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E Rosengren

Publications and source records attributed to E Rosengren.

At least 19 recordsLinked to original sources
PubMed2000

Dissection of the enzymatic and immunologic functions of macrophage migration inhibitory factor. Full immunologic activity of N-terminally truncated mutants.

Macrophage migration inhibitory factor (MIF) is a cytokine with broad regulatory functions in innate immunity. MIF belongs to the few cytokines displaying catalytic activities, i.e. MIF has a Pro2-dependent tautomerase and a Cys-Ala-Leu-Cys (CALC) cysteine-based thiol-protein oxidoreductase activity. Previous studies have addressed the roles of the catalytic site residues and the C-terminus. The two activities have not been directly compared. Here we report on the N-terminal mutational analysis and minimization of MIF and on a dissection of the two catalytic activities by comparing mutants P2AMIF, Delta4MIF, Delta5MIF, Delta6MIF, Delta7MIF, Delta8MIF, and Delta10MIF with the cysteine mutants of MIF. As N-terminal deletion was predicted to interfere with protein structure due to disruption of the central beta sheet, it was surprising that deletion of up to six N-terminal residues resulted in normally expressed proteins with wild-type conformation. Strikingly, such mutants exhibited full MIF-specific immunologic activity. While mutation of Pro2 eliminated tautomerase activity, the CALC cysteine residues had no influence on this activity. However, mutant C81SMIF, which otherwise has full biologic activity, only had 32% tautomerase activity. Deletion of four N-terminal residues did not interfere with insulin reduction by MIF. By contrast, reduction of 2-hydroxyethyldisulfide (HED) was markedly affected by N-terminal manipulation, with P2AMIF and Delta2MIF exhibiting 40% activity, and Delta4MIF completely failing to reduce HED. This study constitutes the first comparison of the two catalytic activities of MIF and should assist in understanding the molecular links between the catalytic and immunologic activities of this cytokine and in providing guidelines for N-terminal protein minimization.

Amino Acid Sequence↗
PubMed1999

Inhibition of serum diamine oxidase discloses a constitutive putrescine release from cultured vascular smooth muscle cells.

OBJECTIVE: To determine if putrescine and the higher polyamines spermidine and spermine are released from cultured vascular smooth muscle cells. MATERIAL: Vascular smooth muscle cell line A7r5. TREATMENT: Cells were treated with aminoguanidine (10 or 100 microM) for 1 to 24 h with or without fetal calf serum (10%) present in the culture medium. METHODS: Cellular and medium concentrations of polyamines were determined by liquid chromatography. Total cellular protein was determined by the Bradford procedure. Student's two-tailed t-test was used for statistical calculations. RESULTS: A constitutive release of putrescine was disclosed within 5 h if serum diamine oxidase was inhibited by 10 microM aminoguanidine. The release was linear with time for 24 h and specific for putrescine in the sense that the higher polyamines spermidine and spermine, despite similar cellular concentrations, were not released. Similar amounts of putrescine were released from the smooth muscle cells whether or not culture medium contained serum. Cells, that had been cultured in medium from which fetal calf serum had been omitted for last 48 h, contained less putrescine, spermidine and protein than those that persisted in medium that contained serum. CONCLUSION: A constitutive putrescine release from vascular smooth muscle cells is disclosed in the presence of aminoguanidine.

Amine Oxidase (Copper-Containing)↗
PubMed1997

The macrophage migration inhibitory factor MIF is a phenylpyruvate tautomerase.

A macrophage migration inhibitory factor (MIF), originally described as a product of activated lymphocytes, has been defined as a 12 kDa protein, expressed in a wide variety of tissues. Here MIF is identified as a phenylpyruvate tautomerase (EC 5.3.2.1) having p-hydroxyphenylpyruvate and phenylpyruvate as its natural substrates. The definition of MIF as an enzyme may yield insight into the mechanism of action of this proinflammatory and immunomodulating cytokine.

Amino Acids↗
PubMed1997

Effects of aminoguanidine and L-NAME on histamine-induced blood pressure drop in the rat.

Mean arterial blood pressure changes in response to i.v. administration of histamine were monitored in the anaesthetized rat in the absence or presence of the diamine oxidase (DAO) inhibitor aminoguanidine (AMG, 10 mg kg-1). AMG prolonged the duration of the transient drop in blood pressure induced by a bolus injection of histamine (0.05 mg kg-1) by 34%. In animals pretreated with AMG, no potentiation of the decrease in pressure in response to a 10 min infusion of histamine was observed. However, when infusion was stopped, the time needed for pressure recovery was twice as long in animals treated with AMG as in controls. Blood samples were taken prior to infusion and during the recovery phase and the quantities of histamine were determined by liquid chromatography. The prolonged recovery phase observed in animals pretreated with AMG was associated with five times higher levels of histamine. The duration of histamine-induced hypotension (0.01 mg kg-1) was 50% shorter in the presence of the nitric oxide synthase inhibitor L-NAME (10 mg kg-1). We suggest that DAO, through elimination of histamine from the bloodstream, is important for the recovery from histamine-induced hypotension, and that the duration of histamine-induced pressure drop is influenced by formation of nitric oxide.

