1.
Greiff, Lennart, et al.
(author)
Effects of topical platelet activating factor on the guinea-pig tracheobronchial mucosa in vivo
1997
In: Acta Physiologica Scandinavica. - 0001-6772. ; 160:4, s. 387-391
Journal article (peer-reviewed) abstract
Platelet activating factor (PAF) has been reported to produce a variety of airway effects including epithelial damage and increased airway-lung absorption of hydrophilic tracers. The present study examines effects of PAF on the guinea-pig tracheobronchial mucosa in vivo. Vehicle with and without PAF (4.0 and 8.0 nmol) was superfused onto the tracheobronchial mucosa. The levels of 125I-albumin, previously given intravenously, were determined in tracheobronchial lavage fluids as an index of mucosal exudation of plasma. The mucosa was also examined by scanning electron microscopy. In separate animals, 99mTc-DTPA (a low molecular weight, 492 Da, hydrophilic tracer) was superfused onto the mucosal surface through an oro-tracheal catheter, together with vehicle or PAF (8.0 nmol). A gamma camera determined the disappearance rate of 99mTc-DTPA from the airways as an index of mucosal absorption. PAF produced dose-dependent mucosal exudation of plasma up to 20-fold greater than control (P < 0.001). However, PAF did not damage the epithelium and the absorption ability of the airway mucosa was unaffected. The results, in contrast to previous reports, suggest that PAF may not readily damage the airway mucosa even at large exudative doses of the agent. The present finding support the view that the plasticity of the epithelial junctions allows the creation of valve-like paracellular pathways for unidirectional clearance of extravasated plasma into the airway lumen. We suggest that endogenous PAF may participate in first line respiratory defence reactions by causing lumenal entry of bulk plasma without harming the epithelium.
2.
Korsgren, Magnus, et al.
(author)
Allergic eosinophil-rich inflammation develops in lungs and airways of B cell-deficient mice
1997
In: Journal of Experimental Medicine. - 1540-9538. ; 185:5, s. 885-892
Journal article (peer-reviewed) abstract
Immunoglobulins (Ig), particularly IgE, are believed to be crucially involved in the pathogenesis of asthma and, equally, in allergic models of the disease. To validate this paradigm we examined homozygous mutant C57BL/6 mice, which are B cell deficient, lacking all Ig. Mice were immunized intraperitoneally with 10 micrograms ovalbumin (OVA) plus alum, followed by daily (day 14-20) 30 min exposures to OVA aerosol (OVA/OVA group). Three control groups were run: OVA intraperitoneally plus saline (SAL) aerosol (OVA/SAL group); saline intraperitoneally plus saline aerosol; saline intraperitoneally plus OVA aerosol (n = 6-7). Lung and large airway tissues obtained 24 h after the last OVA or SAL exposure were examined by light microscopy and transmission electron microscopy (TEM). The Ig-deficient mice receiving OVA/ OVA treatment had swollen and discolored lungs and exhibited marked eosinophilia both in large airway subepithelial tissue (49.2 +/- 12.0 cells/mm basement membrane [BM] versus OVA/ SAL control 1.2 +/- 0.3 cells/mm BM; P < 0.001), and perivascularly and peribronchially in the lung (49.3 +/- 9.0 cells/unit area versus OVA/SAL control 2.6 +/- 0.6 cells/unit area; P < 0.001). The eosinophilia extended to the regional lymph nodes. TEM confirmed the subepithelial and perivascular localization of eosinophils. Mucus cells in large airway epithelium increased from 1.5 +/- 0.8 (OVA/SAL mice) to 39.5 +/- 5.7 cells/mm BM in OVA/OVA treated mice (P < 0.001). OVA/SAL mice never differed from the other control groups. Corresponding experiments in wild-type mice (n = 6-7 in each group) showed qualitatively similar but less pronounced eosinophil and mucus cell changes. Macrophages and CD4+ T cells increased in lungs of all OVA/OVA-treated mice. Mast cell number did not differ but degranulation was detected only in OVA/OVA-treated wild-type mice. Immunization to OVA followed by OVA challenges thus cause eosinophil-rich inflammation in airways and lungs of mice without involvement of B cells and Ig.
3.
Persson, Carl, et al.
(author)
The mouse trap
1997
In: Trends in Pharmacological Sciences. - 0165-6147. ; 18:12, s. 465-467
Journal article (peer-reviewed) abstract
Mouse models of asthma are now being used extensively in drug research. However, the successful unravelling of combinatorial interplays of cells and molecules in the murine airways may not be matched by equally successful demonstrations of an asthma-like pathophysiology. Here, Carl Persson, Jonas Erjefalt, Magnus Korsgren and Frank Sundler discuss the fact that major features of asthma may still need to be demonstrated in the airways of allergic mice.
4.
Elies, Rozenn, et al.
(author)
Immunochemical and functional characterization of an agonist-like monoclonal antibody against the M2 acetylcholine receptor.
1998
In: European journal of biochemistry / FEBS. - 0014-2956. ; 251:3, s. 659-66
Journal article (peer-reviewed) abstract
Monoclonal antibodies were raised against a peptide corresponding to the second extracellular loop of the M2 acetylcholine receptor. One of the monoclonal antibodies, B8E5, was selected for further characterization on the basis of its high yield, its isotype (IgG2a), its dissociation kinetics and its agonist-like activity. The epitope recognized by B8E5 corresponded to the N-terminal part of the second extracellular loop of the receptor (V-R-T-V-E-) as determined by competition immunoassays and epitope scanning. The KA of B8E5 for the target peptide was assessed by surface plasmon resonance (SPR) to be 6.5x10(7) M(-1) by equilibrium and 3.7x10(7) M(-1) by kinetic analysis. B8E5 recognized the M2 acetylcholine receptor on rat cardiac tissue. It only recognized the non-reduced receptor in immunoblots. The antibody had no effect on antagonist binding but decreased the affinity for the agonist carbachol. B8E5 decreased the beating frequency of neonatal rat cardiomyocytes. The effect was specific since it was blocked by the target peptide and the antagonist atropine. The EC50 of the antibody corresponded to the KA measured by surface plasmon resonance. The physiological effect of the antibody did not lead to desensitization. The Fab fragments had no physiological effect; subsequent addition of anti-mouse IgG however restored the physiological effect. These results confirm that the N-terminus of the second extracellular loop is a functional target for antibodies against the M2 acetylcholine receptor. They suggest that the functional epitope is only accessible in the non-reduced receptor. The antibodies act through a functional dimerization of the receptor.
5.
Fu, Michael, 1963, et al.
(author)
Agonist-like activity of antibodies to angiotensin II receptor subtype 1 (AT1) from rats immunized with AT1 receptor peptide.
1999
In: Blood pressure. - 0803-7051. ; 8:5-6, s. 317-24
Journal article (peer-reviewed) abstract
In the present study, rats were immunized with angiotensin II receptor subtype 1 (AT1) receptor peptides for 3 months to see if the immunization produced specific anti-AT1 receptor antibodies and if continuous stimulation for 3 months affected blood pressure or induced morphological changes in the organs containing AT1 receptors. Our results showed that there were constant high levels of circulating antibodies throughout the study period in all rats of the immunized group, but not in the control rats, and that there were almost no significant cross-reactions of antisera with AT2 receptor peptide and alpha1 adrenoceptor peptide, except in four rats, which showed low cross-reactions with alpha1 adrenoceptor and AT2 receptor peptides. When an affinity-purified anti-AT1 receptor antibody was used, it specifically displayed the AT1-stimulatory positive chronotropic effect and also localized AT1 receptors. However, in the immunized group, saturation binding of AT1 in homogenates from kidneys showed no difference either in maximal binding sites (Bmax) or in antagonist affinity (Kd). No difference in mRNA of AT1a was found in either kidney or heart, and no morphological changes in the organs were observed, as compared with the control group. Furthermore, immunization did not cause hypertension. In conclusion, the synthetic peptide corresponding to the second extra-cellular loop of the human AT1 receptor was able to produce highly specific and functionally active anti-AT1 receptor antibodies, but unable to induce pathological structural changes or hypertension.
6.
Fu, Michael, 1963, et al.
(author)
Immunohistochemical localization of angiotensin II receptors (AT1) in the heart with anti-peptide antibodies showing a positive chronotropic effect.
