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T Nilsson

Publications and source records attributed to T Nilsson.

At least 163 records · Page 9Linked to original sources
PubMed1993

Enhanced stimulus-secretion coupling in polyamine-depleted rat insulinoma cells. An effect involving increased cytoplasmic Ca2+, inositol phosphate generation, and phorbol ester sensitivity.

To extend previous observations on the role of polyamines in insulin production, metabolism, and replication of insulin-secreting pancreatic beta cells, we have studied the role of polyamines in the regulation of the stimulus-secretion coupling of clonal rat insulinoma cells (RINm5F). For this purpose, RINm5F cells were partially depleted in their polyamine contents by use of the specific ornithine decarboxylase inhibitor difluoromethylornithine (DFMO), which led to an increase in cellular insulin and ATP contents. Analysis of different parts of the signal transduction pathway revealed that insulin secretion and the increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) after K(+)-induced depolarization were markedly enhanced in DFMO-treated cells. These effects were paralleled by increased voltage-activated Ca2+ currents, as judged by whole-cell patch-clamp analysis, probably reflecting increased channel activity rather than elevated number of channels per cell. DFMO treatment also rendered phospholipase C in these cells more sensitive to the muscarinic receptor agonist carbamylcholine, as evidenced by enhanced generation of inositol phosphates, increase in [Ca2+]i and insulin secretion, despite an unaltered ligand binding to muscarinic receptors and lack of effect on protein kinase C activity. In addition, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate, at concentrations suggested to be specific for protein kinase C activation, evoked an increased insulin output in polyamine-deprived cells compared to control cells. The stimulatory effects of glucose or the cyclic AMP raising agent theophylline on insulin release were not increased by DFMO treatment. In spite of increased binding of sulfonylurea in DFMO-treated cells, there was no secretory response or altered increase in [Ca2+]i in response to the drug in these cells. It is concluded that partial polyamine depletion sensitizes the stimulus-secretion coupling at multiple levels in the insulinoma cells, including increased voltage-dependent Ca2+ influx and enhanced responsiveness to activators of phospholipase C and protein kinase C. In their entirety, our present results indicate that the behavior of the stimulus-secretion coupling of polyamine-depleted RINm5F insulinoma cells changes towards that of native beta cells, thus improving the usefulness of this cell line for studies of beta cell insulin secretion.

Adenosine Triphosphate↗
PubMed1993

Mitotic Golgi clusters are not tubular endosomes.

HeLa cells were incubated with 15 nm BSA-gold for 1 or 2 hours to mark the endocytic pathway and mitotic cells were then isolated by shake-off. Thin, frozen sections were labelled with antibodies against two resident Golgi markers, beta-(1,4)-galactosyltransferase and N-acetylglucosaminyltransferase I. Detection of the latter was aided by the use of a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. The secondary antibodies were coupled to either 5 or 10 nm gold so that the distribution of each of the three markers could be followed. Qualitative and quantitative studies showed that there were two populations of clusters, those described by us earlier and termed Golgi clusters (Lucocq et al. (1987) J. Cell Biol. 104, 865-874), containing either or both Golgi markers, and clusters of tubular endosomes containing BSA-gold. There was very little overlap showing that Golgi clusters cannot be tubular endosomes as concluded by Tooze and Hollinshead (1992) Eur. J. Cell Biol. 58, 228-242.

Antibodies, Monoclonal↗
PubMed1992

Proton transfer during the reaction between fully reduced cytochrome c oxidase and dioxygen: pH and deuterium isotope effects.

The pH dependence of proton uptake and electron transfers during the reaction between fully reduced cytochrome c oxidase and oxygen has been studied using the flow-flash method. Proton uptake was monitored using different pH indicators. We have also investigated the effect of D2O on the electron-transfer reactions. Proton uptake was biphasic throughout the pH range studied (6.3-9.3), and the decrease of the observed rate constants at increasing pH could be described by titration curves with pKa values of 8-8.5. Of the four phases resolved in the redox reaction, the rate constants for the first two were independent of pH, whereas that of the third decreased at increasing pH with a pKa of 7.9. All phases except the first were slower in D2O than in H2O. The values obtained for kH/kD were 1.0 for the first phase, 1.4 for the second and third phases, and 2.5 for the fourth phase. We suggest from these results that the fast phase of proton uptake is initiated by the second phase of the redox reaction and that this step includes a partially rate-limiting internal proton transfer. The third and fourth phases of the redox reaction are suggested to be rate limited by proton uptake from the medium. The pH dependencies of the proton uptake reactions are consistent with the participation of a titrable group in the protein in proton transfer from the medium to the oxygen-binding site.

