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Biomedical subjects

T Nilsson

Publications and source records attributed to T Nilsson.

At least 199 records · Page 11Linked to original sources
PubMed1989

Suppression of insulin release by galanin and somatostatin is mediated by a G-protein. An effect involving repolarization and reduction in cytoplasmic free Ca2+ concentration.

The effects of galanin and somatostatin on insulin release, membrane potential, and cytoplasmic free Ca2+ concentration [( Ca2+]i) were investigated using beta-cells isolated from obese hyperglycemic mice. Whereas insulin release was measured in a column perifusion system, membrane potential and [Ca2+]i were measured with the fluorescent indicators bisoxonol (bis-(1,3-diethylthiobarbiturate)trimethineoxonol) and quin 2, in cell suspensions in a cuvette. Galanin (16 nM) and somatostatin (400 nM) suppressed glucose-stimulated insulin release in parallel to promoting repolarization and a reduction in [Ca2+]i. The reduction in [Ca2+]i comprised an initial nadir followed by a slow rise and the establishment of a new steady state level. The slow rise in [Ca2+]i was abolished by 50 microM D-600, a blocker of voltage-activated Ca2+ channels. Both peptides suppressed insulin release even when [Ca2+]i was raised by 25 mM K+. Under these conditions the inhibition of insulin release was partly reversed by an increase in the glucose concentration. Addition of 5 mM Ca2+ to a cell suspension, incubated in the presence of 20 mM glucose and either galanin, somatostatin, or the alpha 2-adrenergic agonist clonidine (10 nM), induced oscillations in [Ca2+]i, this effect disappearing subsequent to the addition of D-600. The effects of galanin, somatostatin, and clonidine on [Ca2+]i were abolished in beta-cells treated with pertussis toxin. In accordance with measurements of [Ca2+]i, treatment with pertussis toxin reversed the inhibitory effect of galanin on insulin release. The inhibitory action of galanin and somatostatin on insulin release is probably accounted for by not only a repolarization-induced reduction in [Ca2+]i and a decreased sensitivity of the secretory machinery to Ca2+, but also by a direct interaction with the exocytotic process. It is proposed that these effects are mediated by a pertussis toxin-sensitive GTP-binding protein.

Animals↗
PubMed1989

Risk assessment of vibration exposure and white fingers among platers.

The dose-response relationship between vibration exposure and vascular disorders in the hands was examined in platers. The study was based on a cross section of 89 platers and 61 office workers divided according to exposure to vibration into four groups. Vibration exposure was assessed by measuring the acceleration intensity on a sample of tools, together with both subjective rating and objective measurements of the exposure time. The frequency-weighted energy equivalent acceleration for 4 h was 4.6-4.7 m/s2. The point prevalence of white fingers was 42% for the plater category currently exposed with an odds ratio of 85. The time laps before contraction of white fingers (latency time) was four years for the 10th percentile, and was shorter than predicted according to the ISO-5349 standard. The prevalence of white finger symptoms staged according to the Taylor-Pelmear scale was comparable to the prevalences according to the Stockholm Workshop Scale. Vibration exposure was the dominant source of white fingers and each year of vibration exposure increased the odds ratio for white fingers by 11%. Distal circulation in the hands was assessed by a timed Allen test. The odds ratio for a positive Allen test was higher for the workers exposed to vibration compared to the non-exposed workers. The use of the timed Allen test is suggested in the clinical examination for vibration white fingers. The observed high risk for contracting white fingers could be prevented by exposure level reduction and/or restriction of exposure duration.

Adult↗
PubMed1989

Extracellular ATP mobilizes intracellular Ca2+ in T51B rat liver epithelial cells: a study involving single cell measurements.