Amine Oxidase (Copper-Containing)↗
PubMed1996

Inhibition of diamine oxidase promotes uptake of putrescine from rat small intestine.

In blood from the portal vein of anaesthetized rats the levels of histamine and putrescine were 2-3-fold lower compared to arterial blood. Putrescine concentration was increased severalfold and the difference between portal and arterial blood abolished in animals pretreated with the specific diamine oxidase inhibitor aminoguanidine. Histamine concentration was 40% lower in portal compared to arterial blood in animals treated with the mast cell degranulator compound 48/80. In animals pretreated with aminoguanidine, compound 48/80 enhanced the level of histamine and no difference was observed between arterial and portal blood. The amounts of intravenously injected [14C]-labeled histamine was about 15% lower in portal compared to arterial blood. The uptake of [14C]-putrescine from the small intestine was estimated. In urine from animals pretreated with aminoguanidine the concentration of [14C]-putrescine was more than 40-times higher than in control animals corresponding to a calculated uptake of about 7% in aminoguanidine treated animals. Our results suggest that intestinal diamine oxidase clears the blood from diamines and prevents luminal uptake of putrescine.

Amine Oxidase (Copper-Containing)↗
PubMed1996

Enzymatic and non-enzymatic oxygenation of tyrosine.

Tyrosinase isolated from cultured human melanoma cells was studied for tyrosine oxygenation activity. L-Tyrosine and D-tyrosine were used as substrates and dopa was measured with HPLC and electrochemical detection as the product of oxygenation. Incubations were performed in the presence or absence of dopamine as co-substrate. Oxygenation of L-tyrosine occurred only in the presence of dopamine as co-substrate. No oxygenation of D-tyrosine was found, and we conclude that human tyrosinase is characterised by exclusive specificity for the L-isomer of tyrosine in its oxygenase function. It has recently been suggested that superoxide anion is a preferential oxygen substrate for human tyrosinase. Incubations were therefore performed with L- and D-tyrosine, human tyrosine, and xanthine/xanthine oxidase in the system, generating superoxide anion and hydrogen peroxide. Considerable formation of dopa was observed, but the quantity was the same irrespective of whether D-tyrosine or L-tyrosine was used as the substrate. Furthermore, formation of dopa occurred in a xanthine/xanthine oxidase system when bovine serum albumin (BSA) was substituted for tyrosinase. Our results provide no evidence that superoxide anion is an oxygen substrate for human tyrosinase. In the incubate containing xanthine/xanthine oxidase, catalase completely inhibited dopa formation, and superoxide dismutase and mannitol each strongly inhibited dopa formation. The results are compatible with hydroxyl radicals being responsible for the formation of dopa, since such radicals may be secondarily formed in the presence of superoxide anion and hydrogen peroxide.

Animals↗
PubMed1996

Comparison between activation of ornithine decarboxylase and histidine decarboxylase in rat stomach.

We compared the responses of rat stomach ornithine decarboxylase (ODC) and histidine decarboxylase (HDC) to food intake, oral treatment with antisecretagogues, NaHCO3, and hypertonic NaCl, antrectomy, intravenous infusion of gastrin-17, the selective cholecystokinin (CCK)-B/gastrin receptor antagonist L-365,260, and the somatostatin analogue RC-160. The serum gastrin concentration and oxyntic mucosal ODC and HDC activities were higher in freely fed rats than in fasted rats. Food intake in fasted rats raised the serum gastrin concentration and the ODC and HDC activities. Ranitidine, omeprazole, and NaHCO3 raised the serum gastrin concentration and activated ODC and HDC. Hypertonic NaCl raised the ODC activity 200-fold, whereas circulating gastrin and HDC activity were increased only moderately. Infusion of gastrin-17 activated HDC but not ODC. L-365,260 prevented the activation of HDC but not of ODC in response to food intake and treatment with omeprazole, NaHCO3, or hypertonic NaCl. Antrectomy prevented the food- and omeprazole-evoked rise in oxyntic mucosal HDC activity but not the rise in ODC activity. RC-160 suppressed HDC activity after food intake and treatment with omeprazole, NaHCO3, or NaCl. In contrast, RC-160 suppressed omeprazole- and NaHCO3-evoked ODC activation but not that evoked by food intake or NaCl. The results support the view that HDC in the oxyntic mucosa is activated by gastrin and suppressed by somatostatin. The induction of ODC is not mediated by gastrin; ODC activation appears to be related to acid inhibition per se or to mucosal maintenance and repair; somatostatin, or rather the lack of it, might contribute to the induction of ODC after acid blockade. The mechanism behind the activation of rat stomach ODC seems to differ depending on the type of stimulus.

