Biomedical subjects
L Nilsson
Publications and source records attributed to L Nilsson.
Plasticity of contractile endothelin-B receptors in human arteries after organ culture.
1. The pharmacology and mRNA expression of endothelin (ET) receptors in human omental arteries were characterized by use of functional contractile assays and the reverse transcriptase-polymerase chain reaction (RT-PCR). 2. In freshly obtained segments of human omental arteries, ET-1 and ET-3 induced concentration-dependent contractions which were normalized to the response produced by 60 mM K+. ET-1 produced a maximum contraction (Emax) amounting to 151 +/- 17% of the K+ response. The pEC50 for this agonist was 8.64 +/- 0.17. The effect of ET-3 was less pronounced (Emax: 71 +/- 22% and pEC50: 6.69 +/- 0.17) than that of ET-1. The ET receptors involved were characterized with FR139317 (a selective ETA receptor antagonist), PD 145065 (a mixed ETA and ETB receptor antagonist) and BQ 788 (an ETB receptor antagonist). A high concentration of these antagonists (10 microM) abolished the contractile responses to ET-3, and produced a parallel rightward shift of the ET-1 concentration-response curve without changing the maximal effect. FR139317 and PD 145065 were equally effective while BQ 788 was much less effective. This is consistent with ETA receptors mediating contraction in human omental arteries. 3. Arterial segments cultured for 5 days in serum-free Dulbecco's medium at 37 degrees C under sterile and humidified conditions retained contractility although responses to 60 mM K+ were somewhat reduced. ET-3 was significantly more potent in the cultured arteries (pEC50: 8.56 +/- 0.15) and achieved a greater maximum effect (Emax: 116 +/- 19%). Responses were not antagonised by FR139317 but were competitively blocked by PD 145065 and BQ 788 with the latter antagonist being the more potent. In contrast Emax (179 +/- 17%) and pEC50 (8.66 +/- 0.23) values for ET-1 were not significantly different from those obtained with fresh arteries. PD 145065 still demonstrated a rightward shift of the ET-1-induced concentration-response curve, whereas FR139317 and BQ 788 caused non-significant shifts. These findings suggest that functional ETB receptors contribute significantly to the endothelin contractile response in cultured arteries. 4. Two-site analysis of the ET-1 induced concentration-response curve from cultured arteries suggests that ETB receptors, at the high potency component, and ETA receptors, at the low potency component, contribute both to the contractile response in relative proportion of 70% and 30%, respectively. Further analysis suggested that the ETA receptor would be capable of evoking at least 75% of the ET-1 contraction in the absence of ETB receptors, although with a lower potency as compared to fresh arteries. 5. Electrophoresis of RT-PCR products from the smooth muscle layer of freshly obtained human arteries indicated the presence of mRNA for both ETA and ETB receptors. Arteries cultured for 1 and 5 days demonstrated an increase of mRNA for the ETB receptor as compared to the ETA receptor. The identities of the PCR products were verified by restriction enzyme digestion. 6. In freshly obtained human omental arteries, the contractile effects of endothelins appear to be mediated predominantly by the ETA receptor subtype, with a negligible contribution by ETB receptors. Cultured arterial segments, however, exhibited a substantial ETB receptor mediated contractile response and an increase in ETB receptor mRNA content, consistent with an upregulation of functional ETB receptors. These in vitro data suggest plasticity in the smooth muscle cell expression of contractile ETB receptors.
Effects of heparin coating of cardiopulmonary bypass circuits on in vitro oxygen free radical production during coronary bypass surgery.