T51B rat liver epithelial cells were stimulated with extracellular ATP. Changes in cytoplasmic free Ca2+ concentration [( Ca2+]i) were measured by fura-2 both in a large population of cells on coverslips in a cuvette and in single cells in a microscopic system. Extracellular ATP evoked a prompt increase in [Ca2+]i in both the presence and absence of extracellular Ca2+, although the effect was less pronounced in the latter case. These findings indicate that at least part of the [Ca2+]i increase is due to mobilization of intracellularly bound calcium. Stimulation with ATP did not mobilize the total pool of intracellular releasable Ca2+, as evidenced from experiments where subsequent addition of ionomycin evoked a pronounced increase in [Ca2+]i in the absence of extracellular Ca2+. The effect of ATP was maintained at room temperature but was markedly impaired in the absence of continuous stirring of the buffer solution. In the absence of stirring, ATP had to be increased to the millimolar range in order to evoke a pronounced effect. Single cell measurements revealed a heterogenous Ca2+ response to ATP, with some cells failing to respond with a detectable increase in [Ca2+]i. The actual increase in [Ca2+]i was not uniform throughout the cytoplasm, but seemed to start in one part of the cell. Even if part of the [Ca2+]i increase might be accounted for by ATP promoting the hydrolysis of phosphatidylinositol 4,5-bisphosphate and thereby a generation of InsP3 and diacylglycerol, there was no initiation of DNA synthesis under the present conditions. Hence, extracellular growth factors exert either a quantitative difference in second messenger production or additional stimulatory effects by activating intracellular signal pathways beyond these represented by [Ca2+]i and protein kinase C.

Adenosine Triphosphate↗
PubMed1989

Glucose-induced changes in cytoplasmic free Ca2+ concentration and the significance for the regulation of insulin release. Measurements with fura-2 in suspensions and single aggregates of mouse pancreatic beta-cells.

The effects of glucose on cytoplasmic free Ca2+ concentration, [Ca2+]i, and insulin release were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Measurements of [Ca2+]i were performed in cell suspensions in a cuvette and in single cell-aggregates in a microscopic system, using fura 2 and quin 2. Insulin release was studied from indicator loaded cells in a column perifusion system. In the presence of 1.28 mM extracellular Ca2+, an increase in the glucose concentration from 0 to 20 mM had two major effects on [Ca2+]i. Initially there was a decrease, which was immediately followed by a pronounced increase. At reduced extracellular Ca2+, or when Ca2+ influx was blocked, glucose induced only a decrease in [Ca2+]i. With increasing intracellular concentrations of indicator, the effects of glucose on [Ca2+]i were markedly reduced. Changes in [Ca2+]i, similar effects being obtained in the cuvette and microfluorometric measurements, were paralleled by changes in insulin release. Insulin release from indicator loaded cells did not markedly differ from that of non-loaded controls, either with respect to rapidity or size in the response to the sugar. The addition of 20 mM glucose increased the efflux of fura 2, an effect that was not related to insulin release. Permeabilization of indicator loaded cells demonstrated a substantial amount of fura 2 bound intracellularly. Although the effects of glucose on [Ca2+]i seemed to be similar in fura 2 and quin 2 loaded cells, the demonstrated leakage and possible intracellular binding should be considered before using fura 2 for measurements in pancreatic beta-cells.

Animals↗
PubMed1989

Adenovirus proteins and MHC expression.

Adenoviruses are able to specifically down-regulate the cell surface expression of MHC class I antigens. Most viral serotypes achieve these ends by synthesizing a protein that binds to class I antigens in the endoplasmic reticulum (ER) and impedes the transport of these molecules to the cell surface. However, viruses belonging to the highly oncogenic subgenus A do not affect the class I antigen expression during acute infection. Instead, they are distinct from other adenoviruses in that they specifically down-regulate the level of mRNAs, encoding MHC class I antigens, in virally transformed cells. The virus-induced reduction of class I antigen expression drastically diminishes the ability of CTLs to recognize cells infected or transformed by adenovirus. A number of issues concerning these viral mechanisms for class I antigen modulation need to be addressed. The molecular mechanism by which the E1A gene product of subgenus A viruses diminishes class I mRNA levels has not been elucidated. Also, the details of the interaction between the E19 protein and class I molecules should be studied, preferably by X-ray crystallography of the complexes. This would clarify the role of the antigen-binding site as well as other portions of the class I molecule in the binding to the E19 protein. Of general importance for our understanding of the sorting and intracellular transport of proteins is the exact delimitation of the signal for ER localization, which is present in the COOH-terminus of the E19 protein. The putative interaction of this peptide sequence with components of the ER membrane should also be studied. Finally, the study of the pathophysiological role of the MHC class I down-regulation will undoubtedly yield new insights into how the immune system combats virally infected and transformed cells.