1998
In: Receptors & channels. - 1060-6823. ; 6:2, s. 99-111
Journal article (peer-reviewed) abstract
Antibodies were produced against a synthetic peptide corresponding to amino acids (165-191) of the second extracellular loop of the human angiotensin II receptor subtype 1 (AT1) in rabbits. The purified antibodies had an apparent affinity of about 1 nM and were monospecific for the AT1-receptor peptide. Chemical modification of the carboxyl groups (glu at positions 173 and 185) and the sulfhydryl group (cys at position 180) of the AT1-receptor peptide did not alter the relative affinity of the coated AT1-receptor peptide to antibodies. The antibodies specifically stained CHO cells expressing the rat AT1a receptor. Immunoblots on rat kidney revealed that the antibody recognized a protein band of 59 +/- 3 kDa in a dose-dependent manner and this band was no longer detected after preincubating the antibodies with AT1-receptor peptide. Using electron microscopic and immunofluorescence immunocytochemistry techniques, angiotensin II receptors were detected in (1) the sarcolemma, T-tubules and nuclei of rat cardiomyocytes, (2) the transluminal side of endothelial cells and (3) fibroblast cells. These localizations are specific, as the immunostaining did not appear when preimmune rabbit serum was used and was blocked after preincubating antibodies with antigenic peptide. Functionally, these antibodies did not affect the ligand binding properties of the receptors but displayed agonist-like activity as shown by dose-dependent increases in beating frequency in cultured neonatal cardiomyocytes. These results suggest that the antibodies against the second extracellular loop of human AT1 receptors were able to specifically recognize AT1 receptors. In addition, they extend the observation that the second extracellular loop of the G-protein coupled membrane receptors is a specific target for antibodies with agonist-like activity.
7.
Holtbäck, U, et al.
(author)
Receptor recruitment: a mechanism for interactions between G protein-coupled receptors.
1999
In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 96:13, s. 7271-5
Journal article (peer-reviewed) abstract
There is a great deal of evidence for synergistic interactions between G protein-coupled signal transduction pathways in various tissues. As two specific examples, the potent effects of the biogenic amines norepinephrine and dopamine on sodium transporters and natriuresis can be modulated by neuropeptide Y and atrial natriuretic peptide, respectively. Here, we report, using a renal epithelial cell line, that both types of modulation involve recruitment of receptors from the interior of the cell to the plasma membrane. The results indicate that recruitment of G protein-coupled receptors may be a ubiquitous mechanism for receptor sensitization and may play a role in the modulation of signal transduction comparable to that of the well established phenomenon of receptor endocytosis and desensitization.
8.
Liu, H R, et al.
(author)
Screening of serum autoantibodies to cardiac beta1-adrenoceptors and M2-muscarinic acetylcholine receptors in 408 healthy subjects of varying ages.
1999
In: Autoimmunity. - 0891-6934. ; 29:1, s. 43-51
Journal article (peer-reviewed) abstract
Autoantibodies to cardiac beta1-adrenoceptors and M2-muscarinic receptors have mainly been found in the sera of patients with idiopathic dilated cardiomyopathy (DCM). In order to elucidate the pathological significance of these autoantibodies in DCM, it is necessary to understand their characteristic distribution in a healthy population of different genders and ages. The peptides corresponding to the sequences of the second extracellular loops of the human beta1-adrenoceptor and M2-muscarinic receptors were therefore used as antigens to screen the sera of 408 healthy subjects of different ages (ranging from 0.5 to 85 years). Of 408 sera, 41 (10.0%) and 46 (11.3%) recognized the beta1-adrenoceptor and M2-muscarinic receptor peptides respectively. Of the positive sera for beta1-adrenoceptors and M2-muscarinic receptors, up to 63.4% and 56.5% had both anti-beta1-adrenoceptor and anti-M2-muscarinic receptor autoantibodies respectively. The antibody titres of the positive sera of healthy subjects were all of a low level, with a geometric mean titre of 1:42+/-1.9 for anti-beta1-adrenoceptor antibodies and 1:51+/-1.7 for anti-M2-muscarinic receptor antibodies. The frequency of occurrence of autoantibodies to both receptors in the sera of healthy subjects increased significantly with age. In conclusion, the autoantibodies to beta1-adrenoceptors and M2-muscarinic receptors in the sera of healthy subjects are characterized by a low frequency of occurrence and low titre, with the frequency of occurrence increasing with age.
9.
Matsui, S, et al.
(author)
Active immunization of combined beta1-adrenoceptor and M2-muscarinic receptor peptides induces cardiac hypertrophy in rabbits.
1999
In: Journal of cardiac failure. - 1071-9164. ; 5:3, s. 246-54
Journal article (peer-reviewed) abstract
BACKGROUND: The high prevalence of patients with dilated cardiomyopathy (DCM) with anti-beta1-adrenoceptor and/or anti-M2-muscarinic receptor autoantibodies in their sera has been observed. However, the pathophysiological role of these autoantibodies in the development of cardiomyopathy is unknown. We previously reported an experimental model of early-stage DCM-like cardiomyopathy induced by immunizing rabbits for 1 year with synthetic peptides corresponding to the sequence of the second extracellular loop of either beta1-adrenoceptor or M2-muscarinic receptor. Because approximately half the sera of patients with DCM that recognize one of the two receptor sequences also recognize the second sequence, a model was created in rabbits simultaneously immunized with the synthetic peptides corresponding to the second extracellular loop of the beta1-adrenoceptor and M2-muscarinic receptor. METHODS AND RESULTS: All rabbits (n = 8) immunized with both peptides had a high titer of both anti-beta1-adrenoceptor and anti-M2-muscarinic receptor autoantibodies in their sera, whereas none of the sera from control rabbits injected with saline (n = 9) was positive. No significant cross-reaction with peptides other than those used for immunization was found. The weight of the hearts of immunized rabbits increased significantly. The hearts of immunized rabbits showed marked concentric left ventricular hypertrophy with mild inflammatory cell infiltration. In these rabbits, mild or moderate interstitial fibrosis was also observed. In electron micrographs, immunized rabbits showed focal myofibrillar lysis, loss of myofilament, and a marked increase in the number of mitochondria and deposition of dense granules in both sarcoplasm and myofibrils. Conversely, one of the control rabbits showed scant mononuclear cell infiltration. However, in this control rabbit, no significant alteration was found by electron microscopy. CONCLUSION: Our results showed the coexistence of both anti-beta1-adrenoceptor and anti-M2-muscarinic receptor autoantibodies in the sera has pathophysiological importance, shown by their ability to induce cardiac hypertrophy in rabbits.
10.
Matsui, S, et al.
(author)
Myocardial injury due to G-protein coupled receptor-autoimmunity.
1998
In: Japanese heart journal. - 0021-4868. ; 39:3, s. 261-74
Journal article (peer-reviewed) abstract
One of the main mechanisms for dilated cardiomyopathy is likely to be autoimmune mediated myocardial damage. So far, a variety of autoantibodies have been detected against a number of putative autoantigens in the sera of patients with dilated cardiomyopathy. A growing body of studies have confirmed that autoantibodies against the second extracellular loop of beta 1-adrenoceptors and M2-muscarinic receptor are present in 30-40% of patients with dilated cardiomyopathy. These anti-beta 1-adrenoceptor and anti-M2-muscarinic receptor antibodies can not only decrease the binding sites of antagonist but also recognize the target receptors. Moreover, these two autoantibodies possess an 'agonist-like' stimulatory effect on the target receptors. In order to elucidate whether the autoantibodies against these autoimmune epitopes play an important role in the pathogenesis of dilated cardiomyopathy, we immunized rabbits over a period of one year with synthetic peptides corresponding to the second extracellular loop of the beta 1-adrenoceptor and the M2-muscarinic receptor. These peptides induced morphological changes in the heart similar to those found in dilated cardiomyopathy. These clinical and experimental findings suggest that these receptor autoantigens are of pathogenic importance in the development of dilated cardiomyopathy in vivo.
11.
Matsui, S, et al.
(author)
Peptides derived from cardiovascular G-protein-coupled receptors induce morphological cardiomyopathic changes in immunized rabbits.
1997
In: Journal of molecular and cellular cardiology. - : Elsevier BV. - 0022-2828. ; 29:2, s. 641-55
Journal article (peer-reviewed) abstract
An experimental model of early-stage cardiomyopathy was created by immunizing rabbits for 1 year with synthetic peptides corresponding to the sequence of the second extracellular loop of either beta-adrenoceptors or M2-muscarinic receptors. Thirty male rabbits were used and divided into three groups: a control group (n = 10), a group immunized with the peptide corresponding to the beta-adrenoceptor (beta 1 group) (n = 10) and a group immunized with the peptide corresponding to the M2-muscarinic receptor (M2 group) (n = 10). If the sera from both groups of immunized rabbits high-titres of anti-peptide antibodies were found throughout the study period but not in the sera from control rabbits or in the preimmune sera of immunized rabbits. No significant cross-reaction with peptides other than those used for immunization was found. The myocardial receptor density of both immunized groups displayed a strong trend toward receptor up-regulation. This was significant in the beta 1 group but not in the M2 group. Both groups of immunized rabbits displayed significantly enlarged ventricles and thinner walls, as compared with the control group. However, in contrast to the beta 1 group, which showed enlarged cavities in both left and right ventricles, the M2 group was mainly affected in the right ventricles. Moreover, morphological examinations of the hearts of rabbits from both immunized groups demonstrated focal myofibrillar lysis, loss of myofilament, mitochondrial swelling and condensation, sarcoplasmic vacuolation, deposition of dense granules in the sarcoplasm and the myofibrils. One of the sex control rabbit hearts which were examined showed mild degenerative changes in the myocardium and scant mononuclear cell infiltration. However, when all the control rabbit hearts were examined by electron microscopy, no significant alterations were found. These results suggest that immunization by peptides, corresponding to the target sequences for anti-receptor autoantibodies in idiopathic dilated cardiomyopathy, induces morphological changes in the heart similar to those found in the human disease.
