaltered. Furthermore, preliminary data obtained from spontaneously hypertensive rats, whose basal urinary DA excretion is higher than that of normotensive Wistar-Kyoto rats, show a tendency for renal COMT activity to be lower. It is concluded that MAOinhibition by phenelzine does not alter sodium excretion. Furthermore, specific renal cortical COMT activity is reduced during partly D1-like receptor-mediated natriuresis, whereas MAO activity remains unchanged. The results suggest that MAO is less important than COMT in regulating DA-mediated natriuresis in the rat kidney.
Our study probed the effects of the beta-2 adrenergic agonist, formoterol and the macrolide antib... more Our study probed the effects of the beta-2 adrenergic agonist, formoterol and the macrolide antibiotic, roxithromycin, on muscle wasting in a well-characterized animal model of cancer cachexia. Female Wistar rats were inoculated with Yoshida AH130 ascites hepatoma (AH) cells to induce rapid and severe cachexia as demonstrated by wet weight determinations of the hearts, gastrocnemius muscles and carcasses. The control animals received saline (vehicle) inoculations. The AH-inoculated rats were treated once daily for four days by i.p. injection with a vehicle control, 1 mg/kg formoterol, 5 and 50 mg/kg roxithromycin or 1 mg/kg formoterol plus 5, 25, 40 and 50 mg/kg roxithromycin. The saline-inoculated animals were treated by i.p. injection with vehicle control, 1 mg/kg formoterol, 5 and 40 mg/kg roxithromycin. As a result, formoterol alone reduced the loss of muscle mass in the AH-inoculated rats by approximately one-half, consistent with literature reports. Roxithromycin alone at 5 mg/kg did not affect muscle mass in the AH-inoculated rats. Roxithromycin given alone at 50 mg/kg reduced the loss of muscle mass in AH-inoculated animals by approximately one-half. With respect to the antagonizing muscle loss, formoterol combined with either 5 or 25 mg/kg roxithromycin did not reach statistical significance versus formoterol alone, while formoterol plus either 40 or 50 mg/kg roxithromycin enhanced protection against muscle loss versus formoterol alone. The gastrocnemius weights in the AH-inoculated rats treated with formoterol combined with 40 mg/kg roxithromycin were not significantly different from the muscle weights in the saline-inoculated controls. To sum up, formoterol and roxithromycin apparently exert anti-cachectic effects in an additive fashion and may offer the potential for combination therapy in cachexia.
Many studies show that monoamine oxidase B in blood cells is a biological marker for personality ... more Many studies show that monoamine oxidase B in blood cells is a biological marker for personality characteristics such as sensation seeking. The mechanism underlying this association is so far not explored. In the present study we have performed electrophoretic mobility-shift assays to investigate the pattern of protein binding to a 150 bp fragment of the proximal 5'-flanking region of the human monoamine oxidase B gene. We compared the pattern using nuclear extracts from human brain and lymphocytes. Interestingly, a correlation was observed between monoamine oxidase B enzyme activity in blood cells (platelets) and the binding pattern of two uncharacterized transcription factors. These data are well in line with the long-standing notion that interindividual differences in platelet monoamine oxidase may represent differences in expression of the enzyme rather than genotypic variation.
Monoamine oxidase type A (MAOA) has been implicated to be part of mechanisms underlying human tem... more Monoamine oxidase type A (MAOA) has been implicated to be part of mechanisms underlying human temperament and psychiatric disorders. We hypothesised that a functional polymorphism in the 5' untranslated region of the MAOA gene is associated with specific personality traits. In 371 healthy Caucasians, we estimated personality traits by the use of the Karolinska Scales of Personality (KSP), Scandinavian Universities Scales of Personality, Health-Relevant 5-Factor Personality inventory, Temperament and Character Inventory and the revised NEO Personality Inventory. In the same subjects, we analysed the genotype of a polymorphic region consisting of a variable number of a 30-bp repeat sequence located approximately 1.2 kb upstream of the MAOA gene. After correction for multiple testing, no statistically significant differences between MAOA genotype and personality were observed in men (n = 206) nor in women (n = 165). We conclude that the structure of this MAOA promoter region does not have a large impact on the expression of personality characteristics in the present Swedish population.
To find genes that underlie disease susceptibilities, genome-wide single nucleotide polymorphisms... more To find genes that underlie disease susceptibilities, genome-wide single nucleotide polymorphisms (SNPs) have been analyzed using high-throughput matrix assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). As a proof-of-concept for this approach, gene regions have been identified that were previously associated by others with certain diseases or traits. On the same technology platform, accurate and absolute transcriptional profiling can be performed and applied to allele expression analysis. Here, we provide a brief review of the technology and its applications to disease gene discovery.
