Search PubMed⌕ Search

Biomedical subjects

E Rosengren

Publications and source records attributed to E Rosengren.

At least 37 records · Page 2Linked to original sources
PubMed1994

Melanins in IGR 1 melanoma cells.

Information on the composition of melanins is obtained by analysis both of 4-amino-3-hydroxyphenylalanine (AHP) after hydriodic acid degradation and of pyrrole-2,3,5-tricarboxylic acid (PTCA) after potassium permanganate oxidation. Analysis of thiazole-4,5-dicarboxylic acid (TDCA) and pyrrole-2,3-dicarboxylic acid (PDCA) after permanganate oxidation, provides additional information on the composition, TDCA on pheomelanin residues, and PDCA on indolic residues without carboxy groups. Using model melanins formed from dopa and cysteinyldopa in different proportions, we found the TDCA/(PTCA+PDCA) ratio to yield a reliable estimate of the relative proportions of pheomelanin and eumelanin. The PDCA/PTCA ratio reflects the relationship between indole residues with and without carboxy groups. We have analyzed degradation products from cultures of IGR 1, an extensively studied melanoma cell line. Cell cultures were harvested after 2, 4, and 7 days. Culture media were changed after 2 days in all series, and also after 4 days in one series harvested at 7 days. Cells without medium change had seven times the amount of melanin found in cultures with medium change. The PDCA/PTCA ratio decreased with increasing amounts of melanin. With increased melanization, eumelanin is increased relatively more than pheomelanin. The cell content of 5-S-cysteinyldopa (5-S-CD) was similar in all cultures, while 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MICA), a eumelanin precursor metabolite, was found in increased amounts of media of heavily pigmented cultures.

Chromatography, Liquid↗
PubMed1994

The mouse brown (b) locus protein functions as a dopachrome tautomerase.

The mouse b locus controls black/brown coat coloration. Its product, the b-protein or TRP-1, has significant homology to tyrosinase, and this has led to suggestions that the b-protein is itself a melanogenic enzyme. In order to investigate its function, we have used lines of mouse fibroblasts stably expressing the b-protein. We were unable to confirm previous reports that the b-protein has tyrosinase or catalase activity, but detected stereospecific dopachrome tautomerase activity in b-protein-expressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A-Sepharose, and the major product of its action on L-dopachrome is 5,6-dihydroxyindole-2-carboxylic acid, as expected for the mammalian enzyme. Since this activity is not present in untransfected fibroblasts we conclude that the b-protein has dopachrome tautomerase activity. Further supporting evidence comes from the analysis of melanin metabolites produced by fibroblasts expressing tyrosinase alone, or in combination with the b-protein. Culture medium from the line expressing both proteins contains significant amounts of methylated carboxylated indoles, such as 6-hydroxy-5-methoxyindole-2-carboxylic acid, which would be expected in cells with an active dopachrome tautomerase. The levels of these compounds in medium from cells expressing tyrosinase alone are approximately 20-fold lower, and not significantly above background. Hence, it appears that the b-protein acts as a dopachrome tautomerase in vivo as well as in vitro.

3T3 Cells↗
PubMed1993

Isolation of a new tautomerase monitored by the conversion of D-dopachrome to 5,6-dihydroxyindole.

Two membrane bound enzymes which tautomerize L-dopachrome and are specific for the L-isomer of dopachrome have been defined in melanin forming cells. Another enzyme that tautomerizes D-dopachrome with concomitant decarboxylation to give 5,6-dihydroxyindole (DHI) was found in the cytoplasm of human melanoma cells, human liver and in all of the organs studied in rat. The decolorization of D-dopachrome with the formation of DHI was used in monitoring the isolation of a tautomerase from liver of male rats and therefore the enzyme is provisionally called D-dopachrome tautomerase. The molecular weight of D-dopachrome tautomerase monomer was approximately 12 kD and its N-terminal amino acid sequence was P-F-V-E-L-E-T-N-L-P-A-. The Km for D-dopachrome was 1.5 mM and Vmax 0.5 mmol per min and mg protein.