12.
Peukert, S, et al.
(author)
The frequency of occurrence of anti-cardiac receptor autoantibodies and their correlation with clinical manifestation in patients with hypertrophic cardiomyopathy.
1999
In: Autoimmunity. - 0891-6934. ; 29:4, s. 291-7
Journal article (peer-reviewed) abstract
The purpose of this study was to investigate the frequency of occurrence of autoantibodies against G-protein coupled cardiovascular receptors and their relation to the clinical manifestation of hypertrophic cardiomyopathy (HCM). Autoantibodies against beta1-receptors, Muscarin-2-receptors, Angiotensin-II-receptor subtype 1 and alpha1-receptors were determined with ELISA in 52 patients with HCM (37 male, 15 female, mean age 55 +/- 15 years) and 40 healthy, age and sex matched controls. The clinical characterization of the HCM-patients included ECG, 24-h Holter, and echocardiography. The results showed that there is no significant difference in the frequency of a single autoantibody between HCM-patients and controls. However, if the number of patients who have autoantibodies against beta1-receptors and/or Muscarin-2-receptors were counted together, there are significantly more autoantibodies in HCM compared to controls (11 vs. 2, p = 0.035). Analysis of clinical data from this pooled group of patients showed that in patients with autoantibodies, heart rate variability (HRV), ultra low frequency (ULF) and very low frequency (VLF) were decreased (HRV by 20%, ULF by 50%, and VLF by 46%, p < 0.008) whereas the QTc-interval was increased by 8% (p < 0.02 each). The ratio of septal to posterior wall thickness was increased by 23% (p = 0.05), and the preejection period was prolonged by 46% in patients with autoantibodies (p < 0.001). These results suggest that the existence of these autoantibodies could be associated with an advanced stage or a severe manifestation of HCM.
13.
Wang, W Z, et al.
(author)
Effects of anti-peptide antibodies against human M2 muscarinic receptors on the cAMP generating system in guinea pig ventricles.
1996
In: Blood pressure. Supplement. - 0803-8023. ; 3, s. 22-4
Journal article (peer-reviewed) abstract
The effects of anti-peptide antibodies (Ab) against the second extracellular loop of human muscarinic receptor-2 on the cAMP generating system in guinea pig ventricles were studied. These effects were compared with those of the muscarinic receptor agonist carbachol (Carb). It was shown that: (1) both Carb and Ab were able to inhibit the isoproterenol (Iso)-stimulated cAMP production of ventricles in a dose-dependent manner. Carb at 2 microM, 10 microM and 50 microM decreased Iso-stimulated cAMP production by 8.0 +/- 1.1, 15.8 +/- 1.2 and 28.4 +/- 1.7%, respectively; whereas Ab at 50 nM, 100 nM and 400 nM decreased it by 5.8 +/- 0.4, 16.8 +/- 1.4 and 30.6 +/- 2.5%, respectively. (2) Both Carb and Ab could also inhibit the basal cAMP content of ventricles significantly. Carb at 10 microM and Ab at 100 nM decreased it by 46.9 +/- 4.2% and 60.2 +/- 4.6%, respectively. (3) The inhibitory effects of both Ab and Carb on Iso-stimulated cAMP production were significantly prevented by atropine at 1.5 microM. (4) The inhibitory effect of Ab at 100 nM was almost completely abolished by the peptide (700 nM) used as immunogen. These findings suggest that the antibodies exhibit a stimulatory muscarinic activity similar to carbachol in the inhibitory modulation of cAMP production.
14.
Zhao, R, et al.
(author)
Effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic acetylcholine receptors on transmembrane potentials and currents in guinea pig ventricular myocytes.
1996
In: Molecular and cellular biochemistry. - 0300-8177. ; 163-164, s. 185-93
Journal article (peer-reviewed) abstract
The effects of anti-peptide antibodies against the second extracellular loop of human M2 muscarinic receptor on transmembrane potentials and currents in guinea pig single ventricular cells were analyzed using whole-cell patch clamp technique. These effects were compared with those of the muscarinic receptor agonists carbachol and acetylcholine. The antibodies shortened the action potential duration in a dose-dependent manner. By using a ramp or step rectangular pulse protocol, it was found that the antibodies increased the outward K+ current and decreased the inward basal ICa significantly. The reversal potential of both carbachol- and antibody-induced extra currents were close to -80 mV, being in proximity to the calculated Ek of -90 mV. A beta-adrenergic receptor agonist, isoprenaline, prolonged the action potential and increased the overshoot which could be inhibited by both antibody and carbachol. Isoprenaline increased inward ICa and outward Ik simultaneously. Both antibody and carbachol could significantly reduce the isoprenaline-stimulated ICa but not the isoprenaline-stimulated Ik. The antibody- or carbachol-induced outward K+ current and the depressant effects of antibody and carbachol on isoprenaline-stimulated ICa were partially antagonized by atropine. These results suggest that the anti-M2 muscarinic receptor antibodies display a stimulatory activity similar to muscarinic receptor agonist on the receptor-mediated electrophysiological events.
15.
Sadziene, Ariadna, et al.
(author)
A bactericidal antibody to Borrelia burgdorferi is directed against a variable region of the OspB protein
1994
In: Infection and Immunity. - : American Society for Microbiology (ASM). - 0019-9567 .- 1098-5522. ; 62:5, s. 2037-2045
Journal article (peer-reviewed) abstract
Borrelia burgdorferi, an agent of Lyme disease, is killed by some monoclonal antibodies in the absence of complement or phagocytes. In the present study, the bactericidal action of monoclonal antibodies against B. burgdorferi and B. hermsii, a cause of relapsing fever, was further characterized. H6831, an antibody recognizing the OspB proteins of some B. burgdorferi strains, and H4825, an antibody specific for one serotype of B. hermsii, were purified, and Fab fragments of the antibodies were prepared. In time-kill studies, more than 99.9% of strain B31 B. burgdorferi cells were killed after 30 min of exposure to H6831 Fab fragments. The MBC of the Fab fragments was 10 micrograms/ml. Electron microscopy revealed that the bactericidal Fab fragments produced numerous blebs and cell lysis of the borrelias for which they were specific. To identify the epitope for H6831, the OspB sequences of H6831-susceptible and -resistant strains and mutants were determined. The deduced OspB proteins of all H6831-resistant strains and mutants differed from the strain B31 OspB at residue 253. Murine antisera raised against a 21-mer synthetic peptide representing the region around residue 253 were specific for strain B31 by Western blot (immunoblot) and growth inhibition assays. Furthermore, the antipeptide serum inhibited the binding of H6831 to whole borrelias. These findings indicated that the linear component of the bactericidal antibody's epitope was located at or near residue 253.
16.
Ata, K A, et al.
(author)
Expression of transforming growth factor-beta1, 2, 3 isoforms and type I and II receptors in acute focal cerebral ischemia: an immunohistochemical study in rat after transient and permanent occlusion of middle cerebral artery.
1999
In: Acta neuropathologica. - 0001-6322. ; 97:5, s. 447-55
Journal article (peer-reviewed) abstract
Transforming growth factor beta (TGF-beta) is involved in the modulation of cell growth, differentiation and repair following injury of various organs. Previous studies on human autopsy material have indicated that TGF-beta isoforms-beta1, -beta2 and -beta3, and TGF-beta receptor type I are expressed in various cells of necrotizing brain lesions like infarction and abscess. The present immunohistochemical study was designed to investigate changes that may occur with regard to TGF-beta and its receptors type I and II in a rat model of focal brain ischemia induced by transient or permanent occlusion of the middle cerebral artery. Our findings indicate that at days 1 and 3 following such transient and permanent ischemia there is an up-regulation of TGF-beta isoforms -beta1, -beta2 and -beta3, and TGF-beta receptor types I and II mainly in the perifocal neurons, reactive astroglial cells, endothelial cells and macrophages.
17.
Fransson, Lars-Åke, et al.