During cardiopulmonary bypass (CPB) oxygen free radicals (OFR) are formed, which can mediate reactions damaging tissue components. Blood contact with artificial surfaces during CPB leads to an activation of leukocytes, which are one of the sources of the OFR. Heparin coating of the CPB circuit reduces granulocyte activation. In the present study, the heparin-coated circuits with noncoated cardiotomy reservoirs (Group HC) were compared with noncoated, otherwise similar CPB sets (Group C). In each group, 8 patients were operated on for coronary revascularization. The release of granulocyte granule proteins myeloperoxidase (MPO) and lactoferrin (LF) was evaluated. Production of OFR in the whole blood and in the granulocyte suspension were measured by chemiluminescence (CL). In both groups the whole blood CL declined during CPB. The whole blood CL induced by serum-opsonized zymosan, when enhanced by luminol, was significantly lower in Group HC at 45 min after CPB start (68 +/- 6% of initial values in Group HC vs. 87 +/- 6% in Group C, mean +/- SEM) and 30 min after protaminization (54 +/- 6% of initial values in Group HC vs. 72 +/- 6% in Group C, mean +/- SEM), and CL was significantly higher in Group HC at CPB end (83 +/- 5% of initial values in Group HC vs. 67 +/- 5% in Group C, mean +/- SEM) when enhanced by lucigenin. CL of isolated granulocytes showed no significant differences between the groups. Release of MPO at CPB end and of LF 45 min after start of CPB and at CPB end were significantly lower in the heparin-coated CPB circuits.
Plasma cyanate concentrations in chronic renal failure.
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Estimation of skin prick test reactions with a scanning program.
We evaluated a new method of computer-based scanning of skin prick test wheal areas. To test the calibration of the program, we scanned five constructed circles of exactly defined areas between 5 and 255 mm2. One of these circles was scanned in different degrees of contrast (modes), the middle mode being used in the other experiments. We also investigated the inter- and intraoperator variation. Finally, results obtained by measuring diameters of wheals and by the new method of area determination were compared for 82 histamine and 75 egg wheals. The areas found agreed well with the real areas (P < 0.01) (mean 97.4-100.8%), except for the smallest wheal (5mm2) (NS). Areas obtained in the "middle position" closely resembled the real area. The intraoperator coefficient of variation (c.v.) was 1.4% (0.3-4.3), the day-to-day c.v. was 1.9% (0.2-5.3), and the interoperator c.v. was 2.3% (0.7-5.7), with a higher c.v. for small areas. Histamine and egg wheals were significantly larger with the diameter method (126% and 129%, respectively) than with the area method. The difference between the methods was most pronounced for small wheal areas. The c.v. of the scanning method was significantly lower than that of the diameter method. The new program was precise and is recommended for registration of skin test reactions in scientific trials. Although the scanner works well and has high precision, the major problem in skin testing seems still to be the reproducibility of the skin test technique employed.
Beta-amyloid protein protein precursor expression in lacrimal glands and tear fluid.
PURPOSE: Proteases in tear fluid play important roles in the regulation of corneal wound healing. Inhibitors of proteolytic activity are major modulators of the associated events. Although it is known that various enzyme inhibitors exist in tear fluid, it is not known whether certain isoforms of the beta-amyloid protein precursor (beta-APP), a potent inhibitor of serine proteases, are present in tear fluid. The purpose of this study was to investigate whether beta-APP can be detected in human tear fluid and, if so, to determine the isoform composition and cellular origin. METHODS: Tear fluid was collected from healthy volunteers. The beta-APP was identified and characterized by immunoblotting using antibodies specific for domains of the beta-APP. The protein was characterized further by ion exchange chromatography. Expression of the beta-APP gene was studied using in situ hybridization and RNA-RNA solution hybridization assay. RESULTS: beta-APP with protease inhibitory properties was identified in all samples of human tear fluid. Immunologic analysis revealed that it had been processed proteolytically before secretion. Gene expression studies showed that the beta-APP gene was expressed in lacrimal glands, particularly in acinar cells. The gene transcript almost exclusively corresponded to beta-APP containing the protease inhibitor insert. CONCLUSIONS: beta-APP is expressed in lacrimal glands and subsequently is secreted into tear fluid. Because the bulk of the beta-APP contained the protease inhibitor insert, the authors propose that beta-APP is an important regulator of proteolysis in tear fluid and that possibly it plays a role in the events associated with corneal wound healing. This suggests a novel physiological function of beta-APP in addition to those previously described-regulation of blood coagulation and cell growth.
[Old data do not reflect current lower-risk IVF].
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Structure, thermodynamics and cooperativity of the glucocorticoid receptor DNA-binding domain in complex with different response elements. Molecular dynamics simulation and free energy perturbation studies.