Animals↗
PubMed1992

Photoinduced electron transfer from tris(2,2'-bipyridyl)ruthenium to cytochrome c oxidase.

Flash photolysis has been used to effect electron transfer from tris(2,2'-bipyridyl)ruthenium(II) to cytochrome c oxidase in the presence of a sacrificial electron donor, aniline. The observation that photoreduction occurs only at low ionic strength and high pH indicates that an electrostatic complex between the ruthenium compound and the enzyme is the reactive species. The reaction was followed at 830, 605, and 445 nm. The initial absorbance changes observed suggest that the copper ion CuA is the preferred electron acceptor and that electron transfer from the excited ruthenium complex takes place in less than 1 microsecond. Some rapid cytochrome a reduction is also observed. Absorbance changes after the initial transients suggest that the reduced CuA then equilibrates with cytochrome a with a rate constant of 2 x 10(4) s-1. Comparison of the absorbance changes at 605 and 445 nm and the kinetic difference spectrum in the Soret region indicate that no reduction of cytochrome a3 takes place. With the oxidized enzyme, no further reactions were detected, whereas, in the peroxide and ferryl intermediates, cytochrome a reoxidizes on a millisecond time scale. The reaction appears biphasic in both intermediates, with rate constants in the range 2 x 10(2) to 4 x 10(3) s-1. This is considerably slower than the maximal rates observed for electron transfer between cytochrome a and the bimetallic site found in earlier work and suggests rate limitation by other processes. The rates obtained for the slower phases are close to the rate for catalysis of cytochrome c oxidation.

2,2'-Dipyridyl↗
PubMed1992

Ultrastructural and Immunocytochemical Studies on the H(2)O(2)-Producing Enzyme Pyranose Oxidase in Phanerochaete chrysosporium Grown under Liquid Culture Conditions.

The ultrastructural distribution of the sugar-oxidizing enzyme pyranose 2-oxidase (POD) in hyphae of Phanerochaete chrysosporium K-3 grown under liquid culture conditions optimal for the enzyme's production was studied by transmission electron microscopy immunocytochemistry. Using the 3-dimethylaminobenzoic acid-3-methyl-2-benzothiazolinone hydrazone hydrochloride H(2)O(2) peroxidase spectrophotometric assay, POD was detected in mycelial extracts from days 7 to 18, with maximum activity recorded on day 12. Onset of POD activity occurred in the secondary phase of hyphal development at a time of stationary growth, glucose limitation, and pH increase. POD was also detected extracellularly in the culture fluid from days 7 to 18, with maximum activity recorded on day 13. At early stages of development (3 to 4 days), using anti-POD antibodies and immunogold labeling, POD was localized in multivesicular and electron-dense bodies and in cell membrane regions. After 10 to 12 days of growth, at maximum POD activity, POD was concentrated within the periplasmic space where it was associated with membrane-bound vesicles and other membrane structures. At later stages of development (17 to 18 days), when the majority of hyphae were lysed, POD was observed associated with residual intracellular membrane systems and vesicles. Transmission electron microscopy immunocytochemical studies also demonstrated an extracellular distribution of the enzyme at the stationary growth phase, showing its association with fungal extracellular slime. In studies of ligninolytic cultures of the same fungus, POD was found to have a similar intracellular and extracellular distribution in slime as that recorded for cultures grown with cornsteep. POD's peripheral cytoplasmic distribution shows similarities to the cellular distribution of that reported previously for H(2)O(2)-dependent lignin and manganese peroxidases in P. chrysosporium.

Journal Article↗
PubMed1991

Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C.

Glucose-induced changes in cytoplasmic pH (pHi) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Glucose, at concentrations above 3-5 mM, depolarized the beta-cell and increased pHi, cytoplasmic free Ca2+ ([Ca2+]i), and insulin release. This increase in pHi was dependent on the presence of extracellular Na+ and was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, a blocker of Na+/H+ exchange. Stimulation of protein kinase C with phorbol ester also induced an alkalinization. However, when protein kinase C activity was down-regulated, glucose stimulation still induced alkalinization. At 20 mM glucose, 10 mM NH4Cl induced a marked rise in pHi, paralleled by repolarization, inhibition of electrical activity, and decreases in both [Ca2+]i and insulin release. Reduction in [Ca2+]i was prevented by 200 microM tolbutamide, but not by 10 mM tetraethylammonium. At 4 mM glucose, NH4Cl induced a transient increase in insulin release, without changing [Ca2+]i. Exposure of beta-cells to 10 mM sodium acetate caused a persistent decrease in pHi, an effect paralleled by a small transient increase in [Ca2+]i. Acidification per se did not change the beta-cell sensitivity to glucose, not excluding that the activity of the ATP-regulated K+ channels may be modulated by changes in pHi.