Analgesics↗
PubMed1996

The immunoregulatory mediator macrophage migration inhibitory factor (MIF) catalyzes a tautomerization reaction.

BACKGROUND: Recent studies of melanin biosynthesis have uncovered an unusual enzymatic activity which converts the non-naturally occurring D-isomer of 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) into 5,6-dihydroxyindole-2-carboxylic acid (DHICA). The aim of the present investigation was to isolate and characterize the enzyme catalyzing this tautomerization reaction. MATERIALS AND METHODS: After we performed a tissue survey of D-dopachrome tautomerase activity, 10 bovine lenses were homogenized and used as a source of enzyme. A soluble fraction was obtained by high-speed centrifugation and subjected to successive FPLC chromatography on Phenyl-sepharose, Mono S cation-exchange, and Superdex gel-filtration. The isolated enzyme was electrophoresed, blotted onto PVDF membrane, and the N terminus analyzed by gas phase micro-sequencing. RESULTS: The protein catalyzing the conversion of D-dopachrome to DHICA was purified to homogeneity in 14% yield and showed a molecular weight of 12 kD when analyzed by SDS-PAGE. The first 27 amino acid residues of this protein were sequenced and found to be identical with those of bovine macrophage migration inhibitory factor (MIF). The catalytic activity of native MIF was confirmed by studies of purified recombinant human MIF, which showed the same tautomerase activity. While L-dopachrome was not a substrate for this reaction, the methyl esters of the L- and D-isomers were found to be better substrates for MIF than D-dopachrome. CONCLUSIONS: MIF has been described recently to be an anterior pituitary hormone and to be released from immune cells stimulated by low concentrations of glucocorticoids. Once secreted, MIF acts to control, or counter-regulate, the immunosuppressive effects of glucocorticoids on the immune system. Although the tested substrate, D-dopachrome, does not occur naturally, the observation that MIF has tautomerase activity suggests that MIF may mediate its biological effects by an enzymatic reaction. These data also offer a potential approach for the design of small molecule pharmacological inhibitors of MIF that may modulate its potent immunoregulatory effects in vivo.

Amino Acid Sequence↗
PubMed1995

Cloning and sequencing of a cDNA encoding rat D-dopachrome tautomerase.

An enzyme which converts D-dopachrome into 5,6-dihydroxyindole has recently been isolated from rat liver. Enzymatic D-dopachrome conversion has been observed in extracts from all tissues examined of several species, including man. We have now cloned and sequenced a 628 bp long cDNA encoding the enzyme provisionally called D-dopachrome tautomerase. The cDNA was isolated by 3' and 5' rapid amplification and cloning of cDNA ends (RACE) from rat liver cells using degenerate oligonucleotide primers, deduced from the N-terminal peptide sequence of D-dopachrome tautomerase. The cDNA contains an open reading frame encoding 118 amino acids. Edman degradation of intact and of trypsin degraded D-dopachrome tautomerase fragments gave information on and corroborated 67% of the deduced protein sequence. A homology search in the EST database found a human cDNA encoding a peptide sharing 66% homology with the rat enzyme. The rat D-dopachrome tautomerase shares 27% homology with the rat macrophage migration inhibitory factor (MIF).

Amino Acid Sequence↗
PubMed1995

Fibroblasts co-expressing tyrosinase and the b-protein synthesize both eumelanin and phaeomelanin.

Melanin synthesis in the mouse involves the interaction of many pigmentation loci. Tyrosinase, the product of the albino (c) locus, catalyses the first step of the pathway. The brown (b) locus protein has significant homology to tyrosinase and controls black/brown coat coloration, but its function is controversial. To investigate the function of the b-protein and its interaction with tyrosinase, we established cell lines expressing both tyrosinase and the b-protein by transfecting tyrosinase-expressing fibroblasts with a b-protein expression vector. The tyrosinase-expressing parent line does not have L-dopachrome tautomerase activity, but this enzyme is detectable in double transfectants as well as in fibroblasts expressing the b-protein alone. Cells expressing both proteins have a higher steady-state level of tyrosinase than fibroblasts expressing tyrosinase alone, and contain elevated levels of melanin intermediates. This is thought to result from interaction of tyrosinase with the b-protein. Only phaeomelanin is detectable in fibroblasts expressing tyrosinase alone, whereas double transfectants synthesise significantly more phaeomelanin and detectable eumelanin.

