Peroxidases in lignifying xylem of Norway spruce, Scots pine and silver birch

Lignin is a complex plant polymer synthesized through co-operation of multiple intracellular and extracellular enzymes. It is deposited to plant cell walls in cells where additional strength or stiffness are needed, such as in tracheary elements (TEs) in xylem, supporting sclerenchymal tissues and at the sites of wounding. Class III peroxidases (POXs) are secreted plant oxidoreductases with implications in many physiological processes such as the polymerization of lignin and suberin and auxin catabolism. POXs are able to oxidize various substrates in the presence of hydrogen peroxide, including lignin monomers, monolignols, thus enabling the monolignol polymerization to lignin by radical coupling. Trees produce large amounts of lignin in secondary xylem of stems, branches and roots. In this study, POXs of gymnosperm and angiosperm trees were studied in order to find POXs which are able to participate in lignin polymerization in developing secondary xylem i.e. are located at the site of lignin synthesis in tree stems and have the ability to oxidize monolignol substrates. Both in the gymnosperm species, Norway spruce and Scots pine, and in the angiosperm species silver birch the monolignol oxidizing POX activities originating from multiple POX isoforms were present in lignifying secondary xylem in stems during the period of annual growth. Most of the partially purified POXs from Norway spruce and silver birch xylem had highest oxidation rate with coniferyl alcohol, the main monomer in guaiacyl-lignin in conifers. The only exception was the most anionic POX fraction from silver birch, which clearly preferred sinapyl alcohol, the lignin monomer needed in the synthesis of syringyl-guaiacyl lignin in angiosperm trees. Three full-length pox cDNAs px1, px2 and px3 were cloned from the developing xylem of Norway spruce. It was shown that px1 and px2 are expressed in developing tracheids in spruce seedlings, whereas px3 transcripts were not detected suggesting low transcription level in young trees. The amino acid sequences of PX1, PX2 and PX3 were less than 60% identical to each other but showed up to 84% identity to other known POXs. They all begin with predicted N-terminal secretion signal (SS) peptides. PX2 and PX3 contained additional putative vacuolar localization determinants (VSDs) at C-terminus. Transient expression of EGFP-fusions of the SS- and VSD-peptides in tobacco protoplasts showed SS-peptides directed EGFP to secretion in tobacco cells, whereas only the PX2 C-terminal peptide seems to be a functional VSD. According to heterologous expression of px1 in Catharanthus roseus hairy roots, PX1 is a guaicol-oxidizing POX with isoelectric point (pI) approximately 10, similar to monolignol oxidizing POXs in protein extracts from Norway spruce lignifying xylem. Hence, PX1 has characteristics for participation to monolignol dehydrogenation in lignin synthesis, whereas the other two spruce POXs seem to have some other functions. Interesting topics in future include functional characterization of syringyl compound oxidizing POXs and components of POX activity regulation in trees.

Expression of functional GABAc receptors in the brain

γ-aminobutyric acid (GABA) is the main inhibitory transmitter in the nervous system and acts via three distinct receptor classes: A, B, and C. GABAC receptors are ionotropic receptors comprising ρ subunits. In this work, we aimed to elucidate the expression of ρ subunits in the postnatal brain, the characteristics of ρ2 homo-oligomeric receptors, and the function of GABAC receptors in the hippocampus. In situ hybridization on rat brain slices showed ρ2 mRNA expression from the newborn in the superficial grey layer of the superior colliculus, from the first postnatal week in the hippocampal CA1 region and the pretectal nucleus of the optic tract, and in the adult dorsal lateral geniculate nucleus. Quantitative RT-PCR revealed expression of all three ρ subunits in the hippocampus and superior colliculus from the first postnatal day. In the hippocampus, ρ2 mRNA expression clearly dominated over ρ1 and ρ3. GABAC receptor protein expression was confirmed in the adult hippocampus, superior colliculus, and dorsal lateral geniculate nucleus by immunohistochemistry. From the selective distribution of ρ subunits, GABAC receptors may be hypothesized to be specifically involved in aspects of visual image motion processing in the rat brain. Although previous data had indicated a much higher expression level for ρ2 subunit transcripts than for ρ1 or ρ3 in the brain, previous work done on Xenopus oocytes had suggested that rat ρ2 subunits do not form functional homo-oligomeric GABAC receptors but need ρ1 or ρ3 subunits to form hetero-oligomers. Our results demonstrated, for the first time, that HEK 293 cells transfected with ρ2 cDNA displayed currents in whole-cell patch-clamp recordings. Homomeric rat ρ2 receptors had a decreased sensitivity to, but a high affinity for picrotoxin and a marked sensitivity to the GABAC receptor agonist CACA. Our results suggest that ρ2 subunits may contribute to brain function, also in areas not expressing other ρ subunits. Using extracellular electrophysiological recordings, we aimed to study the effects of the GABAC receptor agonists and antagonists on responses of the hippocampal neurons to electrical stimulation. Activation of GABAC receptors with CACA suppressed postsynaptic excitability and the GABAC receptor antagonist TPMPA inhibited the effects of CACA. Next, we aimed to display the activation of the GABAC receptors by synaptically released GABA using intracellular recordings. GABA-mediated long-lasting depolarizing responses evoked by high-frequency stimulation were prolonged by TPMPA. For weaker stimulation, the effect of TPMPA was enhanced after GABA uptake was inhibited. Our data demonstrate that GABAC receptors can be activated by endogenous synaptic transmitter release following strong stimulation or under conditions of reduced GABA uptake. The lack of GABAC receptor activation by less intensive stimulation under control conditions suggests that these receptors are extrasynaptic and activated via spillover of synaptically released GABA. Taken together with the restricted expression pattern of GABAC receptors in the brain and their distinctive pharmacological and biophysical properties, our findings supporting extrasynaptic localization of these receptors raise interesting possibilities for novel pharmacological therapies in the treatment of, for example, epilepsy and sleep disorders.