Amiloride↗
PubMed1991

Measurements of cytoplasmic free Ca2+ concentration in human pancreatic islets and insulinoma cells.

In human pancreatic islets an increase in the glucose concentration from 3 to 20 mM raised the free cytoplasmic Ca2+ concentration [( Ca2+]i), an effect being reversible upon withdrawal of the sugar. Depolarization with a high concentration of K+ or the sulphonylurea tolbutamide also raised [Ca2+]i. Addition of extracellular ATP produced a transient rapid rise in [Ca2+]i. Oscillations in [Ca2+]i were observed in the presence of 10 mM glucose. Insulinoma cells responded to glucose and tolbutamide with increases in [Ca2+]i, whereas the sulphonamide diazoxide caused a decrease in [Ca2+]i. These findings confirm previous results obtained in rodent beta-cells.

Adult↗
PubMed1991

Interaction with the inositol 1,4,5-trisphosphate receptor promotes Ca2+ sequestration in permeabilised insulin-secreting cells.

Electropermeabilised insulin-secreting RINm5F cells sequestered Ca2+, resulting in a steady-state level of the ambient free Ca2+ concentration corresponding to 723 +/- 127 nM (mean +/- SEM, n = 10), as monitored by a Ca(2+)-selective minielectrode. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) promoted a rapid and pronounced release of Ca2+. This Ca2+ was resequestered and a new steady-state Ca2+ level was attained, which was always lower (460 +/- 102 nM, n = 10, P less than 0.001) than the steady-state Ca2+ level maintained before the addition of Ins(1,4,5)P3. Whereas the initial reuptake of Ca2+ subsequent to Ins(1,4,5)P3 stimulation was relatively slow, the later part of reuptake was fast as compared to the reuptake phases of a pulse addition of extraneous Ca2+. In the latter case the uptake of Ca2+ resulted in a steady-state level similar to that found in the absence of Ins(1,4,5)P3. Addition of Ins(1,4,5)P3 under this condition resulted in a further Ca2+ uptake and thus a lower steady-state Ca2+ level. Heparin, which binds to the Ins(1,4,5)P3 receptor, also lowered the steady-state free Ca2+ concentration. In contrast to Ins(1,4,5)P3, inositol 1,3,4,5-tetrakisphosphate was without effect on Ca2+ sequestration. These findings are consistent with the presence of a high-affinity Ins(1,4,5)P3 receptor promoting continuous release of Ca2+ under basal conditions and/or the Ins(1,4,5)P3 receptor being actively involved in Ca2+ sequestration.

Biological Transport↗
PubMed1991

Uptake and release of protons during the reaction between cytochrome c oxidase and molecular oxygen: a flow-flash investigation.

Changes in pH during the reactions of the fully reduced and mixed-valence cytochrome oxidase with molecular oxygen have been followed in flow-flash experiments, using the pH indicator phenol red. Solubilized enzyme as well as enzyme reconstituted into phospholipid vesicles has been studied. With the solubilized enzyme, a biphasic uptake of one proton from the medium was observed, whereas the reconstituted enzyme gave release of 1.3 protons to the extravesicular medium. It is concluded from these results that a total of two to three protons are taken up during oxidation of the fully reduced enzyme. Kinetic analysis suggests that the proton uptake is initiated by the transfer of the third electron to the oxygen binding site. A reaction scheme that integrates proton transfers and oxygen chemistry is presented.

Animals↗
PubMed1991

Outcome of the abduction external rotation test among manual and office workers.

Reactions during the Abduction External Rotation (AER) test were studied among 71 platers and 70 assemblers exposed to vibratory tools and manual work and 45 unexposed white collar workers. The frequency of positive neurological reactions was 31% among the platers, 6% among the assemblers, and 16% among the white collar workers. Platers reported the highest fatigue rates in the shoulder regions during the test. A positive association was noted between a pathological AER test and current problems in the neck/scapula or shoulder/upper arm regions, day- or night-time numbness in the hands, tension neck, and carpal tunnel syndrome. Age, length of daily vibration exposure, and vibration acceleration level were also positively associated with a positive AER test outcome. The results indicate that the outcome of the AER test is sensitive to neck and shoulder disorders but also, to some extent, to the carpal tunnel syndrome.