(author)
Oligosaccharide mapping of proteoglycan-bound and xyloside-initiated dermatan sulfate from fibroblasts
1991
In: Glycoconjugate Journal. - 1573-4986. ; 8:2, s. 108-115
Journal article (peer-reviewed) abstract
The copolymeric structure of dermatan sulfate chains synthesized by skin fibroblasts has been examined. Chains initiated onto exogeneous p-nitrophenyl-beta-D-xylopyranoside or attached to protein in a large proteoglycan, PG-L, and two small proteoglycans, PG-S1 and PG-S2, have been compared by using high resolution electrophoresis and gel chromatography of oligosaccharides generated by specific enzymatic or chemical degradations. The results confirm that chains attached to PG-L are glucuronate-rich, whereas novel findings indicate that chains attached to either of the two PG-S variants yield closely similar oligosaccharide maps, have approximately equal glucuronate and iduronate content and contain over 90% 4-sulfated disaccharide repeating units. Dermatan sulfate chains built onto xyloside at concentrations of 50 microM and below have a copolymeric structure similar to that of chains from the two PG-S variants. These findings indicate that the polymer-modifying machinery can generate chains with extended iduronate-containing repeats also when the xylose primer is not linked to core protein.
18.
Fu, Michael, 1963, et al.
(author)
Increase in functional activity rather than in amount of Gi-alpha in failing human heart with dilated cardiomyopathy.
1992
In: Cardiovascular research. - 0008-6363. ; 26:10, s. 950-5
Journal article (peer-reviewed) abstract
OBJECTIVE: The aim was to investigate whether or not increased pertussis toxin catalysed ADP ribosylation correlates with increased amount of Gi-alpha in failing human heart. DESIGN: Antisera raised against unique synthetic peptides corresponding to alpha subunits of Gs and Gi 1-3 were used in immunoblotting and ELISA to determine amounts of various G proteins. Adenylyl cyclase activity, beta adrenoceptors, and muscarinic receptors were then measured in cardiomyopathic hearts (n = 6) obtained at transplant in order to study whether or not an altered expression of G proteins has relevance to the integrity and function of the receptor--adenylyl cyclase system. Six non-failing control hearts were also studied. RESULTS: No significant differences in the peptide equivalent amounts of either Gs or Gi were found in the failing human heart as compared to the non-failing heart. However, functional activity of Gi was shown to increase significantly since there was a decrease in basal (57%), isoprenaline stimulated (60%), and guanyliminodiphosphate stimulated (52%) adenylyl cyclase activity. In contrast the density of beta adrenoceptors was markedly decreased (51%) in failing human heart in comparison to non-failing hearts. Neither the density nor the affinity of muscarinic receptors changed in the failing human heart. CONCLUSION: These results suggest that in the failing human heart, there is an increase in functional activity rather than in amount of Gi, and an important part of functional expression of Gi-alpha may be regulated at the post-translational level.
19.
20.
Leung, P S, et al.
(author)
Angiotensin II receptors, AT1 and AT2 in the rat epididymis. Immunocytochemical and electrophysiological studies.
1997
In: Biochimica et biophysica acta. - 0006-3002. ; 1357:1, s. 65-72
Journal article (peer-reviewed) abstract
Previous work from our laboratory has provided evidence for the presence of a tissue renin-angiotensin system in the rat epididymis. In the current investigation, the regional localization of angiotensin II receptors, type I (AT1) and type II (AT2) was studied immunocytochemically using specific anti-peptide antibodies against the second extracellular loops of AT1 and AT2 receptors, and pharmacologically using specific receptor antagonists in conjunction with the short-circuit current technique. The immunocytochemical results showed that AT1 and AT2 immunoreactivities were predominantly localized in the basal region of the epididymal epithelium. Electrophysiological studies using the short-circuit current technique demonstrated a stimulatory effect of basolaterally applied angiotensin II on the epididymal electrogenic ion transport. This effect was inhibitable by the addition of AT1 antagonist, losartan but not by AT2 antagonist, PD123177, indicating a functional role of AT1 in epididymal electrolyte transport. The present finding suggests that angiotensin II receptors may play an important role in the regulation of epididymal function.
21.
Nelimarkka, Lassi, et al.
(author)
Expression of small extracellular chondroitin/dermatan sulfate proteoglycans is differentially regulated in human endothelial cells.
1997
In: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 272:19, s. 12730-12737
Journal article (peer-reviewed) abstract
We have examined the expression of the small extracellular chondroitin/dermatan sulfate proteoglycans (CS/DS PGs), biglycan, decorin, and PG-100, which is the proteoglycan form of colony stimulating factor-1, in the human endothelial cell line EA.hy 926. We have also examined whether modulation of the phenotype of EA.hy 926 cells by tumor necrosis factor-alpha (TNF-alpha) is associated with specific changes in the synthesis of these PGs. We demonstrate that EA.hy 926 cells, when they form monolayer cultures typical of macrovascular endothelial cells, express and synthesize detectable amounts of biglycan and PG-100, but not decorin. On SDS-polyacrylamide gel electrophoresis both PGs behave like proteins of the relative molecular weight of approximately 250,000. TNF-alpha that changed the morphology of the cells from a polygonal shape into a spindle shape and that also stimulated the detachment of the cells from culture dish, markedly decreased the net synthesis of biglycan, whereas the net synthesis of PG-100 was increased. These changes were parallel with those observed at the mRNA level of the corresponding PGs. The proportions of the different sulfated CS/DS disaccharide units of PGs were not affected by TNF-alpha. Several other growth factors/cytokines, such as interferon-gamma, fibroblast growth factors-2 (FGF-2) and -7 (FGF-7), interleukin-1beta, and transforming growth factor-beta, unlike TNF-alpha, modulated neither the morphology nor the biglycan expression of EA.hy 926 cells under the conditions used in the experiments. However, PG-100 expression was increased also in response to FGF-2 and -7 and transforming growth factor-beta. None of the above cytokines, including TNF-alpha, was able to induce decorin expression in the cells. Our results indicate that the regulatory elements controlling the expression of the small extracellular CS/DS PGs in human endothelial cells are different.
22.
Skoglund, Thomas, 1969, et al.
(author)
A method for 2D reconstruction of intracellularly labeled neurons from sequential sections.
1994
In: Journal of neuroscience methods. - 0165-0270. ; 53:2, s. 199-202
Journal article (peer-reviewed) abstract
A technique for 2D reconstruction of intracellularly labeled neurons from sequential sections is described. The system consists of a Charged Coupled Device-camera mounted on a microscope, a videomixer and a IBM-compatible PC with a framegrabber. The neurons (interneurons from the spinal cord of the cat) were labeled iontophoretically by horshradish peroxidase and subsequently cut in 60 microns sections. The sections were aligned using the video mixer by fitting the cut dendrites and axon from one section with their counterparts in the following section. The images were then digitized in the PC where they were fused to create a superimposed picture of the aligned parts of the neuron; a 2D reconstruction was created.
23.
Wallukat, G, et al.
(author)
Agonistic effects of anti-peptide antibodies and autoantibodies directed against adrenergic and cholinergic receptors: absence of desensitization.
1996
In: Blood pressure. Supplement. - 0803-8023. ; 3, s. 31-6
Journal article (peer-reviewed) abstract
Affinity-purified autoantibodies and anti-peptide antibodies directed against the second extracellular loop of the beta 1-adrenoceptor increase the beating rate of cultured cardiomyocytes just like the beta-adrenergic agonist isoprenaline. Their positive chronotropic action is blocked by beta-adrenergic antagonists. Affinity-purified autoantibodies and anti-peptide antibodies directed against the muscarinic cholinergic M2 receptor exert in these myocytes, like the muscarinic cholinergic agent carbachol, a negative chronotropic effect that is antagonized by atropine. In contrast to the agonism of isoprenaline and carbachol, the described agonistic effects of the antibodies are not subject to desensitization.
24.
Xu, X, et al.
(author)
Postreceptor events involved in the up-regulation of beta-adrenergic receptor mediated lipolysis by testosterone in rat white adipocytes.