Amino Acid Sequence↗
PubMed1993

Effects of receptor agonists on polyamine concentrations and spermidine/spermine N1-acetyltransferase activity in rat parotid gland.

The polyamine putrescine might be formed via a degradation (catalyzed by spermidine/spermine N1-acetyltransferase, SSAT) of the higher polyamines spermidine and spermine to putrescine. The involvement of different intracellular signal pathways in the regulation of putrescine formation was studied in explants and in cultured cells of rat parotid glands by using receptor agonists that activate separate second messenger systems, and measuring their effects on the concentrations of putrescine, spermidine and spermine and on the SSAT activity. The beta-adrenoceptor agonist isoprenaline, which is an activator of cAMP formation, increased the putrescine concentration and stimulated the SSAT activity. Pilocarpine, a drug that activates the muscarinic receptors and thereby enhances the phosphoinositide turnover, had no effect on either the polyamine concentrations or on the SSAT activity. Epidermal growth factor (EGF), which induces activation of a protein tyrosine kinase, had no effect on the polyamine concentrations or on the SSAT activity. The adenylate cyclase activator forskolin increased the glandular levels of putrescine. Taken together, these findings suggest that increases in putrescine concentration in cultured rat parotid gland cells are accompanied by accumulation of cAMP.

Acetyltransferases↗
PubMed1993

Extracellular gamma-aminobutyric acid levels in the rat caudate-putamen: monitoring the neuronal and glial contribution by intracerebral microdialysis.

Intracerebral microdialysis with high pressure liquid chromatography (HPLC) coupled to electrochemical detection was employed to characterize gamma-aminobutyric acid (GABA) release and the effects induced by a preceding neuron-depleting ibotenic acid (IBO) lesion in the rat caudate-putamen (CPu). Extracellular GABA overflow was monitored in the intact and excitotoxically lesioned CPu, either 7-10 days (acute) or more than 3 months post-lesioning (chronic), using loop type dialysis probes perfused at a rate of 2 microliters/min. In the intact CPuu, basal GABA levels were 0.97 pmol/30 microliters of dialysate in the awake animals and 0.76 pmol/30 microliters under halothane anaesthesia. In both the acute and chronic IBO lesioned CPu the extracellular GABA levels were reduced by 80% and 67%, respectively, under halothane anaesthesia. KCl added to the perfusion fluid at a concentration of 100 mM resulted in dramatic increases in GABA overflow from baseline levels in the intact CPu (60- to 70-fold), which were almost totally abolished (> 95%) in the excitotoxically lesioned CPu. Veratridine administered at 75 microM, produced a 45-fold increase in GABA overflow in the intact CPu, but failed to produce any effect in the lesioned CPu. The addition of nipecotic acid (0.5 mM), a GABA uptake blocker, increased basal extracellular GABA levels 6-15-fold in the intact CPu, while GABA overflow in either the acute or chronic lesioned CPu was not significantly altered. Although Ca(2+)-free conditions (with 20 mM Mg2+ added) or tetrodotoxin (TTX, 1 microM) did not alter the basal GABA overflow in the intact CPU under halothane anaesthesia, the omission of Ca2+ resulted in a 47% reduction in basal extracellular GABA levels in awake, freely moving animals. Nipecotic acid-induced GABA overflow was reduced by 22% under Ca(2+)-free conditions, and by 33% in the presence of 1 microM TTX. Moreover, KCl-evoked GABA overflow was reduced by 86% in Ca(2+)-free conditions and by 40% when administered in the presence of 1 microM TTX. These results indicate that the extracellular GABA levels recorded by intracerebral microdialysis in the CPu are derived predominantly from neuronal sources. Under baseline resting conditions only a small fraction (up to 20-30%) of the neuronal release was Ca(2+)-dependent and TTX-sensitive (i.e. possessing the characteristics of impulse-dependent vesicular release).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Effects of luminal stimuli on polyamine metabolism in the small intestine of the rat: the role of enteric nerves.

