Tyrosinase (monophenol, o-diphenol:oxygen oxidoreductase, EC 1.14.18.1) is a copper-containing metalloprotein that is ubiquitously distributed in nature. Tyrosinases are found in prokaryotic as well as in eukaryotic microorganisms, and in mammals, invertebrates and plants. Tyrosinase is a mono-oxygenase and a bifunctional enzyme that catalyzes the o-hydroxylation of monophenols and subsequent oxidation of o-diphenols to quinones [1,2]. The activities are also referred to as cresolase or monophenolase and catecholase or diphenolase activities, respectively. Tyrosinase thus accepts monophenols and diphenols as substrates, and the monophenolase activity is the initial rate-determining reaction [2,3]. A homology search of the genome database of the filamentous fungus Trichoderma reesei identified a new T. reesei tyrosinase gene tyr2, encoding a protein with a putative signal sequence. The gene was overexpressed in the native host under the strong cbh1 promoter, and the tyrosinase enzyme was secreted into the culture supernatant. This is the first report on a secreted fungal tyrosinase. Expression of TYR2 in T. reesei resulted in good yields, corresponding to approximately 0.3 and 1 gAEL )1 tyrosinase in shake flask cultures and laboratory-scale batch fermentation, respectively. T. reesei TYR2 was purified with a three-step purification procedure, consisting of desalting by gel filtration, cation exchange chromatography and size exclusion chromatography. The purified TYR2 protein had a significantly lower molecular mass (43.2 kDa) than that calculated from the putative amino acid sequence (61.151 kDa). According to N-terminal and C-terminal structural analyses by fragmentation, chromatography, MS and peptide sequencing, the mature protein is processed from the C-terminus by a cleavage of a peptide fragment of about 20 kDa. The T. reesei TYR2 polypeptide chain was found to be glycosylated at its only potential N-glycosylation site, with a glycan consisting of two N-acetylglucosamines and five mannoses. Also, low amounts of shorter glycan forms were detected at this site. T. reesei TYR2 showed the highest activity and stability within a neutral and alkaline pH range, having an optimum at pH 9. T. reesei tyrosinase retained its activity well at 30°C, whereas at higher temperatures the enzyme started to lose its activity relatively quickly. T. reesei TYR2 was active on both l-tyrosine and l-dopa, and it showed broad substrate specificity.Abbreviations TYR2, tyrosinase 2 from Trichoderma reesei; Q-TOF, quadrupole time-of-flight.

ObjectiveTo assess the impact of reference pricing and extension of generic substitution on the daily cost of antipsychotic drugs in Finland during the first year after its launch. Furthermore, the additional impact of reference pricing on prior implemented generic substitution is assessed.MethodsA retrospective analysis was performed between 2006 and 2010. A segmented linear regression analysis of interrupted time series was used to estimate changes in the levels and trends in the cost of one day of treatment. Of the study drugs, clozapine belonged to generic substitution already at the start of the study period while olanzapine and quetiapine were included in generic substitution alongside with reference pricing in 2009. Risperidone was included in generic substitution in 2008, before reference pricing.ResultsA substantial decrease in the daily cost of all four antipsychotic substances was seen after one year of the implementation of reference pricing and the extension of generic substitution. The impact ranged from -29.9% to -66.3%, and it was most substantial on the daily cost of olanzapine. Also in the daily cost of risperidone a substantial decrease of -43.3% was observed. However, most of these savings, -32.6%, were generated by generic substitution which had been adopted prior.ConclusionsReference pricing and the extension of generic substitution produced substantial savings on antipsychotic medication costs during the first year after its launch, but the intensity of the impact differed between active substances. Furthermore, our results suggest that the additional cost savings from reference pricing after prior implemented generic substitution, are comparatively low.Electronic supplementary materialThe online version of this article (doi:10.1186/s13561-014-0009-3) contains supplementary material, which is available to authorized users.

