| 1. |
- Borg, Jörgen, et al.
(författare)
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Amino-terminal anchored surface display in insect cells and budded baculovirus using the amino-terminal end of neuraminidase.
- 2004
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Ingår i: Journal of Biotechnology. - : Elsevier BV. - 1873-4863 .- 0168-1656. ; 114:1-2, s. 21-30
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Tidskriftsartikel (refereegranskat)abstract
- Methods currently used for surface display on insect cells and budded baculovirus, all utilize the sequences from class I transmembrane proteins. This gives rise to some problems when handling unknown genes or cDNAs encoding full-length proteins. First, the stop codon from the cloned gene will be located upstream of the sequence for the transmembrane region. Second, the chance of getting the sequences encoding the signal peptide and the transmembrane region in frame with the cloned gene is small. To minimize these problems, we here present a method by which cDNAs or genes of interest can be cloned and fused to the codons for the signal peptide and transmembrane region of neuraminidase (NA), a class II transmembrane protein of the influenza virus. By placing both the signal peptide and transmembrane region at the amino-terminal, potential problems regarding stop codons are eliminated and errors in frame-shift minimized. To obtain proof of principle, the gene encoding enhanced green fluorescent protein, EGFP, was subcloned into a shuttle vector downstream of the neuraminidase sequence and the fusion product was then transferred to a baculovirus vector and transfected into insect cells (Sf9). Using this method, EGFP was found to be expressed on the surface of both infected cells and budded virus in an accessible manner.
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| 2. |
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| 3. |
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| 4. |
- Blom, Anna M, et al.
(författare)
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Increase of bikunin and alpha1-microglobulin concentrations in urine of rats during pregnancy is due to decreased tubular reabsorption
- 1997
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002. ; 1361:2, s. 198-202
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Tidskriftsartikel (refereegranskat)abstract
- Bikunin and alpha1-microglobulin are two plasma proteins of about 25 kDa which are made in the liver from a common precursor. The concentration of bikunin in human urine has been shown to increase several fold during various conditions of stress. The mechanism behind this increase is unknown. We have studied pregnant rats and found that the bikunin and alpha1-microglobulin levels in their urine increased 3-fold towards the end of the pregnancy, whereas those of albumin and orosomucoid did not. There were no significant changes in either the bikunin/alpha1-microglobulin mRNA level or the concentrations of the two proteins in serum. These findings imply that the synthesis and the clearance rates of bikunin and alpha1-microglobulin are normal during pregnancy but that the tubular reabsorption of these proteins is decreased.
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| 5. |
- Falkenberg, Cecilia, et al.
(författare)
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A study of the physiological consequences of sympathetic denervation of the heart caused by the arterial switch procedure.
- 2010
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Ingår i: Cardiology in the young. - 1467-1107. ; 20:2, s. 150-8
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The arterial switch operation is the corrective operation for transposition of the great arteries, defined as the combination of concordant atrioventricular and discordant ventriculo-arterial connections, but there have been concerns about silent subendocardial ischaemia on exercise and coronary artery growth. The arterial switch divides the majority of the sympathetic nerves entering the heart; we have studied the effects of coronary flow and sensitivity to catecholamine stimulation in an animal model. METHODS: A total of 10 piglets were operated on cardiopulmonary bypass with section and resuturing of aortic trunk, pulmonary artery and both coronary arteries, with 13 sham-operated controls. After 5-7 weeks of recovery, seven simulated switch survivors and 13 controls were studied. RESULTS: Basal heart rate was significantly higher in switch piglets: in vivo mean (standard deviation) 112 (12) versus sham 100 (10) beats per minute, (p = 0.042); in vitro (Langendorff preparation): 89 (9) versus sham 73 (8) beats per minute (p = 0.0056). In vivo maximal heart rate in response to epinephrine was increased in switch piglets, 209 (13) versus 190 (17) beats per minute (p = 0.044). In vitro dose-response curves to norepinephrine were shifted leftward and upwards (p = 0.0014), with an 80% increase in heart rate induced by 0.095 (0.053) norepinephrine micromole per litre perfusate in switch hearts versus 0.180 (0.035) norepinephrine micromole per litre (p = 0.023). Increase in coronary flow on norepinephrine stimulation and maximal coronary flow were significantly reduced in switch hearts: 0.3 (0.2) versus 0.8 (0.4) millilitre per gram heart weight (p = 0.045) and 2.5 (0.4) versus 3.1 (0.4) millilitre per gram heart (p = 0.030), respectively. CONCLUSIONS: A combination of increased intrinsic heart rate, increased sensitivity to chronotropic actions of norepinephrine, and a decreased maximal coronary flow creates potential for a mismatch between perfusion and energy demands.
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| 6. |
- Falkenberg, Cecilia, et al.