Expression of cytochrome P450 enzymes and metabolism of timolol in human ocular tissue

Glaucoma is a group of progressive optic neuropathies causing irreversible blindness if not diagnosed and treated in the early state of progression. Disease is often, but not always, associated with increased intraocular pressure (IOP), which is also the most important risk factor for glaucoma. Ophthlamic timolol preparations have been used for decades to lower increased intraocular pressure (IOP). Timolol is locally well tolerated but may cause e.g. cardiovascular and pulmonary adverse effects due to systemic absorption. It has been reported that approximately 80% of a topically administered eye drop is systemically absorbed. However, only limited information is available on timolol metabolism in the liver or especially in the human eye. The aim of this work was to investigate metabolism of timolol in human liver and human ocular tissues. The expression of drug metabolizing cytochrome P450 (CYP) enzymes in the human ciliary epithelial cells was studied. The metabolism of timolol and the interaction potential of timolol with other commercially available medicines were investigated in vitro using different liver preparations. The absorption of timolol to the aqueous humor from two commercially available products: 0.1% eye gel and 0.5% eye drops and the presence of timolol metabolites in the aqueous humor were investigated in a clinical trial. Timolol was confirmed to be metabolized mainly by CYP2D6 as previously suggested. Potent CYP2D6 inhibitors especially fluoxetine, paroxetine and quinidine inhibited the metabolism of timolol. The inhibition may be of clinical significance in patients using ophthalmic timolol products. CYP1A1 and CYP1B1 mRNAs were expressed in the human ciliary epithelial cells. CYP1B1 was also expressed at protein level and the expression was strongly induced by a known potent CYP1B1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The CYP1B1 induction is suggested to be mediated by aryl hydrocarbon receptor (AHR). Low levels of CYP2D6 mRNA splice variants were expressed in the human ciliary epithelial cells and very low levels of timolol metabolites were detected in the human aqueous humor. It seems that negligible amount of CYP2D6 protein is expressed in the human ocular tissues. Timolol 0.1% eye gel leads to aqueous humor concentration high enough to achieve therapeutic effect. Inter-individual variation in concentrations is low and intraocular as well as systemic safety can be increased when using this product with lower timolol concentration instead of timolol 0.5% eye drops.