Adult↗
PubMed1991

The membrane spanning domain of beta-1,4-galactosyltransferase specifies trans Golgi localization.

Chimeric cDNAs were constructed so as to generate hybrid proteins in which different parts of the N-terminal domain of the human invariant chain were replaced by equivalent sequences from the trans Golgi resident enzyme, beta-1,4-galactosyltransferase. The cytoplasmic and membrane spanning domains of galactosyltransferase were found to be sufficient to retain all of the hybrid invariant chain in trans Golgi cisternae as judged by indirect immunofluorescence, treatment with brefeldin A and immuno-electron microscopy. As few as ten amino acids corresponding to the lumenal half of the membrane spanning domain of the Golgi enzyme sufficed to localize most of the hybrid invariant chain to the trans cisternae. A cytoplasmic domain was necessary for complete retention as assessed by flow cytofluorometry but could be provided either by galactosyltransferase or by invariant chain. This suggests that the cytoplasmic domain plays a role accessory to the membrane spanning domain, the latter mediating compartmental specificity.

Amino Acid Sequence↗
PubMed1991

Retinol-binding protein and transthyretin expressed in HeLa cells form a complex in the endoplasmic reticulum in both the absence and the presence of retinol.

To establish a suitable experimental system for studies of the interaction of retinol-binding protein (RBP) with transthyretin (TTR) we have expressed the corresponding cDNAs in HeLa cells. To investigate whether complex formation might occur already in the endoplasmic reticulum (ER), the C-terminal ER retention signal, KDEL, was attached to TTR. The tetrameric TTR-KDEL fusion protein was retained in the ER of HeLa cells. When RBP was co-expressed with TTR-KDEL, RBP was retained intracellularly. A cDNA-encoding purpurin, a protein which is 50% identical to RBP, was then expressed together with TTR-KDEL. Purpurin was not retained intracellularly and did not bind to TTR coupled to Sepharose. The effect of the vitamin A status on the secretion of TTR and RBP was examined. While TTR expressed alone was not retained intracellularly, TTR was retained in vitamin A-deficient cells when co-expressed with RBP. Addition of retinol stimulated rapid secretion of both proteins. These results demonstrate that TTR can form a complex with RBP in the ER. The data suggest that RBP and TTR are secreted as a complex.

Anthraquinones↗
PubMed1991

Treatment of symptomatic metastatic prostatic cancer with cyproterone acetate versus orchiectomy: a prospective randomized trial.

During a 2-year period, 37 patients with symptomatic metastatic prostatic cancer were included in a prospective randomized phase-III trial. Nineteen patients were randomized to subcapsular orchiectomy, and 18 to cyproterone acetate (CPA) treatment with a dose of 50 mg b.i.d. The median age of the patients was 74 years (range 48-88 years), with no differences between the treatment groups. At 3, 6, and 12 months after initiation of the therapy and then every 6 months, patients were clinically and biochemically examined, and isotope scans and X-rays were performed. All patients were followed until death. Relief from symptoms was found following 3 months of treatment in 70.6% (95% confidence limits = 44.0-89.7%) of the patients treated with CPA, and in 83.3% (95% confidence limits = 58.6-96.4%) of the orchiectomized patients. The median time to relapse was 9 months in the CPA group, and 11 months in the orchiectomy group (p greater than 0.05). The median survival time was 13 months, with no differences between the groups. The treatment of advanced prostatic cancer with CPA is found to be a valuable alternative to orchiectomy.

Adenocarcinoma↗
PubMed1991

Cystic duct remnants and biliary symptoms after cholecystectomy. A randomised comparison of two operative techniques.

To assess the importance of cystic duct remnants in causing biliary symptoms after cholecystectomy 80 patients undergoing routine cholecystectomy were randomised to have conventional cholecystectomy (n = 40) or cholecystectomy with removal of the whole cystic duct (n = 40). Fifteen were withdrawn, leaving 65 (32 who had standard cholecystectomy and 33 who had cholecystectomy by the new technique) for analysis. Thirty of the 32, and 27 of the 33, were available for follow up one, two, three, and eight years after operation; 11 of the 30 (37%) compared with 23 of the 27 (85%) were free of biliary symptoms during the eight year period (p less than 0.001). We conclude that removal of the stump of the cystic duct improves the success rate of cholecystectomy in the treatment of gallstones.