Molecular dynamics simulations and free energy perturbation calculations have been performed on the glucocorticoid receptor DNA-binding domain (GRDBD) in complex with three different half-site response elements (RE). These are the glucocorticoid receptor RE (GREH), the estrogen receptor RE (EREH) and an "intermediate" RE (GRE2H), where the base-pair 6A.T in GREH has been replaced by 6G.C. The molecular dynamics/free energy perturbation calculations reveal the same order of stability of the three complexes, i.e. GREH-GRDBD > GRE2H-GRDBD > EREH-GRDBD, as found experimentally, with reasonable quantitative agreement of the relative stabilities with the experiments. The more favourable entropy in associating GRDBD to GREH than to GRE2H could be explained by the additional non-polar surface that is removed from the solvent in the former complex formation. Part of this additional surface originates from the 6T methyl group in GREH. Sequence-dependent hydration patterns of the DNA major groove, which is interrupted by the GRDBD binding, could also contribute to the relatively more favourable entropy in forming GREH-GRDBD than GRE2H-GRDBD. The tenfold reduced cooperativity in the dimeric GRDBD binding to a partially palindromic ERE (palERE) compared to palGRE/GRE2 could be attributed to conformational changes in the residues of the "D-box" (Cys476 to Cys482) observed in the molecular dynamics simulations of EREH-GRDBD, but not in the other two complexes. Moreover, the conformations of the base-pairs at positions 5 and 6 are distorted (compared to free B-DNA) in GREH and GRE2H, due to the binding of GRDBD. Since the corresponding conformational distortions were not found in EREH-GRDBD, it is plausible that the distortions mediate cooperative binding through a propagation of the distortions to the other half site, thus facilitating binding of the second GRDBD.
[A debate on test-tube fertilization. Conclusions by the medical-ethical committee should not be surprising!].
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Somatostatinergic phenotype markers in the human neuroblastoma cell-line LA-N-2.
We have characterized somatostatinergic phenotype markers of the human neuroblastoma, LA-N-2. A single mRNA-transcript (approximately 850bp) and two cellular somatostatin immunoreactivity forms, a high molecular weight form (M(r) 15,000) and a fragment corresponding to somatostatin-28 was found, while the somatostatin-14 peptide was absent. Saturation binding experiments demonstrated a single class of high-affinity somatostatin receptors with Kd and Bmax of 0.27 +/- 0.03 nM and 45 +/- 1 fmol/mg protein. Partial G-protein uncoupling (30%) was demonstrated, using GTP gamma S, with an affinity of 9.7 nM. The LA-N-2 cell line, previously shown to be cholinergic, may serve as a simplified system to elucidate heterologous neurotransmittor interactions. Such studies are of interest since dysfunctions of the cholinergic basal forebrain neurons and somatostatin immunoreactive interneurons have been consistently observed in Alzheimer's disease.
[Involuntary childlessness should receive higher priority. Knowledge about the "borderline" group is rather unclear].
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pi NN coupling from high precision np charge exchange at 162 MeV.
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Influence of place learning on somatostatin levels in the rat brain following environmental deprivation.
We have previously reported increased somatostatin levels in the cerebral cortex of rats housed in impoverished environment and subsequently subjected to a behavioural testing procedure, consisting of open-field exposure and spatial learning. The aim of the present study was to evaluate the degree of neurochemical specificity of the activation of somatostatin neurotransmission and to examine whether the altered levels were due to learning stimulation. Adult rats, previously housed individually for 30 days, were exposed to repeated sessions of a spatial learning task (2 days or 14 days) or repeated sessions of free swimming (14 days). The training sessions of the 14 day group consisted in recurrently changed position of the platform in a learning-set paradigm. Our data showed increased somatostatin immunoreactivity, and unchanged substance P immunoreactivity in the posterior part of the cerebral cortex. However, somatostatin levels increased to a similar extent following 14 days of repeated spatial learning sessions as free swimming sessions. We conclude that the activity of the cortical somatostatin system appears to be sensitive to environmentally induced sensorimotor stimulation in general, rather than learning per se. Thus, external stimulation of early clinical dementia patients with preserved sensorimotor receptivity, in an attempt to restore cognitive function, might be associated with altered somatostatin levels.
Factor for inversion stimulation-dependent growth rate regulation of serine and threonine tRNA species.