Adenoviruses, Human↗
PubMed1989

Failure of glucose to elicit a normal secretory response in fetal pancreatic beta cells results from glucose insensitivity of the ATP-regulated K+ channels.

Fetal pancreatic beta cells demonstrate a deficient insulin release in response to glucose, but the underlying mechanism at the cellular level is unknown. By using beta cells from 21-day fetal rats we made an attempt to clarify the mechanism(s) behind this reduced glucose response. In addition to measuring insulin release, glucose metabolism, and cellular ATP content, ATP-regulated K+ channels (G channels) and voltage-activated Ca2+ currents were investigated with the patch-clamp technique. It was thus demonstrated that the ATP-regulated K+ channels in fetal beta cells were not sensitive to glucose but otherwise had similar characteristics as those of adult beta cells. Also, the characteristics of the voltage-activated Ca2+ currents were similar in adult and fetal beta cells. However, as judged from measurements of both glucose oxidation and glucose utilization, glucose metabolism was impaired in fetal beta cells. In addition, there was no increase in the ATP content, even when the cells were stimulated for 30 min. It is therefore concluded that the attenuated glucose-induced insulin release in fetal pancreatic beta cells is due to an immature glucose metabolism resulting in impaired regulation of the ATP-sensitive K+ channels. These findings may be relevant to the understanding of the deficient stimulus-secretion coupling associated with non-insulin-dependent diabetes.

Adenosine Triphosphate↗
PubMed1989

Mechanism of action of calcitonin on secretion in rat submandibular gland.

Calcitonin (CT) was found to reduce the initial flow of pilocarpine-stimulated saliva from the submandibular glands in the rat. Although there was a concomitant increase of the concentration of calcium and protein in the saliva, the calcium/protein ratio was not significantly affected. CT also caused a significant increase of the potassium concentration in submandibular saliva. Both in vivo and in vitro, CT inhibited the production of cyclic AMP (cAMP) both in the absence and in the presence of forskolin. This decrease in intracellular cAMP levels could result in an inhibition of mucus secretion, which would explain the previously observed calcitonin-induced intracellular accumulation of mucus in the submandibular gland acinar cells. CT did not affect the cytoplasmic free Ca2+ concentration (as measured with fura 2) in isolated submandibular acini either in the absence or in the presence of cholinergic or adrenergic agonists. These results indicate that the inhibition of fluid secretion in the submandibular gland by calcitonin must be located distal to changes in [Ca2+]i. It can be concluded that CT affects both mucus and fluid secretion in the submandibular gland, but that only the inhibition of mucus secretion can as yet be explained by an effect at the level of the second messenger.

Adenylyl Cyclases↗
PubMed1989

Intra- and Extracellular Localization of Lignin Peroxidase during the Degradation of Solid Wood and Wood Fragments by Phanerochaete chrysosporium by Using Transmission Electron Microscopy and Immuno-Gold Labeling.

The distribution of lignin peroxidase during degradation of both wood and woody fragments by the white rot fungus Phanerochaete chrysosporium was investigated by using anti-lignin peroxidase in conjunction with postembedding transmission electron microscopy and immuno-gold labeling techniques. The enzyme was localized in the peripheral regions of the fungal cell cytoplasm in association with the cell membrane, fungal cell wall, and extracellular slime materials. In solid wood, lignin peroxidase was detected in low concentrations associated with both superficial and degradation zones within secondary cell walls undergoing fungal attack. A similar but much greater level of extracellular peroxidase activity was associated with wood fragments degraded by the fungus grown under liquid culture conditions optimal for production of the enzyme. Efforts to infiltrate degraded wood pieces with high levels of lignin peroxidase showed the enzyme to be restricted to superficial regions of wood decay and to penetrate wood cell walls only where the wall structure had been modified. In this respect the enzyme was able to penetrate characteristic zones of degradation within the secondary walls of fibers to sites of lignin attack. This suggests a possibility for a close substrate-enzyme association during wood cell wall degradation.