The aim of this study was to investigate to what extent polyamine metabolism in the small intestine of the rat is controlled by the enteric nervous system. Polyamine metabolism was followed by measuring the activity of ornithine decarboxylase (ODC) and in some instances also the content of polyamines (putrescine, spermidine and spermine). ODC activity in the intestine was increased when intraluminal pressure was increased and 3 h after placing cholera toxin in the intestinal lumen. Cholera toxin also increased the tissue putrescine content. Atropine or hexamethonium given i.v. did not influence the evoked changes of ODC activity. The pressure induced changes were not decreased by placing lidocaine on the serosal surface. On the other hand, the ODC activity of control segments were decreased by hexamethonium or atropine. The presence of glucose in the intestinal perfusate did not augment tissue ODC activity, neither did the heat stable enterotoxin from Escherichia coli (STa). It is concluded that the effect on polyamine metabolism evoked by luminal pressure or cholera toxin seems not to be mediated via nerves, while nerves seem to influence ODC activity during control conditions. The experiments with enterotoxins suggest that cAMP is the intracellular second messenger controlling intestinal ODC activity.

Animals↗
PubMed1993

Fibroblasts expressing mouse c locus tyrosinase produce an authentic enzyme and synthesize phaeomelanin.

Recent advances in the study of the molecular biology of mouse pigmentation have led to the discovery of a family of proteins involved in the control of melanin synthesis. It has been confirmed that the product of the mouse c (albino) locus is the key melanogenic enzyme tyrosinase, but study of its function and regulation have been hampered by the presence of closely related proteins within melanin-synthesising cells. To overcome these problems, we have established lines of mouse fibroblasts expressing the c locus mouse tyrosinase. Here we describe characterisation of the tyrosinase synthesised by these cells and demonstrate considerable similarity between the expressed tyrosinase and the native enzyme. The expressed tyrosinase is proteolytically cleaved to produce membrane-bound and soluble forms of the expected molecular mass and is rich in N-linked carbohydrate, suggesting that melanocytic differentiation is not a prerequisite for post-translational modification of the protein. The expressed enzyme has tyrosinase activity, but not catalase or dopachrome tautomerase activity, confirming that it is an authentic tyrosinase. Transfected fibroblasts expressing tyrosinase are shown to share several physiological characteristics with melanoma cell lines, including increased pigmentation and tyrosinase activity in response to increased cell density. Since tyrosinase is expressed under a heterologous promoter, these shared characteristics probably reflect translational or post-translational controls that operate in both non-melanocytic and melanocytic cell types. We demonstrate that pigmented fibroblasts contain the melanin synthesis intermediates 5-S-cysteinyldopa and 5-S-glutathionyl-dopa, and produce a phaeomelanin-like pigment, but do not contain detectable eumelanin. Expression of tyrosine is therefore sufficient for the synthesis of a form of melanin pigment in fibroblasts.

3T3 Cells↗
PubMed1993

The mouse brown (b) locus protein has dopachrome tautomerase activity and is located in lysosomes in transfected fibroblasts.

Many genes mapping to pigmentation loci are involved in the regulation of melanin synthesis in the mouse. The brown (b) locus controls black/brown coat coloration, and its product has significant homology to the key melanogenic enzyme tyrosinase. This has led to suggestions that the b-protein is itself a melanogenic enzyme. In order to investigate its function, we have established lines of mouse fibroblasts stably expressing the b-protein by co-transfection of a b-protein expression vector and a plasmid conferring resistance to the antibiotic G418. The b-protein synthesised by these cells has the expected molecular mass of 75 kDa and reacts with three different anti-b-protein antibodies. We were unable to confirm previous reports that the b-protein has tyrosinase or catalase activity, but detected stereospecific dopachrome tautomerase activity in b-protein-expressing fibroblasts. This dopachrome tautomerase binds to Concanavalin A-Sepharose, and the major product of its action on L-dopachrome is 5,6-dihydroxyindole-2-carboxylic acid. Since this activity is not present in untransfected cells we conclude that the b-protein has dopachrome tautomerase activity. Fibroblasts do not contain melanosomes, the specialised organelles in which the b-protein is located in melanocytes. Nevertheless, indirect immunofluorescence localisation of the b-protein in transfected fibroblasts produces a distinctive pattern of intense juxtanuclear staining combined with punctate cytoplasmic staining. Double-labelling shows co-localisation of the b-protein with the late endosomal/lysosomal markers beta-glucuronidase and LAMP-1, both in transfected fibroblasts and in mouse melanoma cells. These findings are consistent with the hypothesis that melanosomes are closely related to lysosomes.