(författare)
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alpha 1-Microglobulin destroys the proteinase inhibitory activity of alpha 1-inhibitor-3 by complex formation
- 1995
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 270:9, s. 4478-4483
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Tidskriftsartikel (refereegranskat)abstract
- The immunoregulatory plasma protein alpha 1-microglobulin (alpha 1-m) and the proteinase inhibitor alpha 1-inhibitor-3 (alpha 1I3) form a complex in rat plasma. In the present work, it was demonstrated that the alpha 1I3.alpha 1-m complex has no inhibitory activity, the bait region was not cleaved by low amounts of proteinases, and it was unable to covalently incorporate proteinases. The results also indicated that the thiolester bond of the alpha 1I3.alpha 1-m complex was broken. The alpha 1I3.alpha 1-m complex was cleared from the circulation much faster than native alpha 1I3, with a half-life of approximately 7 min. Structurally, however, the alpha 1I3.alpha 1-m complex was similar to native alpha 1I3 rather than alpha 1I3 cleaved by proteinases. It is speculated that the role of alpha 1-m is to destroy the function of alpha 1I3 by blocking the bait region and breaking the thiolester and causing its physical elimination by rapid clearing from the blood circulation. It is also possible that the formation of complexes between alpha 1-m and alpha 1I3 may serve as a mean to regulate the function of alpha 1-m since its complex with alpha 1I3 is taken up rapidly by cellular receptors for alpha-macroglobulins.
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| 7. |
- Falkenberg, Cecilia, et al.
(författare)
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Cardiac autonomic function in adolescents operated by arterial switch surgery.
- 2013
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Ingår i: International journal of cardiology. - : Elsevier BV. - 1874-1754 .- 0167-5273. ; 168:3, s. 1887-1893
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Children with transposition of the great arteries, in whom an arterial switch operation (ASO) is performed, have been shown to have an increased incidence of sudden death, which may be due to cardiac autonomic imbalance and repolarisation instability. We hypothesised that i) cardiac norepinephrine (NE) kinetics and ii) arterial baroreflex sensitivity (BRS), reflecting sympathetic activity and vagal function respectively, are altered in this group. METHODS AND RESULTS: 17 children (15.8±1.5years of age) with ASO-surgery in the neonatal period were studied. 17 had cardiac BRS assessed by spontaneous fluctuations of systolic blood pressure and RR-interval, and repolarisation was measured as QT variability index. Matched healthy subjects were controls. Cardiac vagal function and repolarisation pattern were unchanged following ASO-surgery. At cardiac catheterisation, we infused tritiated NE in 8 of these children to examine total body and cardiac sympathetic function at baseline and following 5min of adenosine infusion to induce reflex sympathetic activation. Blood was sampled simultaneously from the aorta and coronary sinus. Cardiac fractional extraction of ([3H])NE was substantially lower in operated children, being 56±10 vs. 82±9% (p=0.0001). Following i.v. adenosine in the operated group, NE total body spillover doubled vs. baseline (p<0.002) and the coronary venous-arterial concentration gradient of ([3H])dihydroxyphenylglycol increased 4-fold (p=0.04). CONCLUSIONS: Arterial switch operation performed neonatally appears to leave cardiac vagal function intact and, although cardiac sympathetic activation in response to adenosine occurs, cardiac neuronal NE reuptake is impaired. This may be pro-arrhythmic by reducing removal capacity of NE from the cardiac synaptic cleft.
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| 8. |
- Falkenberg, Cecilia
(författare)
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Consequences of Arterial Switch Operation in Children Born with Transposition of the Great Arteries
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Abstract Background: The introduction of the arterial switch operation (ASO) made it the procedure of choice for surgical correction of transposition of the great arteries. A majority of the sympathetic nerves innervate the heart alongside the great vessels; these are therefore likely to be damaged during the surgical procedure; imposing new challenges and questions that need to be addressed. The main aim for this thesis was to assess the long-term cardiac consequences on the autonomic nervous system after surgery (paper I and II) and to create an animal model allowing for cardiac physiological studies (paper III and IV). Methods: Long-term follow-up in adolescents who had undergone ASO as neonates (n=17, 1 female, mean fractional shorting 32±5%) was performed. This included sympathetic nervous system function assessed through infusion of tritiated Norepinephrine ([3H]NE) during heart catheterisation (n=8)(controls n=15) and blood samples analysed with high performance liquid chromatography. Samples were obtained both before and after adenosine stimulation as a response to sympathetic excitation. 