Journal Article↗
PubMed1989

Human interleukin 1 beta stimulates islet insulin release by a mechanism not dependent on changes in phospholipase C and protein kinase C activities or Ca2+ handling.

Isolated islets from adult rats or obese hyperglycemic (ob/ob) mice were incubated with human recombinant interleukin 1 beta in order to study whether the acute effects of the cytokine on islet insulin release are associated with changes in islet phospholipase C activity, Ca2+ handling or protein phosphorylation. The cytokine stimulated insulin release both at low and high glucose concentrations during one hour incubations. In short-term incubations (less than 1 min) interleukin 1 beta did not affect the production of inositoltrisphosphate. Addition of interleukin 1 beta affected neither the cytoplasmic free Ca2+ concentration at rest nor that observed subsequent to stimulation with a high concentration of glucose. Furthermore, the endogenous protein kinase C activity, as visualized by immunoprecipitation of a 32P-labelled substrate for this enzyme, was not altered by interleukin 1 beta. Separation of 32P-labelled proteins by means of 2-dimensional gel electrophoresis failed to reveal any specific effects of the cytokine on the total protein phosphorylation activity. These results suggest that the stimulatory effects on insulin release exerted by interleukin 1 beta are not caused by acute activation of phospholipase C and protein kinase C or by an alteration of islet Ca2+ handling of the B-cells.

Animals↗
PubMed1988

Conversion of CuA to a type II copper in cytochrome c oxidase.

When cytochrome c oxidase is incubated at 43 degrees C for approximately 75 min in a solution containing the zwitterionic detergent sulfobetaine 12, the CuA site is converted into a type II copper as judged by changes in the 830-nm absorption band and the EPR spectrum of the enzyme. SDS-PAGE and sucrose gradient ultracentrifugation indicate concomitant loss of subunit III and monomerization of the enzyme during the heat treatment. Comparison of the optical and resonance Raman spectra of the heat-treated and native protein shows that the heme chromophores are not significantly perturbed; the resonance Raman data indicate that the small heme perturbations observed are limited to the cytochrome a3 site. Proton pumping measurements, conducted on the modified enzyme reconstituted into phospholipid vesicles, indicate that these vesicles are unusually permeable toward protons during turnover, as previously reported for the p-(hydroxymercuri)benzoate-modified oxidase and the modified enzyme obtained by heat treatment in lauryl maltoside. The sulfobetaine 12 modified enzyme is no longer capable of undergoing the recently reported conformational transition in which the tryptophan fluorescence changes upon reduction of the low-potential metal centers. Control studies on the monomeric and subunit III dissociated enzymes suggest that the disruption of this conformational change in the heat-treated oxidase is most likely associated with perturbation of the CuA site. These results lend support to the suggestion that the fluorescence-monitored conformational change of the native enzyme is initiated by reduction of the CuA site [Copeland et al. (1987) Biochemistry 26, 7311].

Animals↗
PubMed1988

Heat treatment of cytochrome c oxidase perturbs the CuA site and affects proton pumping behavior.