We have previously shown that the accumulation of 20 tRNA species in Escherichia coli is individually regulated as a function of cellular growth rate. We have also reported that the growth rate regulation of some but not all tRNA species is dependent on the activity of the factor for inversion stimulation (FIS). In present work, we studied the growth rate regulation of the serine- and threonine-accepting tRNA families. We show that the levels of tRNA(3Thr), tRNA(3Ser), tRNA(2Thr), tRNA(3Thr), and tRNA(4Thr) are reduced in fis cells as the growth rate increases. The accumulation of these tRNA species is reduced 2-5-fold at the fastest bacterial growth rate. The strongest effect is observed for the two minor tRNA species; tRNA(2Ser) and tRNA(2Thr). In contrast, we find that the accumulation of tRNA(1Ser), tRNA(5Ser), and tRNA(1Thr) is similar in wild type and fis bacteria. The data presented provide further evidence for the suggestion that FIS is a stimulating factor that is involved, directly or indirectly, in the high expression level of some tRNA genes at fast bacterial growth rates.
Analysis of the cyanide metabolite 2-aminothiazoline-4-carboxylic acid in urine by high-performance liquid chromatography.
The cyanide metabolite 2-aminothiazoline-4-carboxylic acid (ATC) was converted to N-carbamylcysteine and analyzed by high-performance liquid chromatography (HPLC) and fluorometric detection. ATC was first separated from interfering substances by passing through the cation exchanger AG 50W-X8. Interfering disulfides were converted to thiols by reduction of the eluate with thiopropyl-Sepharose 6B. The thiols were then absorbed to the organomercurial absorbent p-acetoxymercurianiline-Sepharose 4B while ATC was obtained in the effluent. ATC was then converted to N-carbamylcysteine by ring opening at high temperature in an alkaline environment. Derivatization of N-carbamylcysteine was with N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide. The imprecision (CV) of the method was 3.6% and the analytical recovery was quantitative. The detection limit was 0.3 microM. ATC in urine was stable for at least 3 months when stored at -196 to +20 degrees C. The ATC concentration in urine was below the limit of detection in health nonsmokers. The utility of the method was demonstrated by the finding of up to 10.5 microM ATC concentrations in urine samples from human subjects in Mozambique living on a high dietary intake of cyanide from the cassava root. In a metabolic study rats were exposed to acetonitrile in the drinking water. After 4 weeks very high ATC levels were found in the urine, representative values from two rats being 195 and 525 microM, respectively.
[Ovulation stimulation. Use and "abuse"].
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Structural basis for pleckstrin homology domain mutations in X-linked agammaglobulinemia.
Deficiencies in a tyrosine kinase, designated Btk, cause X-linked agammaglobulinemia (XLA) in man, a hereditary defect of B-cell differentiation. Mutations in the newly found PH domain located at the N-terminus of Btk have been shown to be the direct cause of XLA, and here two new mutations, T33P and V64F, are presented. Btk is thus far the only protein in which mutations of the PH domain have been found to cause a disease. The three-dimensional structure of the Btk PH domain was modeled on the basis of the dynamin PH structure. Despite a relatively low sequence similarity the Btk PH domain seems to have the same two beta-sheet structure observed in the known structures. The model was used to interpret the structural basis for disease in five independent point mutations and in an insertion in patients with XLA. The mutated residues F25, V64, and V113, and possibly residue(s) around Q103, could form a binding site, since these amino acids are located close to each other on the surface of the molecule.
Effects of N-terminal deletions of the Escherichia coli protein Fis on growth rate, tRNA(2Ser) expression and cell morphology.
The Escherichia coli Fis protein is known to be involved in a variety of processes, including the activation of stable RNA operons. In this paper we study the ability of a set of N-terminal Fis deletion mutants to stimulate transcription of the tRNA(2Ser) gene. The results indicate that the domain of the Fis protein containing residues 1-26 is not required for transcription activation. The Fis mutants that are still active in transcription stimulation can also complement the reduced growth rates of Fis- cells, suggesting that the same activating domain is involved in this phenomenon. In addition, we show that in fast growing cultures in the absence of an active Fis protein, minicells are formed. These minicells seem to arise from septum formation near the cell poles. Suppression of minicell formation by Fis also does not require the presence of the N-terminal domain of the protein.