24-hour heart rate variability (HRV)(n=15 in both groups) was measured both during the day and night using different algorithms. Baroreflex sensitivity and QT variability index (QTVI) (n=17 in both groups) were measured in awake patients. An animal model was developed using complex open heart surgery during cardiopulmonary bypass to mimick the arterial switch operation in piglets 8 weeks of age. The piglets surviving at least 5 to 6 weeks post-operation had follow-up of physiological response to catecholamines and were studied in vivo and in vitro using the Langendorff perfusion system. Results: In both groups the specific activity of [3H]NE decreased from the artery to the coronary sinus, but to a lesser extent in the ASO group. The extraction fraction in the ASO group was 56±10% compared to 82±9% in the healthy subjects (p<0.001). The arterial to coronary sinus plasma concentration of [3H] dihydroxyphenylglycol (DHPG) was significantly increased in the healthy group (70%, p=<0.0001) but was not so in the ASO group (8%, p=0.5). The difference of endogenous DHPG increase from the arterial to the coronary sinus was significantly smaller in the ASO group (p=0.008). After adenosine infusion, the total body NE spillover increased in the ASO group (p=0.002), reflecting major sympathetic activation. [3H]DHPG step-up from the artery to the coronary sinus increased 4-fold following adenosine. HRV frequency-domain at night-time, when cardio-parasympathetic drive is likely to be most pronounced, showed a significant decrease of normalized high frequency in the ASO group (52±20) compared to healthy subjects (68±15)(p=0.018). Time-domain showed no statistical difference between the two groups, neither during day-time nor night-time. Baroreflex sensitivity and QTVI did not show significant differences between groups. The animal model resulted in 14 out of 19 piglets surviving the mimicked ASO. Piglets operated with mimicked ASO had a significantly higher basal heart rate both in vivo (p=0.042) and in vitro (p=0.0056). Conclusion: A disturbed but functioning sympathetic cardiac innervation was found in the ASO patients at long-term follow-up. The vagal tone seemed normal in terms of BRS, however, frequencydomain analysis showed a decreased parasympathetic tone at night time in the ASO group. The surgical challenges due to translocation of the coronary arteries and the consequences of an injured autonomic nervous system impose risks of decreased myocardial perfusion and arrhythmias. Thus, the present data suggest that these patients ought to have follow-up that includes autonomic nervous system assessment.
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| 9. |
- Falkenberg, Cecilia, et al.
(författare)
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Expression of a functional proteinase inhibitor capable of accepting xylose: bikunin
- 2001
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Ingår i: Archives of Biochemistry and Biophysics. - : Elsevier BV. - 0003-9861. ; 387:1, s. 99-106
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Tidskriftsartikel (refereegranskat)abstract
- Bikunin is a Kunitz-type proteinase inhibitor, which is cross-linked to heavy chains via a chondroitin sulfate chain, forming inter-alpha-inhibitor and related molecules. Rat bikunin was produced by baculovirus-infected insect cells. The protein could be purified with a total yield of 20 mg/liter medium. Unlike naturally occuring bikunin the recombinant protein had no galactosaminoglycan chain. Endoglycosidase digestion also suggested that the recombinant form lacked N-linked oligosaccharides. Bikunin is translated as a part of a precursor, alpha1-microglobulin/bikunin, but the functional significance of the cotranslation is unknown. Our results indicate that the proteinase inhibitory function of bikunin is not regulated by the alpha1-microglobulin-part of the alpha1-microglobulin/bikunin precursor since recombinant bikunin had the same trypsin inhibitory activity as the recombinant precursor. Both free bikunin and the precursor were also functional as a substrate in an in vitro xylosylation system. This demonstrates that the alpha1-microglobulin-part is not necessary for the first step of galactosaminoglycan assembly.
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| 10. |
- Olofsson, Peter, et al.
(författare)
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Genetic links between the acute-phase response and arthritis development in rats.
- 2002
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Ingår i: Arthritis and Rheumatism. - 1529-0131 .- 0004-3591. ; 46:1, s. 259-268
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Tidskriftsartikel (refereegranskat)abstract
- OBJECTIVE: The acute-phase inflammatory response is closely correlated with the development of rheumatoid arthritis, but the pathophysiologic role of its specific components is largely unknown. We investigated the genetic control of the acute-phase protein response in pristane-induced arthritis (PIA), which is a chronic erosive arthritis model in rats. METHODS: Plasma levels of the acute-phase proteins interleukin-6 (IL-6), alpha1-acid glycoprotein (orosomucoid), fibrinogen, and alpha1-inhibitor3 were quantified in 3 strains of rats during the development and progression of disease: DA and LEW.1F, which are susceptible to arthritis, and E3, which is resistant. Genetic linkage analysis was performed on an F2 intercross between E3 and DA to determine the genetic control of the acute-phase response in arthritis. Elevated levels of alpha1-acid glycoprotein were associated with acute inflammation, whereas levels of IL-6 were increased during the entire course of the disease. RESULTS: Using these acute-phase markers as quantitative traits in linkage analysis revealed a colocalization of loci controlling the acute-phase response and regions previously shown to control the development of arthritis in chromosomes 10, 12, and 14. In addition, 2 loci that were not associated with arthritis were found to regulate serum levels of the acute-phase protein Apr1 (acute-phase response 1) at the telomeric end of chromosome 12 and Apr2 on chromosome 5. CONCLUSION: The PIA model in rats is a useful tool for understanding some of the pathways leading to chronic erosive arthritis. The analysis of acute-phase proteins in PIA and its application as quantitative traits for studying the genetics of arthritis will promote the understanding of the genetic regulation of the acute-phase response.
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