Betuliinijohdannaisten syntetisointi solunsisäisiä patogeenejä vastaan

White birches (Betula spp.) are utilized in huge quantities in the forest industry in northern latitudes of the world and low-value side-stream birch bark is burnt for energy. Outer birch bark is rich in (up to 30% dry weight) triterpene betulin and it can be easily isolated by solvent extraction. Betulin could be used as such or as starting material for more valuable products, fine chemicals and pharmaceuticals. In this work two sets of betulin derivatives were synthesized and screened as antiviral, antileishmanial and antibacterial agents. First set includes relatively easily synthesizable betulin derivatives, such as esters and various oxidation products. Second set includes novel heterocyclic betulin derivatives, where triazolo ring is fused by Diels-Alder reaction to the lupane skeleton of betulin. In the biological assays against human pathogens Semliki Forest virus; L. donovani parasites, which cause tropical disease leishmaniasis; and Gram-negative bacteria Chlamydia pneumoniae, several betulin derivatives displayed low-micromolar 50% growth inhibition values in vitro. In addition, most of the derivatives showed low cytotoxicity against the host cell lines.Metsäteollisuus käyttää koivua raaka-aineenaan suunnattomia määriä. Sivuotteena syntyvä koivunkuori poltetaan energian tuotantoon. Koivun ulkokuori sisältää runsaasti betuliini-nimistä triterpeeniä jopa 30 % kuivapainostaan. Betuliini voidaan helposti eristää kuoresta liuotinuutolla. Betuliinia voidaan käyttää sellaisenaan tai lähtöaineena muille tuotteille, hienokemikaaleille ja lääkeaineille. Tässä väitöskirjatyössä syntetisoitiin kaksi betuliinijohdannaisryhmää ja yhdisteiden tehokkuutta testattiin alfavirusten, Leishmania-suvun alkueläinten ja keuhkoklamydiabakteerin vastaisina yhdisteinä. Ensimmäinen ryhmä sisältää melko helposti valmistettavissa olevia johdannaisia, kuten betuliinin estereitä ja erilaisia hapetustuotteita. Toinen ryhmä sisältää uusia heterosyklisiä betuliinijohdannaisia, joissa triatsoli-rengas on fuusioitu betuliinin lupaanirakenteiseen hiilivetyrankaan. Testattaessa betuliinijohdannaisia ihmispatogeenejä vastaan, useat johdannaiset estivät taudinaiheuttajien kasvua mikromolaarisella konsentraatiolla. Lisäksi suurin osa betuliinijohdannaisista oli harmittomia isäntäsoluina käytettyjä solulinjoja vastaan

Molecular biology of progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1)

Progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is an autosomal recessively inherited disorder characterized by age of onset at 6-15 years, stimulus-sensitive myoclonus, tonic-clonic epileptic seizures and a progressive course. Mutations in the cystatin B (CSTB) gene underlie EPM1. The most common mutation underlying EPM1 is a dodecamer repeat expansion in the promoter region of CSTB. In addition, nine other mutations have been identified. CSTB, a cysteine protease inhibitor, is a ubiquitously expressed inhibitor of cathepsins, but its physiological function is unknown. The purpose of this study was to investigate CSTB gene expression and CSTB protein function in normal and pathological conditions. The basal CSTB promoter was mapped and characterized using different promoter-luciferase gene constructs. The binding activity of transcription factors to one ARE half, five Sp1 and four AP1 sites in the CSTB promoter was demonstrated. The CSTB promoter activity was clearly decreased using a CSTB promoter with "premutation" repeat expansions and in individuals with alike expansions. The expression of CSTB mRNA and protein was markedly reduced in patient cells. The endogenous CSTB protein localized to the nucleus, cytoplasm and lysosomes, and in differentiated cells merely to the cytoplasm. This suggests that the subcellular distribution of CSTB is dependent on the differentation status of the cells. The proteins representing patient missense mutations failed to associate with lysosomes, implying the importance of the lysosomal association for the proper physiological function of CSTB. Several alternatively spliced CSTB isoforms were identified. Of these CSTB2 was widely expressed with very low levels whereas the other alternatively spliced forms seemed to have limited tissue expression. In patients CSTB2 expression was reduced similarly to that of CSTB. The physiological relevance of CSTB alternative splicing remains unknown. The mouse Cstb transcript was shown to be present in all embryonic stages and adult tissues examined. The expression was highest at embryonic day 7 and in thymus, as well as in postnatal brain in the cortex, caudate putamen, thalamus, hippocampus, and in the Purkinje cell layer of the cerebellum. Our data implies that CSTB expression is tightly temporally and spatially regulated. The data presented in my thesis lay the basis for further understanding of the role of CSTB in health and disease.Unverricht-Lundborg-tyyppinen etenevä myoklonusepilepsia (EPM1) on peittyvästi periytyvä aivoja rappeuttava sairaus, jonka oireisiin kuuluvat epileptiset kohtaukset ja ärsykeherkät tahdottomat lihasnykäykset. Taudin oireet alkavat 6-15 vuoden iässä ja ovat eteneviä. EPM1:n taustalla ovat virheet kystatiini B (CSTB) -geenissä. Yleisin mutaatio on 12 emäksestä muodostuvan toistojakson monistuma CSTB-geenin säätelyalueella, minkä lisäksi tunnetaan yhdeksän muuta mutaatiota. CSTB-proteiini toimii tiettyjen valkuaisaineita pilkkovien entsyymien estäjänä, mutta sen tehtävää elimistössä ei tarkoin tunneta. Tässä väitöskirjatyössä tutkittiin normaalin CSTB-geenin ilmenemistä hiirellä ja ihmisellä, CSTB-proteiinin solulokalisaatiota sekä potilasmutaatioiden vaikutusta näihin. Kartoitimme ihmisen CSTB-geenin säätelyalueen ja osoitimme siihen paikantuvien transkriptiotekijöiden sitoutumisen. CSTB:n ilmeneminen väheni selvästi säätelyalueen sisältäessä toistojakson pidentymän sekä henkilöillä, joilla on normaalin muodon ja tautimutaation välinen muoto toistojakson pidentymästä. EPM1-potilaiden soluissa CSTB-geenin lähetti-RNA:n sekä proteiinin määrä oli merkittävästi alentunut. CSTB-proteiini paikantui jakautuvissa soluissa solunsisällä tumaan, sytoplasmaan ja lysosomeihin sekä erilaistuneissa soluissa pääasiallisesti sytoplasmaan. Potilasmutaatioiden seurauksena CSTB-proteiini menetti lokalisaation lysosomeihin. Tulosten mukaan solun erilaistumistaso vaikuttaa CSTB:n paikantumiseen ja lysosomaalinen sijainti on tarpeellinen sen oikean toiminnan kannalta. CSTB-geenissä havaittiin vaihtoehtoista silmikoitumista. CSTB2-muoto ilmeni laajasti, mutta sen osuus oli vain 0,4-4,1 % koko CSTB-geenin ilmentymisestä. Sen ilmeneminen oli alentunut potilassoluissa. Muut muodot näyttivät ilmenevän kudosspesifisesti. CSTB-geenin vaihtoehtoisen silmikoitumisen fysiologinen merkitys jäi epäselväksi. Havaitsimme hiiren Cstb-geenin ilmenevän laajasti ja olevan korkeinta 7-päivän ikäisessä sikiössä sekä kateenkorvassa. Keskushermostossa Cstb ilmeni isoaivokuorella, aivokuorukassa, talamuksessa ja hippokampuksessa, sekä pikkuaivojen Purkinje-solukerroksessa. Tulostemme perusteella CSTB ilmeneminen on voimakkaasti ajallisesti ja paikallisesti säädeltyä. Väitöskirjassani esitetyt tulokset luovat perustaa jatkotutkimuksille selvitettäessä CSTB-proteiinin normaalia toimintaa sekä sen puuttumisen ja virheellisen toiminnan merkitystä EPM1-taudin synnyssä

Leishmanicidal activity of betulin derivatives in Leishmania amazonensis; Effect on plasma and mitochondrial membrane potential, and macrophage nitric oxide and superoxide production

Herein, we evaluated in vitro the anti-leishmanial activity of betulin derivatives in Venezuelan isolates of Leishmania amazonensis, isolated from patients with therapeutic failure. Methods: We analyzed promastigote in vitro susceptibility as well as the cytotoxicity and selectivity of the evaluated compounds. Additionally, the activity of selected compounds was determined in intracellular amastigotes. Finally, to gain hints on their potential mechanism of action, the effect of the most promising compounds on plasma and mitochondrial membrane potential, and nitric oxide and superoxide production by infected macrophages was determined. Results: From the tested 28 compounds, those numbered 18 and 22 were chosen for additional studies. Both 18 and 22 were active (GI(50) 45 mu M, SI > 20) for the reference strain LTB0016 and for patient isolates. The results suggest that 18 significantly depolarized the plasma membrane potential (p <0.05) and the mitochondrial membrane potential (p <0.05) when compared to untreated cells. Although neither 18 nor 22 induced nitric oxide production in infected macrophages, 18 induced superoxide production in infected macrophages. Conclusion: Our results suggest that due to their efficacy and selectivity against intracellular parasites and the potential mechanisms underlying their leishmanicidal effect, the compounds 18 and 22 could be used as tools for designing new chemotherapies against leishmaniasis.Peer reviewe

Biotransformation of Cyclodextrine-Complexed Semisynthetic Betulin Derivatives by Plant Cells