Although the price competition induced by reference pricing decreased the prices of antipsychotics in Finland in the short-term, the prices had a tendency to stagnate or even to turn in an upward direction in the medium- to long-term. Furthermore, the additional impact of reference pricing over and above previously implemented generic substitution remained quite modest.

Estimating the effects of reforms in advance is an important part of evidence-based and transparent legislative processes. The aim of this study was to describe a microsimulation method created to produce ex ante estimates of pharmaceutical pricing and reimbursement policy reforms. As a case example, the estimates for the 2016 pharmaceutical reimbursement scheme reform, including, e.g., the introduction of a €50 annual deductible, are presented. A static microsimulation model was developed based on the reimbursed purchases of 380,931 individuals drawn at random (10% sample) from the prescription register. The 2016 reform was projected to create savings of €44 million/year for the National Health Insurance (NHI). For patients, the median annual out-of-pocket costs increased from €78 to €96 (by +€18). For 97%, the estimated change was less than €50/year. The majority of patients whose out-of-pocket costs increased had relatively low prior costs. However, >€50/year increases predominantly affected patients entitled to higher reimbursements based on chronic or severe illnesses, among whom older and lower-income individuals were overrepresented. Increases of >€100 were rare (0.003%) and derived from exceptional circumstances. The microsimulation produced prompt but versatile estimates of the effects of legislative reforms by factoring in the entire spectrum of individual situations among affected patients.

Eriarvoisuus terveyspalvelujen laadussa ja saatavuudessa voi vahvistaa sosioekonomisia terveyseroja. Toimeentulotuen saajat sairastavat ja käyttävät julkisia terveyspalveluja muuta saman ikäistä väestöä enemmän. Tutkimuksessa vertailtiin rekisteriaineiston avulla toimeentulotuen saajien (N=37 836) ja ei-toimeentulotuen saajien (N=430 997) lääkekorvausoikeuksia ja lääkkeiden käyttöä Helsingissä vuonna 2010. Menetelminä käytettiin vakiointia ja logistista regressioanalyysia. Kun erot ikä- ja sukupuolirakenteessa, maahanmuuttajataustassa, tulotasossa ja opiskelijoiden osuudessa vakioidaan, toimeentulotuen saajilla on ei-toimeentulotuen saajia useammin lääkekorvausoikeuksia (20 % vs. 15 %) sekä psyykensairauksiin (34 % vs. 14 %), astmaattisiin sairauksiin (11 % vs. 5 %), diabetekseen (6 % vs. 3 %) ja sydän- ja verisuonisairauksiin (21 % vs. 13 %) liittyviä lääkeostoja. Psyykenlääkkeiden käyttö yleistyy toimeentulotuen saantikuukausien määrän kasvaessa, mutta psyyken sairauden vuoksi erityiskorvaukseen oikeutetut saavat toimeentulotukea yleisimmin vain muutaman kuukauden. Psyykenlääkityksen (korvausoikeus tai lääkeosto) ja toimeentulotuen saannin yhteys on samansuuntainen kantaväestöllä ja maahanmuuttajilla (toimeentulotukea 1–3 kuukautta ja 10–12 kuukautta saaneilla ristitulosuhteet (OR) 2,8 [2,7–2,9] ja 5,3 [5,0–5,5] kantaväestöllä; 2,4 [1,9–3,2] ja 7,8 [6,4–9,5] OECD-maista saapuneilla; 2,3 [1,8–3,0] ja 3,6 [2,9–4,3] entisen Neuvostoliiton alueelta saapuneilla; ja 2,0 [1,6–2,6] ja 5,1 [4,4–6,0] muista kuin em. maista saapuneilla, kun vertailuryhmänä ei-toimeentulotuen saajat). Tutkimus vahvistaa käsitystä sairaudesta osana huono-osaisuutta. Terveysongelmien yleisyys on syytä ottaa huomioon toimeentulotuen saajien palveluita suunniteltaessa.