The catastrophic epidemic of subacute myelo-optic neuropathy (SMON) affected Japan around 1970 wi... more The catastrophic epidemic of subacute myelo-optic neuropathy (SMON) affected Japan around 1970 with thousands of victims. The cause was attributed to high doses of locally acting oxyquinolines. It has been speculated that oxyquinoline derivatives of the clioquinol type can disturb the retention of vitamin B12 through chelation of Co2+. In the present paper, possible effects of clioquinol on the uptake and tissue distribution of [57Co]-cyanocobalamin have been studied in mice. In vivo experiments showed markedly decreased accumulation of radiolabelled vitamin B12 in the kidney and skin in animals that were pre-treated with clioquinol. The chloroform:water partition coefficients for [57Co]-cyanocobalamin in the presence or absence of clioquinol were also determined. No statistically significant alterations in the partition coefficient for [57Co]-cyanocobalamin in the presence of clioquinol was evident, indicating that clioquinol does not bind cobalt. In addition, transmethylation reactions in the CNS in mice treated with clioquinol were studied. Specific activities of methionine adenosyltransferase, and S-adenosylhomocysteine levels were not affected. In contrast, clioquinol treatment caused a significant increase in the levels of S-adenosylmethionine in the brain. The data of the present study show that clioquinol treatment can affect the accumulation of vitamin B12 in the kidney and the skin but not in the brain. These results do not support the hypothesis that clioquinol causes its damage to the nervous system by a direct chemical interaction with vitamin B12.
Monoamine oxidases (MAO) play a critical role in the degradation of endogenous and exogenous amin... more Monoamine oxidases (MAO) play a critical role in the degradation of endogenous and exogenous amines throughout the body. There are two distinct MAO isoforms, MAO-A and MAO-B, which both are encoded in genes on the X chromosome. Alterations in MAO-B activity have previously been connected with several neurological disorders. Platelet MAO (trbc-MAO) is exclusively of the B-type and the catalytic activity of this enzyme is under strong, yet unknown, genetic control. Specific trbc-MAO activity has been reported to be increased in certain neurodegenerative diseases and to correlate with personality traits such as sensation seeking and impulsiveness. In the present study, we investigated if trbc-MAO activity is associated with genotype at a variable region (A/G dimorphism) in intron 13 of the human gene encoding MAO-B. The MAOB intron 13 allele status and levels of trbc-MAO were determined for 55 Caucasian non-smoking males.
Plasma activity of the enzyme semicarbazide-sensitive amine oxidase (SSAO) is high in diabetes. P... more Plasma activity of the enzyme semicarbazide-sensitive amine oxidase (SSAO) is high in diabetes. Production of angiotoxic substances (an aldehyde, hydrogen peroxide, and ammonia) in vessel walls is catalysed by SSAO, suggesting a role for SSAO in the development of complications of diabetes. The objective of the present study was to follow up plasma SSAO activity (measured radiometrically), HbA(1c) (using ion exchange chromatography), and retinopathy (by fundus photography) after 2.8 years, in 34 patients with Type 2 diabetes. We also measured urinary levels of an SSAO substrate, methylamine, by fluorometric high-performance liquid chromatography (HPLC). As at baseline, plasma SSAO activity was now higher in subjects with retinopathy (mean 19.5) than in subjects without retinopathy (mean 16.0), 95% confidence interval (CI) for difference 0.6-6.3 nmol benzylamine ml(-1) plasma h(-1). SSAO activity had not changed significantly since baseline, mean difference -1.65 and 95% CI for difference -3.76 to 0.46 nmol benzylamine ml(-1) plasma h(-1). Mean HbA(1c) level remained higher for patients with retinopathy (now 7.9%) compared to those without retinopathy (6.1%), 95% CI for difference 0.6-3.0%. Comparing baseline and the present study, retinopathy was nonproliferative; level had worsened for five and improved for two patients. Urinary methylamine/creatinine ratio was lower in the group of patients with retinopathy (mean 0.99) than in those without retinopathy (mean 1.78), 95% CI for difference 0.1-1.5 microg mg(-1). The results of the present study are compatible with a role for SSAO in the development of diabetic retinopathy.