Zebrafish as a model of Parkinson's disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease among the elderly. Its etiology is unknown and no disease-modifying drugs are available. Thus, more information concerning its pathogenesis is needed. Among other genes, mutated PTEN-induced kinase 1 (PINK1) has been linked to early-onset and sporadic PD, but its mode of action is poorly understood. Most animal models of PD are based on the use of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP is metabolized to MPP+ by monoamine oxidase B (MAO B) and causes cell death of dopaminergic neurons in the substantia nigra in mammals. Zebrafish has been a widely used model organism in developmental biology, but is now emerging as a model for human diseases due to its ideal combination of properties. Zebrafish are inexpensive and easy to maintain, develop rapidly, breed in large quantities producing transparent embryos, and are readily manipulated by various methods, particularly genetic ones. In addition, zebrafish are vertebrate animals and results derived from zebrafish may be more applicable to mammals than results from invertebrate genetic models such as Drosophila melanogaster and Caenorhabditis elegans. However, the similarity cannot be taken for granted. The aim of this study was to establish and test a PD model using larval zebrafish. The developing monoaminergic neuronal systems of larval zebrafish were investigated. We identified and classified 17 catecholaminergic and 9 serotonergic neuron populations in the zebrafish brain. A 3-dimensional atlas was created to facilitate future research. Only one gene encoding MAO was found in the zebrafish genome. Zebrafish MAO showed MAO A-type substrate specificity, but non-A-non-B inhibitor specificity. Distribution of MAO in larval and adult zebrafish brains was both diffuse and distinctly cellular. Inhibition of MAO during larval development led to markedly elevated 5-hydroxytryptamine (serotonin, 5-HT) levels, which decreased the locomotion of the fish. MPTP exposure caused a transient loss of cells in specific aminergic cell populations and decreased locomotion. MPTP-induced changes could be rescued by the MAO B inhibitor deprenyl, suggesting a role for MAO in MPTP toxicity. MPP+ affected only one catecholaminergic cell population; thus, the action of MPP+ was more selective than that of MPTP. The zebrafish PINK1 gene was cloned in zebrafish, and morpholino oligonucleotides were used to suppress its expression in larval zebrafish. The functional domains and expression pattern of zebrafish PINK1 resembled those of other vertebrates, suggesting that zebrafish is a feasible model for studying PINK1. Translation inhibition resulted in cell loss of the same catecholaminergic cell populations as MPTP and MPP+. Inactivation of PINK1 sensitized larval zebrafish to subefficacious doses of MPTP, causing a decrease in locomotion and cell loss in one dopaminergic cell population. Zebrafish appears to be a feasible model for studying PD, since its aminergic systems, mode of action of MPTP, and functions of PINK1 resemble those of mammalians. However, the functions of zebrafish MAO differ from the two forms of MAO found in mammals. Future studies using zebrafish PD models should utilize the advantages specific to zebrafish, such as the ability to execute large-scale genetic or drug screens.

Effects of oral calcium sensitizers on experimental heart failure

Suun kautta annosteltava kalsiumherkistäjä parantaa sydämen vajaatoimintaan liittyvää pumppausvajetta kokeellisissa sydämen vajaatoimintamalleissa Huolimatta viime vuosikymmenien lääketieteellisestä kehityksestä krooninen sydämen vajaatoiminta on silti edelleen vakava, elämänlaatua voimakkaasti rajoittava sairaus. Kalsiumherkistäjät ovat uusi, sydämen pumppausvoimaa lisäävä lääkeryhmä. Levosimendaani, kotimaista alkuperää oleva kalsiumherkistäjä, on kliinisessä käytössä akuutin vajaatoiminnan hoitoon suonensisäisesti ja lyhytaikaisesti annosteltavana valmisteena. Levosimendaanilla on aktiivinen metaboliitti, OR-1896, jonka oletetaan olevan vuorokauden mittaisen levosimendaani-infuusion jälkeen havaittujen useita päiviä kestävien hyödyllisisten vaikutuksisten takana. Levosimendaanin kroonisen, suun kautta tapahtuvan annostelun vaikutuksista tieto on vähäisempää, mutta sillä näyttää olevan positiivisia vaikutuksia potilaiden raportoimana. FM Marjut Louhelainen on selvittänyt väitöskirjassaan suun kautta annosteltavan levosimendaanin ja sen pitkäkestoisen aktiivisen metaboliitin vaikutuksia kroonisen vajaatoiminnan hoidossa käyttämällä sekä hypertensiivisen sydäntaudin että 2 tyypin diabeteksen komplisoimaan sydäninfarktin kokeellisia malleja. Tutkimuksessa selvitettiin lisäksi vajaatoimintaan johtavia molekyylitason tapahtumia sydänlihaksessa. Tutkimuksessa osoitettiin, että krooninen suun kautta annosteltu hoito sekä kalsiumherkistäjä levosimendaanilla että sen aktiivisella metaboliitilla estää hypertensiiviseen sydämen vajaatoiminnan aikaasaamaa sydämen uudelleenmuovaantumista ja siihen liittyvää kuolleisuutta. Nämä vaikutukset välittyivät vähentyneen sydänlihassoluhypertrofian, solukuolleisuuden ja neurohumaraalisen aktivaation kautta. Levosimendaanin ja OR-1896:n osoitettiin myös parantavan sydämen pumppausfunktiota tyyppi 2 diabeteksen komplisoimassa sydäninfarktissa. Ei-diabeettiseen tilanteeseen verrattuna diabetekseen liittyvä infarktin jälkeinen vajaatoiminnan kehitys oli yhteydessä lisääntyneeseen tulehdukseen, fibroosiin, solukuolemaan, neurohumoraaliseen aktivaatioon ja ennenaikaiseen kudoksen vanhenemiseen. Sekä levosimendaani, että OR-1869 vähensivät tulehduksen, fibroosin ja solukuoleman merkkejä ja vaimensi neurohumoraalista aktivaatiota. OR-1896 myös vähensi solujen vanhenemiseen liittyvien merkkiaineiden ilmentymistä. Väitöskirjassa todettiin, että suun kautta annosteltuna sekä levosimendaani, että sen aktiivinen metaboliitti OR-1896, omaavat terapeuttista potentiaalia sekä hypertensiivisen sydäntaudin hoitoon että sydäninfarktin jälkeisen vajaatoiminnan estoon. FM Marjut Louhelaisen farmakologian alaan kuuluva väitöskirja Effects of oral calcium sensitizers on experimental heart failure tarkastetaan Helsingin yliopiston Lääketieteellisessä tiedekunnassa perjantaina 29.01.2010 klo 12 (Biomedicum Helsinki, luentosali 2, Haartmaninkatu 8, Helsinki). Vastaväittäjänä toimii professori Raimo Tuominen, Helsingin yliopiston Farmasian tiedekunnasta ja kustoksena professori Eero Mervaala Helsingin yliopiston Lääketieteellisestä tiedekunnasta.

