| 1. |
- Larsson, Gunilla, et al.
(författare)
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Kinetic Characterization of dUTPase from Escherichia coli
- 1996
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 271:39, s. 24010-24016
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Tidskriftsartikel (refereegranskat)abstract
- The enzyme dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, thereby preventing a deleterious incorporation of uracil into DNA. The best known dUTPase is that from Escherichia coli, which, like the human enzyme, consists of three identical subunits. In the present work, the catalytic properties of the E. coli dUTPase were investigated in the pH range 5-11. The enzyme was found to be highly specific for dUTP and discriminated both base and sugar as well as the phosphate moiety (bound dUDP was not hydrolyzed). The second best substrate among the nucleotides serving as building blocks for DNA was dCTP, which was hydrolyzed an astonishing 105 times less efficiently than dUTP, a decline largely accounted for by a higher Km for dCTP. With dUTP·Mg as substrate, kcat was found to vary little with pH and to range from 6 to 9 s1. Km passed through a broad minimum in the neutral pH range with values approaching 107 M. It increased with deprotonation of the uracil moiety of dUTP and showed dependence on two ionizations in the enzyme, exhibiting pKa values of 5.8 and 10.3. When excess dUTPase was reacted with dUTP·Mg at pH 8, the two protons transferred to the reaction medium were released in a concerted mode after the rate-limiting step. The Mg2+ ion enhances binding to dUTPase of dUTP by a factor of 100 and dUDP by a factor of 10. Only one enantiomer of the substrate analog 2-deoxyuridine-5-(-thio)-triphosphate was hydrolyzed by the enzyme. These results are interpreted to favor a catalytic mechanism involving magnesium binding to the -phosphate, rate-limiting hydrolysis by a shielded and activated water molecule and a fast ordered desorption of the products. The results are discussed with reference to recent data on the structure of the E. coli dUTPase·dUDP complex.
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| 2. |
- Larsson, Jan-Olov
(författare)
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Aspects of health surveillance at child welfare centres
- 1996
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Objective: To evaluate aspects of the health surveillance at Child Welfare Centres (CWC) particularly the promotion of mental development and health. Method, Subjects: In the main study, which this thesis is a part of, a cohort of children from the general population was studied with regards to their health and development during the preschool years. This part of the study was designed to compare measures of the children's health and development using standard Child Welfare Centre (CWC) methods with prospectively collected information from the longitudinal project. Results: Study I. The first home visit to the newborn baby by the nurse at the CWC was evaluated. Her assessments seemed valid in identifying families with stressful psychosocial conditions. When the general home situation was judged as 'poor' or 'dubious' the boys hadan increased risk to have a delayed mental development at 4-5 years of age. Study II. The four-year check-up at CWC was studied by means of a score. The score was useful in identifying children at risk of delayed mental and behavioural development on a group level. Study III. A model combining the methods used in I and II together with other information at CWC was presented. The results indicate the usefulness of the concept of developmental surveillance. The results indicated that it was possible to identify children at risk of development delay on an individual level. Study IV. Screening at CWC for Minimal Brain Dysfunction at 6 years of age showed that children with an positive screening result at school-start may have exhibited signs of delayed psychoneurological development and symptoms of psychopathology already at 4 years of age. Study V. Children experiencing psychosocial stress and exhiting psychopathological symptoms were found to run a higher risk of having accidents than other children. This information could be useful in the accident prevention work at CWC. Study VI. Behavioural problems and psychopathology in preschool children were evaluated with both a categorical classification in the DSM-III, and a dimensional approach. High rates of certain diagnoses, especially 'oppositional defiant disorder' and 'undersocialised conduct disorder' was found. In future studies, the results will be used in order to develop a CWC-questionnaire with the objective of identifying behavioural problems. Conclusions: The health surveillance programme at CWC, with regards to developmental delay and behavioural problems, has components that seem to be valid in identifying current, and to some extent future, health problems. However, the programme may need revision, e.g. adding tools to identify behavioural problems, and more clear cut advice to the staff on when and how to intervene in a family where the child needs support and stimulation. The significance of the observations in this thesis for the children's health during the school age will be the object for future studies.
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| 3. |
- Nord, Johan, et al.
(författare)
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dUTPase from the retrovirus equine infectious anemia virus: specificity, turnover and inhibition
- 1997
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Ingår i: FEBS Letters. - 1873-3468. ; 414:2, s. 271-274
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Tidskriftsartikel (refereegranskat)abstract
- The kinetic properties of dUTPase from equine infectious anemia virus (EIAV) were investigated. KM (1.1 [plusmn] 0.1 [mu ]M) and kcat (25 s[minus ]1) were found to be independent of pH in the neutral pH range. Above pH 8.0, KM increases slightly. Below pH 6.0, the enzyme is rapidly deactivated. Detergent was found to enhance activity, leaving KM and kcat unaffected. Compared to the Escherichia coli dUTPase, the EIAV enzyme is equally potent in hydrolyzing dUTP, but less specific. Inhibition of the viral enzyme by the nucleotides dTTP, dUMP and a synthetic analogue, 2[prime ]-deoxyuridine 5[prime ]-([alpha ],[beta ]-imido)triphosphate, is stronger by one order of magnitude.
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