In this study, three semisynthetic betulonic acid-based compounds, 20(29)-dihydrolup-2-en[2,3- d ]isoxazol-28-oic acid, 1-betulonoylpyrrolidine, and lupa-2,20(29)-dieno[2,3- b ]pyrazin-28-oic acid, were studied in biotransformation experiments using Nicotiana tabacum and Catharanthus roseus cell suspension cultures. Biotransformation was performed using cyclodextrin to aid dissolving poorly water-soluble substrates. Several new derivatives were found, consisting of oxidized and glycosylated (pentose- and hexose-conjugated) products.Peer reviewe

Leishmanicidal Activity of Betulin Derivatives in Leishmania amazonensis; Effect on Plasma and Mitochondrial Membrane Potential, and Macrophage Nitric Oxide and Superoxide Production

Herein, we evaluated in vitro the anti-leishmanial activity of betulin derivatives in Venezuelan isolates of Leishmania amazonensis, isolated from patients with therapeutic failure. Methods: We analyzed promastigote in vitro susceptibility as well as the cytotoxicity and selectivity of the evaluated compounds. Additionally, the activity of selected compounds was determined in intracellular amastigotes. Finally, to gain hints on their potential mechanism of action, the effect of the most promising compounds on plasma and mitochondrial membrane potential, and nitric oxide and superoxide production by infected macrophages was determined. Results: From the tested 28 compounds, those numbered 18 and 22 were chosen for additional studies. Both 18 and 22 were active (GI50 ≤ 2 µM, cytotoxic CC50 > 45 µM, SI > 20) for the reference strain LTB0016 and for patient isolates. The results suggest that 18 significantly depolarized the plasma membrane potential (p < 0.05) and the mitochondrial membrane potential (p < 0.05) when compared to untreated cells. Although neither 18 nor 22 induced nitric oxide production in infected macrophages, 18 induced superoxide production in infected macrophages. Conclusion: Our results suggest that due to their efficacy and selectivity against intracellular parasites and the potential mechanisms underlying their leishmanicidal effect, the compounds 18 and 22 could be used as tools for designing new chemotherapies against leishmaniasis

Leishmanicidal Activity of Betulin Derivatives in Leishmania amazonensis; Effect on Plasma and Mitochondrial Membrane Potential, and Macrophage Nitric Oxide and Superoxide Production

Herein, we evaluated in vitro the anti-leishmanial activity of betulin derivatives in Venezuelan isolates of Leishmania amazonensis, isolated from patients with therapeutic failure. Methods: We analyzed promastigote in vitro susceptibility as well as the cytotoxicity and selectivity of the evaluated compounds. Additionally, the activity of selected compounds was determined in intracellular amastigotes. Finally, to gain hints on their potential mechanism of action, the effect of the most promising compounds on plasma and mitochondrial membrane potential, and nitric oxide and superoxide production by infected macrophages was determined. Results: From the tested 28 compounds, those numbered 18 and 22 were chosen for additional studies. Both 18 and 22 were active (GI50 ≤ 2 µM, cytotoxic CC50 > 45 µM, SI > 20) for the reference strain LTB0016 and for patient isolates. The results suggest that 18 significantly depolarized the plasma membrane potential (p < 0.05) and the mitochondrial membrane potential (p < 0.05) when compared to untreated cells. Although neither 18 nor 22 induced nitric oxide production in infected macrophages, 18 induced superoxide production in infected macrophages. Conclusion: Our results suggest that due to their efficacy and selectivity against intracellular parasites and the potential mechanisms underlying their leishmanicidal effect, the compounds 18 and 22 could be used as tools for designing new chemotherapies against leishmaniasis

Cystatin B: mutation detection, alternative splicing and expression in progressive myclonus epilepsy of Unverricht-Lundborg type (EPM1) patients

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Noggin null allele mice exhibit a microform of holoprosencephaly

Holoprosencephaly (HPE) is a heterogeneous craniofacial and neural developmental anomaly characterized in its most severe form by the failure of the forebrain to divide. In humans, HPE is associated with disruption of Sonic hedgehog and Nodal signaling pathways, but the role of other signaling pathways has not yet been determined. In this study, we analyzed mice which, due to the lack of the Bmp antagonist Noggin, exhibit elevated Bmp signaling. Noggin−/− mice exhibited a solitary median maxillary incisor that developed from a single dental placode, early midfacial narrowing as well as abnormalities in the developing hyoid bone, pituitary gland and vomeronasal organ. In Noggin−/− mice, the expression domains of Shh, as well as the Shh target genes Ptch1 and Gli1, were reduced in the frontonasal region at key stages of early facial development. Using E10.5 facial cultures, we show that excessive BMP4 results in reduced Fgf8 and Ptch1 expression. These data suggest that increased Bmp signaling in Noggin−/− mice results in downregulation of the hedgehog pathway at a critical stage when the midline craniofacial structures are developing, which leads to a phenotype consistent with a microform of HP

Heterocycle-fused Lupane Triterpenoids Inhibit Leishmania donovani Amastigotes

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