Expression and function of angiotensins in the regulation of intraocular pressure - an experimental study

Glaucoma is a multifactorial long-term ocular neuropathy associated with progressive loss of the visual field, retinal nerve fiber structural abnormalities and optic disc changes. Like arterial hypertension it is usually a symptomless disease, but if left untreated leads to visual disability and eventual blindness. All therapies currently used aim to lower intraocular pressure (IOP) in order to minimize cell death. Drugs with new mechanisms of action could protect glaucomatous eyes against blindness. Renin-angiotensin system (RAS) is known to regulate systemic blood pressure and compounds acting on it are in wide clinical use in the treatment of hypertension and heart failure but not yet in ophthalmological use. There are only few previous studies concerning intraocular RAS, though evidence is accumulating that drugs antagonizing RAS can also lower IOP, the only treatable risk factor in glaucoma. The main aim of this experimental study was to clarify the expression of the renin-angiotensin system in the eye tissues and to test its potential oculohypotensive effects and mechanisms. In addition, the possible relationship between the development of hypertension and IOP was evaluated in animal models. In conclusion, a novel angiotensin receptor type (Mas), as well as ACE2 enzyme- producing agonists for Mas, were described for the first time in the eye structures participating in the regulation of IOP. In addition, a Mas receptor agonist significantly reduced even normal IOP. The effect was abolished by a specific receptor antagonist. Intraocular, local RAS would thus to be involved in the regulation of IOP, probably even more in pathological conditions such as glaucoma though there was no unambiguous relationship between arterial and ocular hypertension. The findings suggest the potential as antiglaucomatous drugs of agents which increase ACE2 activity and the formation of angiotensin (1-7), or activate Mas receptors.