In the present report we describe the astrocytic localization and content of monoamine oxidase-B ... more In the present report we describe the astrocytic localization and content of monoamine oxidase-B (MAO-B) by means of a 3H-L-deprenyl emulsion autoradiography in primary cultures of rat astrocytes, in cryosectioned astrocytoma surgical specimen, and in cryosections of human spinal cords from patients dying in amyotrophic lateral sclerosis (ALS) and controls. The occurrence of MAO-B enzyme protein depends on the degree of cellular differentiation as demonstrated by studies on astrocytes in primary cultures analyzed at two different stages of maturation. Highly differentiated cells exhibited high relative enzyme concentration whereas glioblasts lacked or showed very low contents of MAO-B enzyme. This was further substantiated by studies performed on human astrocytoma tissue using 3H-L-deprenyl emulsion autoradiography in combination with immunohistochemical detection of glial fibrillary acidic protein (GFAP). Regional increases of MAO-B concentration were found in ALS lumbar sections with quantitative 3H-L-deprenyl autoradiography. On the basis of results obtained from double staining for GFAP and MAO-B, the increase in MAO-B seemed to be due to an increased number of astrocytes as well as an increased content of MAO-B in reactive species of astrocytes. A cell culture model has been used that produces cells with morphology and GFAP-content similar to reactive cells. These astrocytes exhibited high relative content of the MAO-B enzyme protein. In the light of the presented data, taking into account the finding that a subpopulation of reactive cells contained low levels of MAO-B, a heterogeneity among reactive astrocytes was observed.
In the present study, levels ofS-adenosylmethionine (SAM) andS-adenosylhomocysteine (SAH) in whol... more In the present study, levels ofS-adenosylmethionine (SAM) andS-adenosylhomocysteine (SAH) in whole blood as well asl-methionineS-adenosyltransferase (MAT) activity in erythrocytes were assayed in a series of 20 patients with Parkinson's disease and 12 healthy control subjects. A significant difference was found with regard to SAM levels between patients and controls, with the detected levels being 383.1 ± 41.5 nMfor the parkinsonian
To measure plasma semicarbazide-sensitive amine oxidase (SSAO) activities and detect retinopathy ... more To measure plasma semicarbazide-sensitive amine oxidase (SSAO) activities and detect retinopathy in Type 2 diabetes mellitus (DM). Cross-sectional, population-based study of 65 diabetes patients (61 diagnosed from the age of 30 years) with or without retinopathy as determined by fundus photography in primary care. HbA1c was analysed by ion exchange chromatography on a Mono S for HbA1c column. SSAO activities were assayed radiometrically and formaldehyde-albumin adducts by ELISA in plasma samples from patients and 136 healthy controls. Subjects with diabetes had higher plasma SSAO activity, measured as nmol benzylamine x mlplasma(-11) x h(-1)(mean 20.6), than controls (mean 14.3), P<0.0001; 95% confidence interval (CI) for difference 4.9-7.7. SSAO activity was higher in patients with retinopathy (mean 23.2) than in those without (mean 18.9), P=0.012; 95% CI for difference 1.0-7.5, and related to the HbA1c value. No statistically significant relationship between diabetes duration and SSAO activity was found. With HbA1c values and insulin treatment entered into a multiple logistic regression model, SSAO activity no longer predicted retinopathy, P increasing from 0.025 to 0.17. SSAO activity and the presence of any retinopathy were unrelated to titres of antibodies against formaldehyde-treated human serum albumin. SSAO activity, earlier found to be elevated in Type 1 DM, is also elevated in Type 2 DM. The SSAO family of enzymes may be involved in the development of diabetic retinopathy, possibly by catalysing the formation of toxic metabolites. A potent and specific inhibitor of human SSAO might help prevent retinopathy in Type 1 and Type 2 DM.