3T3 Cells↗
PubMed1993

Hydrogen peroxide as an inducer of elevated tyrosinase level in melanoma cells.

The effects of systems generating active oxygen species (superoxide anion, hydrogen peroxide, hydroxyl radical) on tyrosinase have been studied in cultured human melanoma cells. Tyrosinase activity was determined by measuring the quantity of 5-S-L-cysteinyl-L-dopa (5-S-CD) formed in the presence of D,L-dopa and L-cysteine. In some experiments, the enzyme protein was determined by radio immunoassay [RIA]. Exposure of cells to xanthine/xanthine oxidase or glucose/glucose oxidase resulted in a dose-related elevation of tyrosinase. Catalase, but not superoxide dismutase, prevented this increase indicating that hydrogen peroxide may be the agent responsible for the action, whereas superoxide anion is not involved. Hydroxyl radicals formed by the Haber-Weiss or Fenton type reactions were not found to produce elevation of tyrosinase. Catalase determinations showed no enzyme in the medium but a high concentration in the cells. Inhibition of intracellular catalase by 3-amino-1,2,4-triazole caused an increase in the tyrosinase level. The effects of dopac, xanthine/xanthine oxidase, and glucose/glucose oxidase all producing hydrogen peroxide, and increasing tyrosinase, were enhanced by the inhibition of catalase. It is concluded that hydrogen peroxide, formed by the systems, accounts for the elevation of tyrosinase level. When tyrosinase activities determined by 5-S-CD formation were compared to enzyme amounts found by RIA, the ratios of these values were always constant. This fact indicates that the increase in the tyrosinase activities was not due to an activation of the enzyme, but mirrored the quantities of enzyme protein present in the samples. On the basis of our findings, it is assumed that hydrogen peroxide is a regulator of tyrosinase in normal melanocytes and melanoma cells.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1992

The role of Mg2+ on the formation of the ternary complex between agonist, beta-adrenoceptor, and GS-protein and an interpretation of high and low affinity binding of beta-adrenoceptor agonists.

In the absence of GTP or its analogues, the beta-adrenoceptor agonist (H), the receptor (R) and the stimulatory guanine nucleotide-binding regulatory protein (GS-protein) form a slowly dissociable complex (HRGSGDP). It is presumed that the agonist in this ternary complex is bound to the receptor with high affinity while its binding to the receptor in the HR-complex is of low affinity. We have studied the effect of Mg2+ on the formation of these two complexes by the displacement of (-)-[3H]CGP-12177 with (-)-isoprenaline in cell membranes prepared from guinea-pig lung parenchyma. In the absence of Mg2+, only monophasic displacement curves were obtained with a dissociation constant not differing from that obtained in the presence of Gpp(NH)p and Mg2+. In the presence of Mg2+ and absence of Gpp(NH)p, a shallow binding curve was obtained that fitted a two site model best. One of the dissociation constants agreed well with that obtained in the presence of Gpp(NH)p and Mg2+ but the other one was 27 times smaller. It was concluded that Mg2+ is necessary for the coupling between the HR-complex and the GS-protein. The two dissociation constants obtained in the absence of Gpp(NH)p represent the dissociation constants of the HR- and HRGSGDP-complexes and may correspond to those named KL and KH in the literature. According to our study, there may be no difference in affinity between the agonist and receptor in the HR- and HRGSGDP-complexes. The two dissociation constants obtained in the absence of GTP or its analogues refer to two consecutive reactions.

Adrenergic beta-Agonists↗
PubMed1991

The neuromelanin of the human substantia nigra.