Androgen receptor in transcriptional regulation and carcinogenesis

Androgens control a variety of developmental processes that create the male phenotype and are important for maintaining male fertility and normal functions of tissues and organs that are not directly involved in procreation. Androgen receptor (AR) that mediates the biological actions of androgens is a member of the nuclear receptor superfamily of ligand-inducible transcription factors. Although AR was cloned over 15 years ago, the mechanisms by which it regulates gene expression are not well understood. A growing body of in vitro experimental evidence suggests that a complex network of proteins is involved in the androgen-dependent transcriptional regulation. However, the process of AR-dependent transcriptional regulation under physiological conditions is largely elusive. In the present study, a series of experiments were performed, including quantitative chromatin immunoprecipitation (ChIP) assays, to investigate AR-mediated transcription process using living prostate cancer cells. Our results show that the loading of AR and recruitment of coactivators and RNA polymerase II (Pol II) to both the promoter and enhancer of AR target genes are a transient and cyclic event that in addition to hyperacetylation, also involves dynamic changes in methylation, phosphorylation of core histone H3 in androgen-treated LNCaP cells. The dynamics of testosterone (T)-induced loading of AR onto the proximal promoters of the genes clearly differed from that loaded onto the distal enhancers. Significantly, more holo-AR was loaded onto the enhancers than the promoters, but the principal Pol II transcription complex was assembled on the promoters. By contrast, the pure antiandrogen bicalutamide (CDX) complexed to AR elicited occupancy of the PSA promoter, but was unable to load onto the PSA enhancer and was incapable of recruiting Pol II, coactivators and following changes of covalent histone modifications. The partial antagonist cyproterone acetate (CPA) and mifepristone (RU486) were capable of promoting AR loading onto both the PSA promoter and enhancer at a comparable efficiency with androgen in LNCaP cells expressing mutant AR. However, CPA- and RU486-bound AR not only recruited Pol II and coactivator p300 and GRIP1 onto the promoter and enhancer, but also recruited the corepressor NCoR onto the promoter as efficiently as CDX. In addition, we demonstrate that both proteasome and protein kinases are implicated in AR-mediated transcription. Even though proteasome inhibitor MG132 and protein kinase inhibitor DRB (5, 6-Dichlorobenzimidazole riboside) can block ligand-dependent accumulation of PSA mRNA with same efficiency, their use results in different molecular profiles in terms of the formation of AR-mediated transcriptional complex. Collectively, these results indicate that transcriptional activation by AR is a complicated process, which includes transient loading of holo-AR and recruitment of Pol II and coregulators accompanied by a cascade of distinct covalent histone modifications; This process involves both the promoter and enhancer elements, as well as other general components of the cell machineries e.g. proteasome and protein kinase; The pure antiandrogen CDX and the partial antagonist CPA and RU486 exhibit clearly different profiles in terms of their ability to induce the formation of AR-dependent transcriptional complexes and the histone modifications associated with the target genes in human prostate cancer cells. Finally, by using quantitative RT-PCR to compare the expression of sixteen AR co-regulators in prostate cancer cell lines, xenografts, and clinical prostate cancer specimens we suggest that AR co-regulators protein inhibitor of activated STAT1 (PIAS1) and steroid receptor coactivator 1(SRC1) could be involved in the progression of prostate cancer.

Kidney Induction: control by Notch, Wnt and GDNF/Ret signalling

The permanent mammalian kidney (metanephros) develops as a result of complex reciprocal tissue interactions between a ureteric epithelium and the renal mesenchyme. The overall goal of the research in this thesis was to gain data that will eventually help in elucidating the formation of congenital renal malformations. The experiments in my thesis aimed to reveal the mechanisms by which Notch, Wnt and GDNF/Ret signalling pathways regulate the development of functional kidney. The function of Notch pathway was studied by a transgenic mouse model, where it was shown that overactivation of Notch signalling disturbs kidney development and alters the expression of Gdnf and Ret/GFRa1. This indicates that Notch signalling interplays with GDNF/Ret in the regulation of the primary ureteric budding and its subsequent branching. The data also suggested that strict spatio-temporal regulation of these two pathways is required for determination of ureteric tip-identity, which appeared to be crucial for the branch formation. The function of Wnt signalling in the ureteric morphogenesis was studied by in vivo and in vitro methods to show that a canonical pathway is required for ureteric branching. Stabilisation and deletion of the canonical pathway mediator, b-catenin specifically in the ureteric epithelium result in renal aplasia/hypodysplasia. These defects originate from severe blockage of ureteric branching due to the disrupted Ret signalling. Consequently, ureteric tip specific markers are lost and ureteric stalk identity is expanded throughout the whole epithelium. Thus, the data demonstrates that the Wnt/b-catenin pathway plays an essential role in the patterning and branching of the ureteric epithelium. A novel in vitro method was generated and utilised in nephron induction studies to reveal the mechanisms through which nephrogenesis is induced. Transient GSK3 inhibition results in stabilisation of b-catenin in the isolated renal mesenchyme, which efficiently triggers nephron formation. Also genetic stabilisation of b-catenin specifically in the mesenchyme results in spontaneous nephrogenesis. The results show that activation of the canonical Wnt pathway is sufficient to initiate nephrogenesis, and suggest that this pathway mediates the nephron induction in murine kidney mesenchymes. Taken together, this thesis demonstrates Notch and Wnt signalling pathways as novel regulators of ureteric branching morphogenesis, and that activation of the canonical Wnt pathway is sufficient for nephron induction. The studies also indicate that the Notch and Wnt pathways cross-talk with GDNF/Ret signalling in the patterning of ureteric epithelium.