1993
In: Endocrinology. - 0013-7227. ; 132:4, s. 1651-7
Journal article (peer-reviewed) abstract
In the previous studies we have shown that testosterone increases lipolytic responsiveness to catecholamines in rat white adipocytes, and that is associated with an up-regulation of beta-adrenergic receptor density. However, the postreceptor events involved in the testosterone induced enhancement of beta-adrenergic receptor activated lipolysis in these cells have not been adequately studied, and were therefore investigated in the present study. Male Sprague Dawley rats were divided into three groups: control, castrated, and castrated treated with testosterone. The beta-adrenergic receptor-mediated cAMP accumulation, measured with RIA after isoproterenol (a beta-adrenergic agonist) stimulation was decreased in castrated rats, and reversed by testosterone treatment, suggesting a testosterone effect at or proximal to adenylate cyclase. However, no differences between the groups were found in abundance of G alpha protein messenger RNAs (G alpha s, G alpha i-1, and G alpha i-2) as analyzed by Northern blot and a solution hybridization RNase protection assay, or in G protein mass measured with a quantitative enzyme-linked immunosorbent assay in fat cell membrane preparation. Lipolysis stimulated by N6-monobutyryl-cAMP was reduced in castrated rats and recovered by testosterone treatment, suggesting that components distal to the adenylate cyclase, i.e. protein kinase A (PKA) and/or hormone sensitive lipase (HSL) also are involved in testosterone regulation of lipolysis. In conclusion, these and previous results suggest that the testosterone-induced increase in lipolytic response to catecholamines in rat white adipocytes is mediated through several events including an increased beta-adrenergic receptor density, probably an increased adenylate cyclase activity and an increased protein kinase A/hormone sensitive lipase activity at the postreceptor level with apparent absence of effect on the expression of G-proteins.
25.
Vujic, Mihailo, 1945, et al.
(author)
Localization of a gene for autosomal dominant Larsen syndrome to chromosome region 3p21.1-14.1 in the proximity of, but distinct from, the COL7A1 locus.
1995
In: American journal of human genetics. - 0002-9297. ; 57:5, s. 1104-13
Journal article (peer-reviewed) abstract
Larsen syndrome (LS) is a skeletal dysplasia (osteochondrodysplasia) in which multiple dislocations of the large joints are the major feature. Nosology in this group of diseases, which constitutes 8% of Mendelian disorders in man, is primarily based on clinical and radiographic features. Hopes for more accurate classification grounds are currently being met by progress in elucidation of underlying genetic defects. We have performed linkage analysis in a large Swedish kindred with autosomal dominant LS and found the gene (LAR1) to be strongly linked to chromosome 3p markers (Zmax = 13.4 at (theta = .00). Recombination analysis indicates that the LAR1 locus is located in a region defined distally by D3S1581 and proximally by D3S1600, which cytogenetically maps to chromosome region 3p21.1-14.1. Linkage and recombination analysis of a COL7A1 PvuII intragenic polymorphism versus LS and chromosome 3 markers indicate that COL7A1 is located close to, but distinct from, the LAR1 locus.
26.
27.
Erjefält, Jonas, et al.
(author)
Microcirculation-derived factors in airway epithelial repair in vivo
1994
In: Microvascular Research. - : Elsevier BV. - 1095-9319 .- 0026-2862. ; 48:2, s. 161-178
Journal article (peer-reviewed) abstract
Airway epithelial repair, by cell migration over a denuded, intact basement membrane, occurs rapidly in vivo. The present study examines microcirculation-derived factors in the reepithelialization process in the guinea pig. A well-defined tracheal zone was gently deepithelialized; no bleeding occurred and the basement membrane was left intact. Plasma exudation was visualized by use of iv colloidal gold (diameter: 5 nm) or fluoresceinisothiocyanate-labeled dextran. Scanning and transmission electron microscopy confirmed the migration of epithelial cells and, additionally, allowed us to examine the presence of an extracellular matrix gel and leukocytes on the denuded basement membrane. Fibronectin was analyzed by immunocytochemistry. Following epithelial removal plasma promptly extravasates and produces a fibrin-fibronectin gel to cover the denuded basement membrane. Epithelial cells dedifferentiate, flatten, and migrate rapidly (several micron/min) beneath the plasma-derived gel. Within 30 min the gel contains numerous leukocytes, some of which are eosinophils. Plasma exudes into the gel until about 8 hr by which time the entire denuded zone (800 microns) is covered by squamous epithelium. The fibrin-fibronectin gel is suggested to be exclusively plasma-derived. In conclusion, reepithelialization in vivo occurs beneath a gel containing adhesive plasma proteins and leukocytes. We suggest that a plasma exudate provides immediate cover of denuded airway basement membrane and that plasma- and leukocyte-derived factors contribute essentially to reepithelialization in vivo.
28.
Fu, Michael, 1963, et al.
(author)
Agonist-like activity of anti-peptide antibodies directed against an autoimmune epitope on the heart muscarinic acetylcholine receptor.
1994
In: Receptors & channels. - 1060-6823. ; 2:2, s. 121-30
Journal article (peer-reviewed) abstract
A synthetic peptide corresponding to amino acids 169-193 of the second extracellular loop of the M2 human muscarinic receptor was used to raise antibodies in rabbits. Affinity purified antibodies specifically recognized a major band with a molecular weight of about 80 kDa on the electrotransferred membrane proteins of both rat ventricles and chinese hamster ovary cells expressing recombinant muscarinic receptors. Incubation of these antibodies with rat myocardial membranes resulted not only in a decrease in the maximal binding capacity, but also in a decrease in receptor antagonist affinity. These antibodies could also mimic the effects of agonist stimulation as demonstrated by inhibition of isoproterenol-stimulated cAMP accumulation and by a negative chronotropic effect on cultured cardiomyocytes. These results suggest that the second extracellular loop of the M2 muscarinic receptor is an immunologically and functionally important domain with properties comparable to those found for autoantibodies against the same domain in idiopathic dilated cardiomyopathy. It strengthens the hypothesis that the second extracellular loop of the members of the superfamily of G-protein coupled membrane receptors could be the main immunogenic region responsible for a pathogenic autoimmune response.
29.
Fu, Michael, 1963, et al.
(author)
Localization of a functional autoimmune epitope on the muscarinic acetylcholine receptor-2 in patients with idiopathic dilated cardiomyopathy.
1993
In: The Journal of clinical investigation. - 0021-9738. ; 91:5, s. 1964-8
Journal article (peer-reviewed) abstract
A peptide corresponding to the sequence 169-193 of the second extracellular loop of the human muscarinic acetylcholine receptor-2 was used as an antigen to screen sera from patients with idiopathic dilated cardiomyopathy (DCM, n = 36) and healthy blood donors (HBD, n = 40). The sera from 14 patients with DCM (38.8%) and 3 HBD (7.5%) recognized the muscarinic receptor peptide at dilutions varying from 1:20 to 1:160 in ELISA. A highly significant correlation (P = 0.006) was found between the presence of antimuscarinic receptor-2 autoantibodies and anti-beta-adrenoceptor-1 autoantibodies in the patients' sera. Affinity-purified autoantibodies from positive sera of patients with DCM recognized on the electrotransferred protein of rat ventricular membrane a major band of about 80 kD. Incubation of autoantibodies with membrane resulted not only in a decrease in the maximal binding sites (Bmax) but also in an increase in Kd of radioligand binding in a concentration-dependent manner. This suggests a mixed-type of inhibition. Moreover, preincubation with atropine abolished the inhibitory effect of autoantibodies on the receptor binding whereas carbachol appeared to have no effect on the activity of the autoantibodies. These data define a subgroup of patients with idiopathic DCM who have in their sera functionally active autoantibodies against muscarinic receptor-2.
30.
Fåhraeus, R, et al.
(author)
Response to cAMP levels of the Epstein-Barr virus EBNA2-inducible LMP1 oncogene and EBNA2 inhibition of a PP1-like activity.
1994
In: The EMBO journal. - 0261-4189. ; 13:24, s. 6041-51
Journal article (peer-reviewed) abstract
The expression of the Epstein-Barr virus LMP1 oncogene is regulated by viral and non-viral factors in a tissue dependent fashion. The virus encoded transcription factor EBNA2 induces its expression in human B-cells. However, this induction also requires the contribution of cellular and/or other viral factors. In nasopharyngeal carcinoma cells and in cells from Hodgkin's lymphoma, LMP1 gene transcription is independent of viral products. Here we show that the effect of a factor binding to a cAMP responsive-like element (CRE) in the LMP1 gene transcription regulatory sequence (LRS) is essential for efficient promoter activity in the DG75 B-cell line and that elevation of cAMP levels in the cells induces LRS-derived CAT activity in a CRE dependent fashion. Incubation of two EBV-immortalized B-cell lines expressing endogenous EBNA2A with 8-Br cAMP increased the levels of the latency associated 66 kDa LMP1 within 2 h. Interestingly, LMP1 expression in DG75 cells conferred resistance to the inhibitory effect of 8-Br cAMP on cell proliferation. The protein phosphatase 1 and 2A (PP1 and PP2A, respectively) inhibitor okadaic acid also stimulated LRS-CAT activity in DG75 cells. EBNA2A from an EBV-immortalized B-cell line co-immunopurified with a PP1-like protein. An EBNA2A fragment spanning residues 324-436 fused to the GST protein specifically rescued a PP1/PP2A-like component from DG75 cell extracts. This GST-EBNA2A fusion product inhibited a PP1-like activity in nuclear extracts from these cells.
31.
Hedman, H., et al.