The pigment of the human substantia nigra was isolated after extraction of lipids and proteins with 2% sodium cholate in 30% ethanol followed by 2% sodium dodecyl sulfate in 10% glycerol. The pigment was hydrolysed with HI or degraded by treatment with KMNO4 and the samples were examined for compounds known to derive from pheomelanin (4-amino-3-hydroxyphenylalanine, AHP and 4-amino-3-hydroxyphenylethylamine, AHPEA), or from eumelanin (pyrrole-2,3,5-tricarboxylic acid, PTCA). The HI hydrolysis yielded AHPEA in large quantities, indicating cysteinyldopamine as the main source of the pheomelanin moiety of the neuromelanin, but also trace amounts of AHP, derived from cysteinyldopa oxidation products. Dopamine and small quantities of dopa were also obtained by HI hydrolysis of the neuromelanin. The yield of PTCA was low, but the amounts observed show that part of the neuromelanin is of the eumelanin type, a fact compatible with an occasional exhaustion of the glutathione-cysteine reduction system at the site of neuromelanin formation.

Chromatography, High Pressure Liquid↗
PubMed1991

Hydrogen peroxide as a mediator of dopac-induced effects on melanoma cells.

Dopac increases tyrosinase activity and exerts cytotoxic effects in cultures of human melanoma cells. The possible role of hydrogen peroxide in these actions was examined. Catalase (100 micrograms/ml) completely reversed the cytotoxic action of 0.3 mM dopac and reduced its tyrosinase-stimulating effect by approximately one half. The results show that extracellular hydrogen peroxide is a mediator of both the tyrosinase-stimulating and cytotoxic actions of dopac. Analysis of the degradation products of melanin from dopac-treated melanoma cells after hydriodic acid (HI) hydrolysis revealed the presence of aminohydroxy-phenylacetic acid (AHPAc). This substance is obtained by HI hydrolysis of melanin formed by oxidation of cysteinyl-dopac. Thus, the presence of AHPAc indicates that dopac is transported into the melanocytes where it serves as a substrate for tyrosinase.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1991

Effects of stimulation of the parasympathetic and sympathetic innervations in bursts on the syntheses of polyamines, DNA and protein in salivary glands of the rat: non-adrenergic, non-cholinergic responses.

The increase in the activity of ornithine decarboxylase, the key enzyme in the formation of polyamines, in the sublingual glands of the chloralose-anaesthetized rat in response to stimulation of the parasympathetic innervation was enhanced by employing an intermittent pattern of nerve stimulation. 20 Hz in bursts delivered for 1 second every 10th second over a period of 3 hours increased the activity 1000-fold, whereas a frequency of 2 Hz delivered continuously, and thus giving rise to the same total number of impulses as the burst stimulation, increased the enzyme activity only 50-fold. Already 2 Hz in bursts caused the ornithine decarboxylase activity to increase. A parasympathetic non-adrenergic, non-cholinergic regulation of ornithine decarboxylase activity was shown in response to the two modes of stimulation. The increase to 2 and 20 Hz in bursts was 2- and 80-fold, respectively, whereas it was 29-fold to 2 Hz continuously. Increases in the concentration of the polyamine putrescine in response to burst stimulation of the parasympathetic nerve were found both in the absence and in the presence of atropine. The ornithine decarboxylase activity of the submaxillary gland was not affected by the parasympathetic stimulation. Stimulation of the sympathetic innervation was more effective in both types of glands, as to increases in the ornithine decarboxylase activity, when employing the continuous mode. In cultured tissue of the sublingual glands which, in vivo, had been subjected to parasympathetic stimulation in bursts (20 Hz), the syntheses of DNA and protein were increased as judged by the incorporation of radio-labelled thymidine and leucine; in the presence of atropine the stimulation resulted only in increased synthesis of protein.

Animals↗
PubMed1991

The role of the antrum and the vagus nerve in the metabolism of histamine in the human gastric mucosa.