3T3 Cells↗
PubMed1995

Tyrosinase activity in the serum of patients with malignant melanoma.

We have recently described a new method for measurement of tyrosinase activity in small amounts of human serum (100 microliters), where the purification of tyrosinase is obtained by adsorption of the enzyme to concanavalin A sepharose. The method, which measures stereospecific dopa oxidation, was used in the winter of 1992-93 for the measurement of activity in serum obtained from 30 healthy subjects and from 10 patients with melanoma metastases. The serum tyrosinase values in the 30 subjects ranged from 0.1 to 1.0 nkatal/l, and the mean value and standard deviation was 0.4 +/- 0.2 nkatal/l. In the 10 patients with melanoma, the tyrosinase serum values ranged from 1.1 to 10.6 nkal/l, and the mean value was 3.1 nkatal/l.

Adult↗
PubMed1995

Seasonal variation of tyrosinase activity in serum.

Serum samples for the analysis of tyrosinase activity were obtained from 10 healthy subjects in autumn, winter and summer. Tyrosinase was purified from 100 microl serum by adsorption to concanavalin A sepharose, the tyrosinase adsorbed to the gel being separated from other components by centrifugation. The gel was suspended in a buffer containing 5-hydroxy-indole-3-acetic acid as an antioxidant and incubated for 2 min with L-cysteine and D-L-dopa at 37 degrees C. The 5-S-L-cysteinyl-L-dopa formed was measured by HPLC and electrochemical detection. Tyrosinase has high stereo-specificity for the L-enantiomer of dopa, and correction for non-specific oxidation was made by simultaneous measurement of 5-S-L-cysteinyl-D-dopa formed from D-dopa. Whereas the oxidation of L-dopa catalysed by tyrosinase ws inhibited by L-tyrosine, the non-specific oxidation of D-dopa was not. Mean serum tyrosinase activity was 0.9 nkatal/l in summer, 0.8 nkatal/l in autumn and 0.4 nkatal/l in winter. The range of tyrosinase activity was much higher in summer and autumn than in winter.

Chromatography, High Pressure Liquid↗
PubMed1994

Amphetamine induces excess release of striatal acetylcholine in vivo that is independent of nigrostriatal dopamine.

The effect of amphetamine on striatal acetylcholine (ACh) release was studied by an in vivo intrastriatal microdialysis technique. Although we expected systemic amphetamine to inhibit baseline striatal ACh release, the opposite was found. In addition, we found that the amphetamine-induced striatal ACh release did not depend on nigrostriatal DA since 6-hydroxydopamine (6-OHDA) lesions had no effect on amphetamine-induced ACh release. Local intrastriatal injection of amphetamine via the microdialysis probe had no effect on striatal ACh release even when the probe was located more laterally in striatum to take advantage of the medial to lateral gradient of striatal ACh and D2 receptors. The hypothesis that amphetamine increased extracellular striatal ACh by increasing the release of biogenic amines besides dopamine was tested by pharmacological manipulations designed to specifically increase local striatal norepinephrine or serotonin levels. The serotonergic and noradrenergic manipulations had no effect on striatal ACh levels. These results indicate that amphetamine-induced release of ACh in striatum is mediated via distal brain regions that are functionally connected with the striatum.

Acetylcholine↗
PubMed1994

Neuromelanin of the human substantia nigra: a mixed-type melanin.

Model melanins, synthesized with different cysteinyldopamine/dopamine ratios in the incubates, were oxidized with KMnO4 and the resulting compounds were analyzed by HPLC. The ratios between a phaeomelanin-derived compound, thiazole-4,5-dicarboxylic acid (TDCA), and a compound derived from eumelanin, pyrrole-2,3,5-tricarboxylic acid (PTCA), reflected the composition of the model melanins. The neuromelanin of the human substantia nigra was isolated, and the pigment, as well as intact brain tissue from human substantia nigra was oxidized with KMnO4 and the TDCA/PTCA ratios were determined. Analysis of the isolated neuromelanin showed it to contain 2.3% sulfur and 8.1% nitrogen. The sulfur content indicates the pigment is a mixed-type melanin, and the TDCA/PTCA ratio indicates that it consists of units derived from benzothiazines and from indoles in about equal amounts.

Autopsy↗