(author)
Defective expression of beta 1-integrins in cells with constitutively active alpha L beta 2-integrins
1997
In: Experimental Cell Research. - : Elsevier Science B.V., Amsterdam. - 0014-4827 .- 1090-2422. ; 232:2, s. 270-276
Journal article (peer-reviewed) abstract
We have investigated a potential relationship between expression of beta 1-integrins and adhesiveness of the beta 2-integrin LFA-1 (alpha L beta 2, CD11a/CD18). By an approach of random mutagenesis and selection we established clones from the human acute lymphatic leukemia cell line HPB-ALL with (i) constitutively active LFA-1 and (ii) with no apparent integrin-beta 1 cell surface expression. Thirty seven of 42 clones selected for activated LFA-1 were found to have lost apparent integrin-beta 1 expression. Conversely, 7 of 21 clones selected for lack of beta 1 expression were found to have activated LFA-1. Since this pointed toward a possible coupling between beta 1 expression and LFA-1 activity, we further analyzed at which level beta 1 expression was blocked. We focused on one clone, HAP4, with activated LFA-I and no detectable beta 1 cell surface expression and found, surprisingly, that it expressed wild-type levels of beta 1 mRNA and, in Western blots of whole cell lysates, apparently normal levels of beta 1 protein. However, in addition to beta 1 of the expected molecular weight, HAP4 expressed a unique 48-kDa band recognized by the polyclonal anti-beta 1 antiserum. Immunoprecipitation experiments revealed that the epitope recognized by the anti-beta 1 antibody 4B4 was hidden or lost. The alpha 4-chain was found in its precursor form but it did not associate with any beta-chain, and it was not processed to its mature form. Instead alpha 4-chains were eventually degraded. Taken together this showed that beta 1-chains were produced but not properly processed in HAP4. From this we propose that HAP4 is deficient in a gene product required both for proper beta 1 folding and for repression of LFA-1 adhesiveness.
32.
Holmberg, Niklas, et al.
(author)
Transgenic tobacco expressing Vitreoscilla hemoglobin exhibits enhanced growth and altered metabolite production
1997
In: Nature Biotechnology. - : Springer Science and Business Media LLC. - 1087-0156 .- 1546-1696. ; 15:3, s. 7-244
Journal article (peer-reviewed) abstract
The gene for Vitreoscilla hemoglobin (VHb) has been introduced and expressed in Nicotiana tabaccum (tobacco). Transgenic tobacco plants expressing VHb exhibited enhanced growth, on average 80-100% more dry weight after 35 days of growth compared to wild-type controls. Furthermore, germination time is reduced from 6-8 days for wild-type tobacco to 3-4 days and the growth phase from germination to flowering was 3-5 days shorter for the VHb-expressing transgenes. Transgenic plants contained, on average, 30-40% more chlorophyll and 34% more nicotine than controls. VHb expression also resulted in an altered distribution of secondary metabolites: In the trangenic tobacco plants anabasine content was decreased 80% relative to control plants.
33.
Larsson, P G, et al.
(author)
Bacterial vaginosis and the vaginal leucocyte/epithelial cell ratio in women attending an outpatient gynaecology clinic.
1991
In: European journal of obstetrics, gynecology, and reproductive biology. - : Elsevier BV. - 0301-2115. ; 42:3, s. 217-20
Journal article (peer-reviewed) abstract
In order to investigate the usefulness of vaginal leucocytosis as a sign of genital infection, vaginal wet smears were obtained from 230 consecutive non-pregnant women attending an outpatient gynaecology clinic. Although 52 (22.6%) of the women were diagnosed as having bacterial vaginosis, none had symptoms of cervicitis or genital infection. Of the patients with bacterial vaginosis 19 (36.5%) had vaginal leucocytosis compared to 37.1% of the women without bacterial vaginosis. In a further study, bacterial vaginosis was also diagnosed in 104 of 384 women (33.3%) attending for first trimester legal abortions. Of the women with bacterial vaginosis 37.5% had vaginal leucocytosis compared to 31.8% for those without bacterial vaginosis (P greater than 0.05). There was no correlation between vaginal leucocytosis and the presence of cervical Chlamydia trachomatis; however, the presence of C. trachomatis did correlate with bacterial vaginosis. Vaginal leucocytes varies with factors unrelated to genital infection. This fact must be taken into consideration when wet smears from patients with suspected genital tract infection are evaluated.
34.
Matsui, S, et al.
(author)
Distribution of serum autoantibodies against G-protein-coupled cardiovascular receptors in Japanese patients with cardiomyopathy.
1996
In: Blood pressure. Supplement. - 0803-8023. ; 3, s. 28-30
Journal article (peer-reviewed) abstract
The prevalence of anti-receptor autoantibodies in the sera of patients with dilated cardiomyopathy has been previously demonstrated. This article will briefly review the distribution of autoantibodies against a series of G-protein-coupled cardiovascular receptors such as human beta 1 and beta 2 adrenoceptors, alpha 1 adrenoceptors, M2 muscarinic acetylcholine receptors and angiotensin II-I (AT1) receptors in a well-defined population of patients with dilated cardiomyopathy from Japan.
35.
Platz-Christensen, Jens Jörgen, et al.
(author)
A longitudinal follow-up of bacterial vaginosis during pregnancy.
1993
In: Acta obstetricia et gynecologica Scandinavica. - : Wiley. - 0001-6349 .- 1600-0412. ; 72:2, s. 99-102
Journal article (peer-reviewed) abstract
Bacterial vaginosis (BV) has been considered by many investigators to be a risk factor for preterm labor. We have followed vaginal pH and the persistence of clue cells in Papanicolaou stained smears in 119 pregnant women during the course of pregnancy. Of 19 patients with clue cells in their smears during the first trimester, 11 (58%) still had clue cells at the second visit during the third trimester. Of the 100 patients without clue cells during their first trimester, none exhibited clue cells during the third trimester. If the persistence of clue cells is truly a risk factor for adverse pregnancy outcome, screening in the first trimester would identify a risk group of 15%. This risk group diminishes to 9% at the time of the third trimester. Vaginal pH > 4.5 had a recovery sensitivity of 76% and specificity of 83%. If clue cells can be considered as the identifying standard for bacterial vaginosis, the sensitivity and specificity of pH is 89% and 94%, respectively. The establishment of the diagnosis of BV during pregnancy and, in some cases, the treatment of the condition may be important as routine procedures in the antenatal center.
36.
Schulze, W, et al.
(author)
Immunocytochemical localization of muscarinic, adrenergic and AT1 receptors.
1996
In: Blood pressure. Supplement. - 0803-8023. ; 3, s. 15-8
Journal article (other academic/artistic) abstract
By indirect immunofluorescence and post-embedding EM gold technique, the localization of alpha 1-adrenergic, M2-muscarinic and angiotensin II receptor-I (AT1) were determinated. With antipeptide antibodies directed against the second extracellular loops of all three receptors, these receptors were found to be localized at the sarcolemma of adult rat cardiomyocytes and at the surface membranes of cultivated neonatal heart cells. Additionally, M2 receptors were localized along T-tubule membranes of both rat and human adult cardiomyocytes. alpha 1-Adrenergic receptors were found intracellular near the surface of atrial granules (ANF-granules). By using M2 and alpha 1-adrenergic receptor antibodies the strongest fluorescence was found in the right atrium of the rat. Besides the localization in cardiomyocytes, AT1 receptors were also localized in outer plasma membranes and the endoplasmic reticulum of fibroblasts, and the surface of smooth muscle cells of the major arteries and veins. Likewise, the muscarinic M2 receptors were found along the outer membranes of endothelial cells from capillaries and endocardium.
37.
Shoberg, Russel J., et al.
(author)
Identification of a highly cross-reactive outer surface protein B epitope among diverse geographic isolates of Borrelia spp. causing Lyme disease
1994
In: Journal of Clinical Microbiology. - : American Society for Microbiology (ASM). - 0095-1137 .- 1098-660X. ; 32:2, s. 489-500
Journal article (peer-reviewed) abstract
The outer surface lipoprotein B (OspB) of Borrelia burgdorferi is a major component of the borrelial protein profile and has been shown to be highly immunogenic in experimentally immunized and infected mammals. However, the ospB loci of different strains show considerable heterology at the nucleic acid sequence level, and the progeny of a clonal strain of B. burgdorferi exhibited OspB polymorphisms with respect to apparent molecular weights and reactivities with monoclonal antibodies. These data suggest that OspB is not a good candidate for vaccination or diagnostic purposes. The present study describes a monoclonal antibody, designated 84C, directed against a very highly conserved domain of the OspB lipoprotein. Western immunoblot analysis with 84C demonstrated reactivity in 84.2% of human, tick, and other vertebrate isolate strains examined from widely diverse geographic regions, including strains of B. burgdorferi sensu stricto and two closely related species, B. garinii and B. afzelii. The 84C-binding region was delimited to a highly conserved 11-amino-acid region in the carboxyl terminus of OspB as demonstrated by (i) DNA sequence analysis of wild-type and 84C-resistant mutant ospB alleles and (ii) deletion mutagenesis of a recombinant ospB gene in Escherichia coli. Finally, the 84C epitope was demonstrated to be exposed on the borrelial surface in situ as (i) the monoclonal antibody 84C was able to agglutinate borrelias in culture and (ii) 84C-resistant escape variants were isolated. These data suggest that the potential value of OspB as a vaccine candidate or diagnostic tool be examined more closely, in the context of the 84C-reactive domain.
