The activity of FH, reductase, an enzyme intimately related to cell duplication, has been studied in synchronously growing yeast cells. The method of synchronisation consists of (a) a period of growth in a rich medium during which the temperature of the culture is changed a t regular intervals between 30" and 38", (b) aeration in a starvation medium, (c) repetition of (a), followed by fractionation of the yeast cells by low-speed centrifugation.After the appearance of buds, enzyme activity doubles rapidly and then remains constant, so that in each generation increase in activity follows a step-wise pattern. Since periodic fluctuations in the specific activity and stability of the enzyme could not be detected, the periodicity of the increase in activity must be attributed to discontinuous de novo enzyme synthesis. This periodic enzyme synthesis is not subject to induction or repression. Thus FH2, the natural substrate of FH, reductase, has no influence on enzyme formation. Moreover, periodic synthesis persists even in the presence of lop3 M amethopterin which has been shown to specifically inhibit FH, reductase intracellularly. Quantitative removal of the inhibitor leads to complete reactivation of the enzyme.Yeast cells are made permeable to actinomycin C for several hours by being grown in a medium containing 0.09 Olio deoxycholic acid. Addition of actinom ycin C, to a final concentration of 0.8 x 10-4 M, to a culture during a period of activity increase causes quite rapid and complete inhibition of the synthesis of FH, reductase. This implies a direct connection between the synthesis of the enzyme and the fromation of RNA. The increase in activity occurring after the addition of actinomycin C is attributable to the translation of existing amounts of the messenger RNA which codes for FH, reductase. From the duration of the increase in enzyme activity after the addition of inhibitor the half-life of this mRNA is estimated to be about 5 minutes.Enzyme is synthesised only once in a cell cycle when the cells are growing logarithmically.Retardation of DNA synthesis by addition of amethopterin to a concentration of 1.6 x lo-, M -the intracellular concentration was determined experimentally to be < iO-5 M -leads to a slackening of the rate of enzyme synthesis but the step-wise pattern is maintained. On the other hand, the synthesis of the total soluble cell protein proceeds as rapidly as in the controls, This finding points to a direct connection between DNA replication and the synthesis of FH, reductase.I n contrast to logarithmically growing cells, the first generation in the lag phase shows two phases of synthesis, one occurring before, and the other after, the onset of DNA replication.The model which has been developed to interpret these observations is based on the assumption that the structural gene for FH, reductase exists in two different functional states, in only one of which can the short-lived messenger be replicated. The change from one state to the other gives rise simultaneously to the step-wise pattern...
The course of synthesis of dihydrofolate reductase (5,6,7,8-tetrahydrofolate : NADP oxidoreductase, EC 1.5.1.3) was followed i n synchronized yeast cells in the presence of 2,3,5-trisethyleneimino-1,4-benzoquinone (TrenimonB) as an inhibitor of DNA replication. The following dependency was found between stages of DNA-synthesis and the time course of enzyme formation : whereas in the absence of the inhibitor, enzyme synthesis takes place in a discontinuous, stepwise fashion during one generation cycle [7], after inhibition of DNA replication by 0.01 mg/ml Trenimon, synthesis sets in a t the normal interval. However, its increase is continuous and almost linear ; or exponential, following the pattern of the synthesis of RNA and total protein.A regulatory mechanism is envisaged in which the synthesis of dihydrofolacte reductase is influenced solely by the replication of DNA and independent on metabolites of the enzyme. On the other hand, the level of free thymine-derivatives increases two-fold in the Trenimon-inhibited cells, although the overall synthesis of thymine is greatly retarded. This is probably due to regulation of thymine synthesis by the end-product of the reaction chain.
PurposeTo examine prescriptions of valproate and oral antiepileptic drugs (OAED) in Germany irrespective of the indication in women in general and particularly in women of childbearing age (13–49 years) and during pregnancy between 2010 and 2020, that is, before, during and after the implementation of the EU risk minimization measures (RMMs).MethodsAnalysis of claims data. Study population: all women continuously insured with the AOK health insurance fund in the respective observation year (2010–2020) and the previous year. OAED were identified by ATC code N03. Period of pregnancy was calculated based on birth information in claims data. Main outcomes measures: (i) prevalent use of valproate/OAED: number of women with at least one prescription of valproate/OAED per year divided by all women of the study population (rate per 1000 women); (ii) percentage of OAED recipients with at least one valproate prescription during pregnancy (13–49 years) in the respective observation year.ResultsPrevalence rate/1000 women for valproate use decreased by −31.33% across all age groups (2010–2014: −7.48%; 2014–2018: −16.47%; 2018–2020: −11,17%) with a strong reduction in women 13–49 years between 2014 and 2018 (−28.74%). The rate for OAED across all age groups rose from 33.43/1000 women in 2010 to 41.03/1000 (+22,73%). Valproate use during pregnancy of women with OAED declined from 1.29% in 2010 to 0.59% in 2020 (−54,26%) (2010–2014: −5.14%; 2014–2018: −42.31%; 2018–2020: −16.69%).ConclusionEven if, due to the descriptive nature of the study, no causal relationship can be postulated between the RMMs and the strong decrease in valproate prescriptions, our results are compatible with the hypothesis that the measures have improved drug therapy safety.
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