The effects of antrectomy and proximal gastric vagotomy on the metabolism of histamine in the human gastric mucosa were studied in the basal state and during pentagastrin stimulation in patients with duodenal or gastric ulcer disease. Mucosal biopsy specimens were taken from the antral and oxyntic gland areas, whereafter histamine content, histidine decarboxylase activity, and histamine methyltransferase activity were simultaneously assayed. Vagotomy was followed by a decrease in the acid secretory capacity and an increase in basal serum gastrin levels. Histamine content of the oxyntic mucosa increased after vagotomy, but the ability of pentagastrin to form new amounts of the amine was impaired. Antrectomy caused a decrease in acid secretion and a fall in gastrin concentrations. Basal histamine content and rate of amine formation in the remaining oxyntic mucosa were unaffected by antrectomy. Antrectomy impaired the ability of pentagastrin to release histamine. Histamine methyltransferase was not affected by pentagastrin, vagotomy, or antrectomy. In conclusion, both antral gastrin and the vagus nerve seem to exert a regulatory influence on the metabolism of histamine in the human oxyntic mucosa. The withdrawal of these factors either causes impaired ability of pentagastrin to release histamine from its storage site or counteracts the ability of pentagastrin to accelerate histamine synthesis.

Analysis of Variance↗
PubMed1991

A sensitive tyrosinase method for human skin.

A sensitive competitive radioimmunoassay for quantitative analysis of tyrosinase in biological tissues has been developed. The binding sites of antityrosinase antibodies were competed for by 125I-labelled human tyrosinase and a known (standard) or unknown (sample) amount of tyrosinase in solution. A sensitivity range of about 5-500 pmol/l was obtained when a solubilized melanoma tyrosinase with a molecular weight of 66,000 D was used as a standard. The method was used on 0.5 cm2 skin samples from which tyrosinase was solubilized with Nonidet P-40. The method is useful for measuring the content of tyrosinase in crude samples where the enzyme activity may be affected by inhibitors or by unspecific oxidation.

Animals↗
PubMed1990

The neuropeptides vasoactive intestinal polypeptide, peptide histidine isoleucine and neuropeptide Y modulate [3H]noradrenaline release from sympathetic nerves in the choroid plexus.

The neurotransmitter peptides vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI) and neuropeptide Y (NPY) are located in nerve fibers supplying the pig choroid plexus, which receives an abundant sympathetic innervation. We characterized the release of [3H]noradrenaline ([3H]NA) from this tissue elicited by electrical field stimulation and studied the effects of the above-mentioned peptides on this release. The release of [3H]NA was found to be almost exclusively of neurogenic origin, despite there being a marked non-neuronal uptake of [3H]NA into the epithelium of the choroid plexus. NPY significantly decreased the release of [3H]NA by approximately 10% while VIP and PHI enhanced release by up to 25 and 35%, respectively, indicating a possible synergistic role of the two latter peptides and NA in the choroid plexus.

Animals↗
PubMed1990

Histamine metabolism in human gastric mucosa. Effect of pentagastrin stimulation.

The metabolism of histamine in the human gastric mucosa was studied in the basal state and during pentagastrin stimulation. Studies were made in healthy volunteers and in patients with peptic ulcer disease. Mucosal biopsies were taken from antral and oxyntic gland areas whereupon histamine content, histidine decarboxylase activity, and histamine methyltransferase activity were simultaneously assayed. Histamine content of the oxyntic gland mucosa was decreased as a consequence of pentagastrin administration in all groups studied, and this decrease was numerically largest in patients with duodenal ulcer disease. Pentagastrin induced a significant increase in histidine decarboxylase activity of the oxyntic gland mucosa with the most profound increase seen in patients with duodenal ulcer. The highest rates of histamine formation were present in the oxyntic mucosa of patients with Zollinger-Ellison syndrome. The activity of histamine methyltransferase was the same in all groups studied and was not changed by pentagastrin. In conclusion, pentagastrin administration in humans is followed by a significant mobilization of histamine only from the oxyntic gland mucosa, an effect that is more pronounced in patients with duodenal ulcer disease.

Adult↗