Cell culture and siRNA-mediated gene silencing MDA-MB-231 and MDA-MB-435 breast cancer cell lines... more Cell culture and siRNA-mediated gene silencing MDA-MB-231 and MDA-MB-435 breast cancer cell lines were obtained from the Developmental Therapeutics Program, NCI/NIH (Frederick, MD) and were cultured in DMEM and RPMI media, respectively, supplemented with 10% fetal calf serum (FCS, Omega Scientific, Tarzana, CA). MCF-7, T47D, NCI-H460, A375 and PC3 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and were cultured according to ATCC recommendations. Normal human dermal fibroblasts (NHDF) were obtained from BioWhittaker (Walkersville, MD) and cultured according to the manufacturer's instructions. Small-inhibitory RNA duplexes were designed according to the guidelines of Elbashir et al. (23) and were synthesized by Dharmacon Research Inc. (Lafayette, CO). Oligonucleotide siRNA sequences for human ICAM1 were designed as follows: siICAM1-1, 5 0 -AAACAACCGGAAGGU-GUAUGA; siICAM1-2, 5 0 -AAGCCAACCAAUGUGCUAUUC; siICAM1-3, 5 0 -AAGAUCACCAUGGAGCCAAUU; siICAM1-4, 5 0 -AACUGUCACUCG-AGAUCUUGA. A control siRNA siGL2 (5 0 -AACGUACGCGGAAUACUU-CGA), which is non-homologous to any human sequence, was obtained from Dharmacon. Additional control siRNA were designed to target human ICAM5
The human neuroblastoma cell line SH-SY5Y was used to study the regulation of methionine adenosyl... more The human neuroblastoma cell line SH-SY5Y was used to study the regulation of methionine adenosyltransferase (MAT II; E.C.2.5.1.6.) catalytic activity and transcript levels in cells of neuronal origin. The cells were exposed for 24 hr to a medium containing different concentrations of methionine (MAT substrate) as well as medium deficient of methionine. Furthermore, cells were treated with hydroxycobalamin, SAM, and the competitive MAT inhibitor cycloleucine. The MAT catalytic activity was inversely correlated to methionine concentrations, e.g. MAT Vmax increased 2-fold in cells grown in methionine-deficient medium as compared with cells cultured under standard conditions. Interestingly, MAT Km also increased from 9.04 +/- 0.44 to 12.08 +/- 0.83 in the methionine-deficient medium. Hydroxycobalamin caused an increase in activity at 40 microM while a decrease was observed at higher concentrations (100, 200, and 400 microM). Cycloleucine caused a significant inhibition of MAT catalytic activity, i.e. the inhibition was approximately 50% in the presence of 4 mM cycloleucine. The relevance of these results for the understanding of observations on MAT catalytic activity in brains of patients with Alzheimer's disease is discussed.
Biological markers for acquisition and extinction of fear conditioning were studied in 40 individ... more Biological markers for acquisition and extinction of fear conditioning were studied in 40 individuals selected for displaying either good or poor acquisition of fear conditioning. as estimated by the skin conductance response. Participants with a short serotonin transporter (5-HTT) promoter allele or low monoamine oxidase activity in platelets (trbc-MAO) displayed better acquisition than those with only long alleles or high trbc-MAO, whereas participants with a long dopamine D4 receptor (D4DR) exon III allele showed delayed extinction compared with those with only short alleles. The findings, that D4DR exon III and 5-HTT promoter genotypes and trbc-MAO activity are related to human fear conditioning, a basic form of associative learning, are consistent with animal studies suggesting a genetic contribution to fear conditioning. The authors suggest that in humans these genetic mechanisms are partly dopaminergic and serotonergic in origin.
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2003
Semicarbazide-sensitive amine oxidases (SSAO) are widely distributed enzymes, with as yet not ful... more Semicarbazide-sensitive amine oxidases (SSAO) are widely distributed enzymes, with as yet not fully elucidated functions and roles, present in many tissues but also circulating in plasma. The enzyme also functions as an adhesion molecule, the vascular adhesion protein-1. In healthy humans, plasma SSAO activity is constant from birth until 16 years of age, when it drops to lower values, gradually increasing again at advanced ages. When measuring SSAO activity, care should be taken to ensure proper preparation and storage conditions, and it should be realized that quite a few drugs unintentionally are good inhibitors, and sometimes even substrates, of SSAO. Under normal conditions SSAO activity is constant and inter-individual variation is small. In various pathophysiological conditions plasma SSAO activities are increased, most notably in diabetes mellitus (both type I and type II), in congestive heart failure and in cirrhotic liver inflammation. In patients with other vascular and inflammatory diseases plasma SSAO is normal, while it is low in children with congenital lung diseases. Interpretation of these changes is speculative, since source and regulation of plasma SSAO are as yet unknown. However, in two situations where the disease-causing process was ended (transplantation, delivery), plasma SSAO returned to normal. Many questions remain to be answered.
Coprrghl 0 Muirksguard 1994 hCTA NEUROI.OGICh OKAZAWA H. MURATA M, WATANABE M, KAMEI M. KANAZAWA ... more Coprrghl 0 Muirksguard 1994 hCTA NEUROI.OGICh OKAZAWA H. MURATA M, WATANABE M, KAMEI M. KANAZAWA I. Dopaminergic stimulation up-regulates the iri vivv expression of brain-derived neurotrophic factor (BDNF) in the striatum. FEBS Lett, 1992: 313: 138-142.
Uploads
Papers by Jonas Ekblom