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- Kasrayan, Alex, et al.
(author)
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Enhancement by effectors and substrate nucleotides of R1-R2 interactions in Escherichia coli class Ia ribonucleotide reductase.
- 2004
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In: J Biol Chem. - 0021-9258. ; 279:30, s. 31050-7
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Journal article (other academic/artistic)abstract
- Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis. The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components harboring the active site region in each class are evolutionarily related. With a few exceptions, ribonucleotide reductases are allosterically regulated by nucleoside triphosphates (ATP and dNTPs). We have used the surface plasmon resonance technique to study how allosteric effects govern the strength of quaternary interactions in the class Ia ribonucleotide reductase from Escherichia coli, which like all class I enzymes has a tetrameric alpha(2) beta(2) structure. The component alpha(2)called R1 harbors the active site and two types of binding sites for allosteric effector nucleotides, whereas the beta(2) component called R2 harbors the tyrosyl radical necessary for catalysis. Our results show that only the known allosteric effector nucleotides, but not non-interacting nucleotides, promote a specific interaction between R1 and R2. Interestingly, the presence of substrate together with allosteric effector nucleotide strengthens the complex 2-3 times with a similar free energy change as the mutual allosteric effects of substrate and effector nucleotide binding to protein R1 in solution experiments. The dual allosteric effects of dATP as positive allosteric effector at low concentrations and as negative allosteric effector at high concentrations coincided with an almost 100-fold stronger R1-R2 interaction. Based on the experimental setup, we propose that the inhibition of enzyme activity in the E. coli class Ia enzyme occurs in a tight 1:1 complex of R1 and R2. Most intriguingly, we also discovered that thioredoxin, one of the physiological reductants of ribonucleotide reductases, enhances the R1-R2 interaction 4-fold.
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- Westman, Anna-Karin, et al.
(author)
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TEM study of the interface between HIPed silicon nitride and encapsulation borosilicate glass
- 2000
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In: Journal of Materials Science. - 0022-2461 .- 1573-4803. ; 35:11, s. 2847-2854
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Journal article (peer-reviewed)abstract
- A transmission electron microscope study has been made of a silicon nitride component with 6 w/o yttrium oxide as a sintering aid hot isostatically pressed (HIP) with an encapsulation glass of borosilicate. The TEM study concentrated on the interface region between ceramic and glass. Two different types of hexagonal boron nitride were formed near the interface. One, with a textured structure, seemed to nucleate heterogeneously on the surfaces of silicon oxynitride grains. The (001) planes of the crystals extended outwards, giving a thickness of approximately 0.5 microns. The other type formed as hexagonally shaped grains separate from the first type and appeared to have grown as several segments in different directions around a nucleus. In each segment BN layers are parallel to each other and perpendicular to their common [001]BM direction. This second type of BN crystal was also detected a little further from the surface within the silicon nitride. The volume fraction of additive glassy phase tended to be lower in this surface region than in the bulk. Possible mechanisms of prevention of encapsulation glass penetration into the porous ceramic component during HIP were discussed
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- Westman, Bo, et al.
(author)
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Effects on skeletal muscle glutathione status of ischemia and reperfusion following abdominal aortic aneurysm surgery.
- 2006
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In: Annals of Vascular Surgery. - : Elsevier BV. - 0890-5096 .- 1615-5947. ; 20:1, s. 99-105
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Journal article (peer-reviewed)abstract
- Glutathione (GSH) is an important endogenous scavenger against reactive oxygen species. Elective abdominal surgery without ischemia and reperfusion leads to decreased muscle GSH concentrations 4-72 hr postoperatively without altering GSH redox status. In the present study, we investigated to what extent muscle GSH status was affected during and following elective abdominal aortic aneurysm repair. From patients (n = 10) undergoing abdominal aortic repair, thigh muscle specimens were taken preoperatively, at maximal ischemia, and at 10 min and 4, 24, and 48 hr of reperfusion. Specimens were analyzed for GSH, amino acids, and energy-rich compounds. At maximal ischemia, phosphocreatine decreased by 37% (p < 0.05) and lactate and creatine increased by 274% and 57% (p < 0.001 and 0.05), respectively, indicating ischemia during the clamping of aorta. Adenosine triphosphate, on the other hand, remained unaltered during the entire study period. Total GSH (tGSH) decreased by 46% at 24 hr and by 43% at 48 hr of reperfusion (p < 0.001), while reduced GSH decreased by 48% at 24 hr and by 44% at 48 hr (p < 0.001). The redox status (GSH/tGSH) of GSH and oxidized GSH remained unaltered. Among the constituent amino acids of GSH, glycine and cysteine remained unaltered while glutamine and glutamate decreased by 55% and 55%, respectively (p < 0.001). Abdominal aortic aneurysm repair induces metabolic alterations characteristic for ischemia. The antioxidative capacity in terms of muscle levels of GSH was decreased. However, the oxidative stress during reperfusion did not change GSH status more than what has been reported following abdominal surgery without ischemia and reperfusion. The results indicate that the oxidative stress elicited by elective abdominal aortic aneurysm repair is outbalanced by a compensated GSH metabolism not giving rise to an increased amount of oxidized GSH or an altered GSH redox status.
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- Yin, Hong, et al.
(author)
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Complete nucleotide sequence of a Coxsackievirus B-4 strain capable of establishing persistent infection in human pancreatic islet cells : effects on insulin release, proinsulin synthesis, and cell morphology
- 2002
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In: Journal of Medical Virology. - : Wiley. - 0146-6615 .- 1096-9071. ; 68:4, s. 544-57
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Journal article (peer-reviewed)abstract
- The aim of the present investigation was to study the effect of infection of human pancreatic islet cells with a strain (VD2921) of Coxsackie B virus serotype 4 capable of establishing persistent infection in these cells, as well as to sequence the strain, to study the determinants of virulence and persistence. Groups of islets were infected and assessments of proinsulin, insulin content, and virus replication were made. Insulin release in response to high glucose was measured. Infected and control islets displayed a strong insulin response to high glucose 3-4 days as well as 7-8 days post-infection (dpi). At 11-17 dpi, the infected islets did not respond at all to high glucose, and the response of the control islets was at this late time point somewhat reduced. The insulin and proinsulin content of the infected islets did not differ significantly from that of the control islets. TCID(50) titrations showed that the VD2921 strain replicated in the islet cells during the whole study. Electron microscopic examination of infected islets did not reveal any virus-induced changes of cell morphology compared with the controls, although higher magnifications of the infected beta-cells showed virus-like particles in the cytoplasm. These results show that certain strains of Coxsackievirus B-4 in vitro can establish a persistent infection that might mimic, the more gradual loss of beta-cell function seen during the clinical course of autoimmune diabetes. The ability of this Coxsackievirus B-4 strain to establish a persistent infection might be due to substitution of 11 amino acids located at the surface of the structural protein VP1, adjacent to the predicted receptor binding canyon of the virus.
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