38.
Sjöblom-Hallén, A, et al.
(author)
An ATF/CRE element mediates both EBNA2-dependent and EBNA2-independent activation of the Epstein-Barr virus LMP1 gene promoter.
1998
In: Journal of virology. - 0022-538X. ; 72:2, s. 1365-76
Journal article (peer-reviewed) abstract
The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is a viral oncogene whose expression is regulated by both viral and cellular factors. EBV nuclear antigen 2 (EBNA2) is a potent transactivator of LMP1 expression in human B cells, and several EBNA2 response elements have been identified in the promoter regulatory sequence (LRS). We have previously shown that an activating transcription factor/cyclic AMP response element (ATF/CRE) site in LRS is involved in EBNA2 responsiveness. We now establish the importance of the ATF/CRE element by mutational analysis and show that both EBNA2-dependent activation and EBNA2-independent activation of the promoter occur via this site but are mediated by separate sets of factors. An electrophoretic mobility shift assay (EMSA) with specific antibodies showed that the ATF-1, CREB-1, ATF-2 and c-Jun factors bind to the site as ATF-1/CREB-1 and ATF-2/c-Jun heterodimers whereas the Sp1 and Sp3 factors bind to an adjacent Sp site. Overexpression of ATF-1 and CREB-1 in the cells by expression vectors demonstrated that homodimeric as well as heterodimeric forms of the factors transactivate the LMP1 promoter in an EBNA2-independent manner. The homodimers of ATF-2 and c-Jun did not significantly stimulate promoter activity. In contrast, the ATF-2/c-Jun heterodimer had only a minor stimulatory effect in the absence of EBNA2 but induced a strong transactivation of the LMP1 promoter when coexpressed with this protein. Evidence for a direct interaction between the ATF-2/c-Jun heterodimeric complex and EBNA2 was obtained by EMSA and coimmunoprecipitation experiments. Thus, our results suggest that EBNA2-induced transactivation via the ATF/CRE site occurs through a direct contact between EBNA2 and an ATF-2/c-Jun heterodimer. EBNA2-independent promoter activation via this site, on the other hand, is mediated by a heterodimeric complex between the ATF-1 and CREB-1 factors.
39.
Vural, Gürcan, et al.
(author)
Inflammatory signs in wet smear and Pap-smear compared with the histopathology from the female lower genital tract.
1995
In: Acta obstetricia et gynecologica Scandinavica. - : Wiley. - 0001-6349 .- 1600-0412. ; 74:6, s. 451-4
Journal article (peer-reviewed) abstract
In order to define the relationship between various criteria of female lower genital tract inflammation, we examined wet smears, cervical smears and biopsies from 131 patients. The presence of clue cells in rehydrated dry smears showed a positive correlation to the presence of clue cells in Papanicolaou stained smears, and to some extent with cytological evidence of inflammation (Kappa 0.48). Cytological inflammatory findings correlated well with the presence of clue cells in rehydrated dry smears, but signs of inflammation diagnosed by histopathology did not correspond to findings in cytological smears, probably because these methods reveal inflammation at different sites.
40.
Wallukat, G, et al.
(author)
Autoantibodies against M2 muscarinic receptors in patients with cardiomyopathy display non-desensitized agonist-like effects.
1999
In: Life sciences. - 0024-3205. ; 64:6-7, s. 465-9
Journal article (peer-reviewed) abstract
Circulating autoantibodies against the human M2 muscarinic receptors have been previously shown in 38% of patients with idiopathic dilated cardiomyopathy. The functional properties of these autoantibodies are reported herein. They were able to decrease the cell beating frequency of myocytes in cultured neonatal rat heart cells in a dose-dependent manner without desensitization over a period of more than 5 hours whereas the non-specific muscarinic receptor agonist carbachol also inhibited the heart cell beating frequency but was desensitized within 1 hour. In the same cell culture, anti-M2 muscarinic receptor autoantibodies were not able to induce internalization of muscarinic receptor whereas carbachol did. These results demonstrate for the first time that anti-M2 muscarinic receptor autoantibodies from patients with idiopathic dilated cardiomyopathy have stimulatory muscarinic activity in vitro, which differ from normal muscarinic agonists by non-desensitization.
41.
Wang, W Z, et al.
(author)
Effects of anti-peptide antibodies against human M2 muscarinic receptors on cardiac function in rats in vivo.
1996
In: Blood pressure. Supplement. - 0803-8023. ; 3, s. 25-7
Journal article (peer-reviewed) abstract
The effects of anti-peptide antibodies (Ab) against the second extracellular loop of human muscarinic receptor-2 on cardiac function in rats in vivo were studied. These effects were compared with those of the muscarinic receptor agonist, carbachol (Carb). It was shown that: (1) both Carb and Ab administered intravenously in the same doses of 0.4 nmol, 1.0 nmol, and 2.0 nmol were able to inhibit the maximal rate of rise of ventricular pressure (+dp/dt max) in a dose-dependent manner. (2) The isoproterenol (Iso)-induced increase in HR and +dp/dt max were also markedly inhibited by Carb and Ab. Administration of 1.0 nmol Carb decreased the Iso-stimulated increase of HR from 13.6 +/- 2.0 to 4.9 +/- 0.7% and decreased the increase of +dp/dt max from 27.9 +/- 3.2 to 4.8 +/- 0.6%, respectively; whereas Ab decreased the HR to 6.5 +/- 1.1% and +dp/dt max to 13.5 +/- 1.2%, respectively. (3) Both the inhibitory effects of Carb and Ab could be significantly blocked by atropine (1.4 nmol). These findings suggest that the Ab display a stimulatory muscarinic activity similar to Carb in inhibiting the cardiac function as evidenced by its negative chronotropic and inotropic effects.
42.
Zhu, L, et al.
(author)
Immunocytochemical studies of M2 muscarinic receptors in guinea pig atria and ventricles using anti-peptide antibodies.
1996
In: Blood pressure. Supplement. - 0803-8023. ; 3, s. 19-21
Journal article (other academic/artistic) abstract
The localization and density of M2 muscarinic receptors in guinea pig atria and ventricles were studied by immunocytochemical techniques, with anti-peptide antibodies against the second extracellular loop of human muscarinic receptor-2. It was shown that the positive immunoreaction of M2 muscarinic receptor was characterized by brown-yellow granules of variable size and shape, which were located mainly on the cell surface and in the cytoplasm far from nuclei. The positive immunoreactive substances appeared in both atrial and ventricular myocytes, with the density being greater in atria than in ventricles. Pre-incubation with atropine, but not carbachol, significantly reduced the intensity of positive immunoreactivity. This indicates that the binding of antibody to receptor site can be partially blocked by M2 muscarinic receptor antagonist.
43.
Agardh, Carl-David, et al.
(author)
Glutathione levels are reduced in diabetic rat retina but are not influenced by ischemia followed by recirculation
1998
In: Metabolism, Clinical and Experimental. - 1532-8600. ; 47:3, s. 269-272
Journal article (peer-reviewed) abstract
Free radicals have recently been proposed to play a role in the development of diabetic retinopathy. The aim of the present study was to examine whether the abnormal metabolism caused by diabetes and by ischemia followed by recirculation interferes with a free radical enzyme defense system in the retina, ie, glutathione. Diabetes mellitus was induced by injecting streptozotocin ([STZ] 60 mg/kg body weight [BW] intraperitoneally). After 2 and 6 months, respectively, glutathione levels were measured in the retina and compared against those of age-matched normal control rats. Retinal ischemia was induced by careful ligation of the vessels and the accompanying optic nerve behind the left eye bulb. The right eye served as a control. After 90 minutes of ischemia, retinal circulation was reestablished by removing the ligature. Two-month-old diabetic rats were kept for an additional 3 days and normal rats for 5 minutes, 15 minutes, or 3 days before they were killed for measurement of glutathione. Retinal levels of glutathione were significantly lower in 6-month diabetic compared with 2-month diabetic rats (16.6 +/- 2.9 v 19.0 +/- 2.2 nmol/mg protein, P < .05) and 6-month normal control rats (16.6 +/- 2.9 v 21.0 +/- 2.1 nmol/mg protein, P < .001). Ischemia followed by recirculation did not influence the total tissue level of glutathione either in 2-month-old diabetic rats or in normal rats. The present study indicates that the abnormal metabolism caused by diabetes, rather than by changes in retinal circulation, results in an impaired defense mechanism against free radicals, a factor that may be of importance for the development of diabetic retinopathy. However, since glutathione levels in the present study were measured in the whole retina, it cannot be excluded that particular cell types, such as vascular cells, show an alteration in glutathione that is masked by the glutathione levels in the other nonvascular cells of the retina. Studies using other techniques are needed to further explore this subject.