It has been previously reported that mild heat treatment (43 degrees C for ca. 60 min) abolishes the proton pumping activity of cytochrome c oxidase while leaving the oxidase activity and cytochromes a and a3 unperturbed [Sone, N., & Nicholls, P. (1984) Biochemistry 23, 6550-6554]. We herein describe the effects of this heat treatment on the electron paramagnetic resonance (EPR) and optical absorption signatures of the redox-active metal centers in the enzyme. We find that heat treatment of the oxidized enzyme causes a local structural perturbation at the CuA site. After heat treatment, the enzyme sample contains three subpopulations, each of which has a different structure at CuA. These include (i) native CuA, (ii) a type 2 copper species similar to the one produced by chemical modification by p-(hydroxymercuri)benzoate (pHMB) [Gelles, J., & Chan, S. I. (1985) Biochemistry 24, 3963-3972], and (iii) a novel type 1 copper species. In addition to changes at the CuA site, we find that heat treatment results in accelerated cyanide binding and the removal of subunit III. If the cytochrome c oxidase is heat treated while fully reduced, none of these changes are observed except for subunit III depletion. Furthermore, partial (CO mixed-valence derivative) reduction of the enzyme as well as ligand binding to cytochrome a3 also protects the enzyme against the heat-induced changes, indicating that the oxygen binding site plays a role in stabilizing the CuA site against structural perturbations.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1988

The possible role of the closed-open transition in proton pumping by cytochrome c oxidase: the pH dependence of cyanide inhibition.

The rate of oxidation of reduced cytochrome c catalyzed by cytochrome oxidase in the presence and absence of cyanide has been measured spectrophotometrically at pH 5.5, 6.4, 7.4 and 8.3. At the cytochrome c concentration used (272 microM), the uninhibited rate is maximal at pH 6.4 and drops to a value about one sixth of this maximum at pH 8.3. In the presence of cyanide, the rate initially drops rapidly, but with the cyanide concentration used (5.5 microM) there is still a measurable rate of oxidation when maximal inhibition has been reached. This inhibited rate decreases as the pH increases, whereas the apparent rate constant for cyanide binding is almost independent of pH. The results have been analyzed on the basis of a model in which two-electron reduction of the oxidized enzyme triggers a transition from a closed to an open conformation. It is assumed that cyanide can only bind to the open conformation and, furthermore, that rapid internal electron transfer to the dioxygen-reducing site occurs in this state alone. The analysis shows that the true constant for cyanide binding decreases with decreasing pH to a constant value at low pH. It also indicates that the increase in the catalytic constant with decreasing pH is associated with an increase in the rate of the closed-open conformational transition on protonation of the enzyme, and it is proposed that this transition is operative in electron gating in the proton-pump function of the enzyme.

Animals↗
PubMed1988

Dual effect of glucose on cytoplasmic free Ca2+ concentration and insulin release reflects the beta-cell being deprived of fuel.

Influence of basal glucose concentration on the response evoked by subsequent stimulation with the sugar, was evaluated by investigating changes in free cytoplasmic Ca2+ concentration, [Ca2+]i, and insulin release, using beta-cells isolated from obese hyperglycemic mice. When increasing the glucose concentration from 0 to either 11 or 20 mM, there was a transient decrease in both [Ca2+]i and insulin release. The decrease was followed by a pronounced increase in both of the parameters. When increasing the basal glucose concentration, the initial decrease gradually disappeared, being abolished already at 5 mM of the sugar and the subsequent increase appeared more rapidly. It is suggested that the observed decrease in [Ca2+]i and thereby insulin release reflects a phenomenon associated with fuel deprived beta-cells.

Animals↗
PubMed1988

Heparin inhibits IP3-induced Ca2+ release in permeabilized pancreatic beta-cells.

Heparin was found to inhibit the Ca2+ release induced by inositol 1,4,5-trisphosphate (IP3) in permeabilized pancreatic beta-cells obtained from obese hyperglycemic mice. The effect of heparin was dose-dependent and not due to inhibition of Ca2+ uptake into the IP3-sensitive pool. The effect appeared specific for heparin and was not reproduced by other polysaccharides such as chondroitin sulfates. Heparin might consequently be a useful tool when investigating the molecular mechanism whereby IP3 mobilizes Ca2+.

Animals↗
PubMed1988

Inhibition of glucose-stimulated insulin release by alpha 2-adrenoceptor activation is parallelled by both a repolarization and a reduction in cytoplasmic free Ca2+ concentration.