Abdominal Pain↗
PubMed1990

Intracellular transport of class II MHC molecules directed by invariant chain.

Three structural motifs in the invariant chain (li) control the intracellular transport of class II major histocompatibility complex molecules. An endoplasmic reticulum retention signal in the full-length li suggests a role for li in the alpha-beta heterodimer assembly. Another signal motif directs a truncated li, alone or associated with individual class II chains, to a degradation compartment by a pathway circumventing the Golgi. When this truncated li binds alpha-beta dimers, a third signal dominates, directing the complex by way of the Golgi to vesicles in the cell periphery, which may represent a subcompartment of recycling endosomes.

Antibodies, Monoclonal↗
PubMed1990

Glucose-stimulated efflux of indo-1 from pancreatic beta-cells is reduced by probenecid.

Indo-1 loaded pancreatic beta-cells, isolated from obese hyperglycaemic mice, were studied with respect to cytoplasmic free Ca2+ concentration ([Ca2+]i), efflux of indicator and insulin release. In the absence of glucose there was a continuous efflux of indo-1 which increased upon stimulation with 20 mM of the sugar. The anion exchange inhibitor probenecid reduced both basal efflux of indo-1 and prevented that promoted by glucose. Measurements of [Ca2+]i and insulin release revealed similar results as previously reported with quin-2 and fura-2. Furthermore, probenecid did not influence the [Ca2+]i responses. It is thus possible to reduce efflux of indo-1 probenecid and thereby improve the measurements of [Ca2+]i in pancreatic beta-cells.

Animals↗
PubMed1990

Extracellular ATP increases cytoplasmic free Ca2+ concentration in clonal insulin-producing RINm5F cells. A mechanism involving direct interaction with both release and refilling of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool.

Effects of extracellularly applied ATP (added as disodium salt) on stimulus-secretion coupling were investigated in clonal insulin-producing RINm5F cells. Cytoplasmic free Ca2+ concentration [( Ca2+]i), electrical activity, membrane potential, formation of InsP3 and insulin release were measured. Addition of ATP in a Ca2(+)-containing medium promoted a rapid rise in [Ca2+]i, which was followed by a slow decline towards the basal level. In a Ca2(+)-free medium, the ATP-induced increase in [Ca2+]i was smaller, but still enough to elicit insulin secretion. Upon normalization of the extracellular Ca2+ concentration, the response to ATP recovered instantaneously. The presence of glucose in the incubation medium was a prerequisite to obtain a pronounced effect of ATP in the absence of extracellular Ca2+. However, glucose did not enhance the response to ATP in a Ca2(+)-containing medium. The effect of ATP was dose-dependent, with a clearly detectable increase in [Ca2+]i at 1 microM and a maximal response being obtained at 200 microM-ATP. The response to ATP was unaffected by activating adenylate cyclase by forskolin, but was abolished by 10 nM of the phorbol ester phorbol 12-myristate 13-acetate. The effects of ATP on [Ca2+]i could not be accounted for by a generalized increase in plasma-membrane permeability, as evident from the failure of the nucleotide to increase the fluorescence of the nuclear stain ethidium bromide. After stimulation with ATP there was an increase in membrane potential, in both the absence and the presence of extracellular Ca2+. Blockage of the voltage-activated Ca2+ channals with D-600, in a Ca2(+)-containing medium, decreased the effect of ATP on [Ca2+]i slightly. Patch-clamp measurements using the cell-attached patch configuration revealed that the RINm5F cells produce spontaneous action potentials, the frequency of which increased markedly on addition of ATP. Whole-cell recordings demonstrated that the increase in spike frequency was not associated with the development of an inward current, but was rather accountable for by a decrease in the activity of the ATP-regulated K+ channels. Addition of 200 microM-ATP stimulated phospholipase C activity, as evident from the formation of InsP3, both in the absence and in the presence of extracellular Ca2+. Thus in the absence of extracellular Ca2+ the stimulatory effect of ATP on insulin release can be explained by InsP3-induced mobilization of intracellularly bound Ca2+. Hence, in the RINm5F cells extracellular ATP acts in a manner similar to other Ca2(+)-mobilizing agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