44.
Ahmadian, Afshin, et al.
(author)
Genetic instability in the 9q22.3 region is a late event in the development of squamous cell carcinoma.
1998
In: Oncogene. - : Springer Science and Business Media LLC. - 0950-9232 .- 1476-5594. ; 17:14
Journal article (peer-reviewed) abstract
Squamous cell carcinoma (SCC) of the skin represents a group of neoplasms which is associated with exposure to UV light. Recently, we obtained data suggesting that invasive skin cancer and its precursors derive from one original neoplastic clone. Here, the analysis were extended by loss of heterozygosity (LOH) analysis in the chromosome 9q22.3 region. A total of 85 samples, taken from twenty-two sections of sun-exposed sites, corresponding to normal epidermis, morphological normal cells with positive immuno-staining for the p53 protein (p53 patches), dysplasias, cancer in situ (CIS) and squamous cell carcinomas (SCC) of the skin were analysed. Overall, about 70% of p53 patches had mutations in the p53 gene but not LOH in the p53 gene or 9q22.3 region. Approximately 70% of the dysplasias showed p53 mutations of which about 40% had LOH in the p53 region but not in the 9q22.3 region. In contrast, about 65% of SCC and CIS displayed LOH in the 9q22.3 region, as well as frequent (80%) mutations and/or LOH in the p53 gene. These findings strongly suggest that alterations in the p53 gene is an early event in the progression towards SCC, whereas malignant development involves LOH and alterations in at least one (or several) tumor suppressor genes located in chromosome 9q22.3.
45.
Akerström, B, et al.
(author)
An intriguing member of the lipocalin protein family : alpha 1-microglobulin
1990
In: Trends in Biochemical Sciences. - 0968-0004. ; 15:6, s. 3-240
Research review (peer-reviewed) abstract
The plasma protein alpha 1-microglobulin is a member of the lipocalin protein superfamily. In the last few years, the work on alpha 1-microglobulin has given unexpected and promising new results. Of particular interest are its molecular association with immunoglobulin A and with proteinase inhibitors, and its interactions with the immune system.
46.
Akerström, B, et al.
(author)
On the interaction between single chain Fv antibodies and bacterial immunoglobulin-binding proteins
1994
In: Journal of Immunological Methods. - 0022-1759. ; 177:1-2, s. 63-151
Journal article (peer-reviewed) abstract
Using four bacterial immunoglobulin-binding proteins, we have analyzed the binding characteristics of a panel of 34 human single chain Fv antibodies, expressed in E. coli and with known specificity and sequence. Several of the single chain Fv antibodies showed affinity for staphylococcal protein A and peptostreptococcal protein L, but not for the streptococcal proteins G or H. The affinity of the binding was higher for protein L (4.5 and 1.4 x 10(9) M-1) than for protein A (7.7 and 6.7 x 10(8) M-1), using the two single chain Fv antibodies displaying the strongest binding activity to these ligands. The binding was shown to be specific by Western blotting, and the single chain Fv antibodies could be purified from crude bacterial culture media by affinity chromatography on protein L- or A-Sepharose. Protein A, which has affinity for the VH domain of the scFv antibodies, was tested against scFv antibodies containing VH1, VH3, VH4 and VH5 domains, and its binding was restricted to approximately half of the scFv antibodies with a VH3 domain. Protein L, which has affinity for the VL domain, was tested against kappa 1, kappa 4, lambda 1, lambda 2 and lambda 3 domains, and it bound all kappa 1 domains, one lambda 2 and one lambda 3 domain. Comparison of the amino acid sequences of binding and non-binding VL domains demonstrated that amino acid residues crucial to the binding of protein L were distributed over a large area outside the hypervariable antigen-binding regions.
47.
Aldenborg, F, et al.
(author)
Mast cells and biogenic amines in radiation-induced pulmonary fibrosis
1993
In: American Journal of Respiratory Cell and Molecular Biology. - 1535-4989. ; 8:1, s. 112-117
Journal article (peer-reviewed) abstract
Sprague-Dawley rats were exposed to a single X-ray dose of 30 Gy over the lungs and examined at 1-wk intervals during the following 3 to 8 wk. Mast cells were counted after specific staining with toluidine blue at a low pH and the mast-cell amines, histamine (Hi) and serotonin (5-HT), were measured using high-performance liquid chromatography. Irradiation induced pneumonitis followed by pulmonary mast-cell hyperplasia and progressive fibrosis 4 to 8 wk after irradiation. By week 4, immature-looking mast cells with a few granules started to appear, followed by a gradual increase in mast cells that reached very high levels after 8 wk, up to 40 to 200 times the normal. The pulmonary Hi and 5-HT content increased concomitantly from 6 and 1 micrograms/g to a maximum of 200 and 18 micrograms/g, respectively. These high levels of amine content and mast-cell densities greatly exceed those of any normal tissue. There was a strong correlation between the Hi and 5-HT content in both normal (r = 0.87) and irradiated (r = 0.93) lung tissue, as well as between the mast-cell density and amine content after irradiation (r = 0.86), thereby indicating that both amines derived from mast cells. The Hi/5-HT quotients were much lower in both normal and irradiated lung tissue (5 and 9, respectively) than in other tissues where these amines are stored in mast cells, or in isolated peritoneal mast cells (43). This relatively higher 5-HT content in pulmonary mast cells suggests that this amine performs a specific function in the lung.
48.
Amandusson, Åsa, et al.
(author)
Colocalization of oestrogen receptor immunoreactivity and preproenkephalin mRNA expression fo neurons in the superficial laminae of the spinal and medullary dorsal horn of rats
1996
In: Eur J Neurosci. - : Wiley InterScience. ; 8:11, s. 2440-2445
Journal article (peer-reviewed) abstract
A double-labelling procedure combining immunohistochemical staining with in situ hybridization using a radiolabelled cRNA probe was employed to demonstrate oestrogen receptor-like immunoreactivity and preproenkephalin-A mRNA in the medullary and spinal dorsal horn of female rats. Both markers labelled large numbers of neurons in the substantia gelatinosa and its trigeminal homologue. Many of these neurons were double-labelled, displaying both oestrogen receptor-like-immunoreactivity and preproenkephalin-A mRNA; cell counts showed that 40-60% of the of the oestrogen receptor-like-immunoreactive cells in the superficial laminae also were labelled for preproenkephalin-A mRNA, and that 60-70% of the preproenkephalin-A mRNA-labelled neurons in the same laminae displayed oestrogen receptor-like immunoreactivity. Previous studies have shown that oestrogen receptors can bind to the promoter region of the preproenkephalin-A gene, and studies on the hypothalamus have demonstrated that oestrogen regulates enkephalin expression in select neuronal populations. The present results demonstrate that enkephalinergic neurons in the superficial dorsal horn contain oestrogen receptors and suggest that oestrogen may play an important role in the modulation of sensory and nociceptive processing in the lower medulla and spinal cord.
49.
Amandusson, Åsa, 1974-, et al.
(author)
Estrogen-induced alterations of spinal cord enkephalin gene expression
1999
In: Pain. - : Elsevier. - 0304-3959 .- 1872-6623. ; 83:2, s. 243-248
Journal article (peer-reviewed) abstract
Enkephalin-synthesizing neurons in the super®cial laminae of the spinal and trigeminal dorsal horn are critical components of the endogenous pain-modulatory system. We have previously demonstrated that these neurons display intracellular estrogen receptors, suggesting that estrogen can potentially influence their enkephalin expression. By using Northern blot, we now show that a bolus injection of estrogen results in a rapid increase in spinal cord enkephalin mRNA levels in ovariectomized female rats. Thus, 4 h after estrogen administration the enkephalin mRNA-expression in the lumbar spinal cord was on average 68% higher (P , 0:05) than in control animals injected with vehicle only. A small increase in the amount of enkephalin mRNA was also seen after 8 h (P , 0:05), whereas no difference between estrogen-injected and control animals was found after 24 h or at time periods shorter than 4 h. Taken together with the previous anatomical data, the present findings imply that estrogen has an acute effect on spinal opioid levels in areas involved in the transmission of nociceptive information.
50.