Effects of the alpha 2-adrenergic agonist clonidine on insulin release, membrane potential, and cytoplasmic free Ca2+ concentration ([Ca2+]i) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Addition of 2 microM clonidine promptly inhibited glucose-stimulated insulin release, an effect accompanied by a lowering in both membrane potential and [Ca2+]i. Within minutes, the effect on Ca2+ was partly reversed, [Ca2+]i attaining a new level, although still significantly lower than in the absence of agonist. This late increase in [Ca2+]i was inhibited by 50 microM D-600, a blocker of voltage-activated Ca2+ channels. The inhibitory effects of clonidine on membrane potential, [Ca2+]i, and insulin release were abolished by 5 microM of the alpha 2-adrenergic antagonist yohimbine. Depolarization with high K+ increased [Ca2+]i also in the presence of clonidine, conditions accompanied by only a minute release of insulin. Secretion was, however, partly restored by subsequent addition of 20 mM glucose. Addition of 5 mM Ca2+ transiently reversed the effects of clonidine on both membrane potential and [Ca2+]i. Although the clonidine-induced repolarization should be enough for closing the voltage-activated Ca2+ channels with a resulting decrease in [Ca2+]i, a direct interaction of the agonist with these channels cannot be excluded. The fact that it was possible to increase [Ca2+]i with only a minor effect on insulin release suggests that the inhibitory effect of clonidine not only is due to a reduction in [Ca2+]i, but also involves interference with some more distal step in the insulin secretory machinery.

Adrenergic alpha-Agonists↗
PubMed1988

Chemical modification of the CuA site affects the proton pumping activity of cytochrome c oxidase.

Cytochrome c oxidase in which the CuA site has been perturbed by extensive modification of the enzyme with the thiol reagent p-(hydroxymercuri)benzoate has been reconstituted into phospholipid vesicles. The reconstituted vesicles lack respiratory control, and the orientation of the enzyme in the vesicles is similar to that of the native cytochrome c oxidase. In the proton translocation assay, the vesicles containing the modified enzyme behave as if they are unusually permeable to protons. When the modified and native proteins were coreconstituted, a substantial portion of the latter became uncoupled as revealed by low respiratory control and low overall proton pumping activity. These results suggest that the modified enzyme catalyzes a passive transport of protons across the membrane. When milder conditions were used for the chemical modification, a majority of the thiols reacted while the CuA site remained largely intact. Reconstitution of such a partially modified cytochrome c oxidase produced vesicles with respiratory control and proton translocating activity close to those of reconstituted native enzyme. It thus appears that the appearance of a proton leak is related to the perturbation of the CuA site. These observations suggest that the structure of CuA may be related to the role of this site in the proton pumping machinery of cytochrome c oxidase.

Animals↗
PubMed1988

Stimulation of pancreatic islet beta-cell replication by oncogenes.

Although the growth potential of the pancreatic islet beta cells is limited, glucose, cAMP, and certain polypeptide growth factors have been reported by other workers to exert modest stimulatory effects on beta-cell replication. To further assess means through which beta-cell growth can be stimulated, selected oncogene constructs linked to a rat insulin promoter were introduced by means of electroporation into free islet cells prepared from fetal rats and adult hyperglycemic obese (ob/ob) mice. The uptake and expression of the added oncogenes were sufficiently efficient to exert effects on beta-cell physiology in short-term experiments (less than or equal to 4 days). Stimulation of islet cell [3H]thymidine incorporation was observed after transfection with src alone or the combination of myc and ras. The effect observed in the fetal islet cells with src was more pronounced than any effect previously reported. Transfection with the src oncogene resulted in phosphorylation of lipocortin I and was paralleled by an increased immunofluorescence against src-like immunoreactivity in a majority of the electroporated cells. It is concluded that electroporation can induce sufficiently efficient expression of added oncogene constructs to study their effects on cells that are not readily transformable into continuously growing cell lines. Furthermore, the results suggest that beta-cell replication might be manipulated extrinsically by inserting appropriate growth-promoting genes into these cells.

Animals↗