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.

p53 and DNA damage checkpoint responses in the human prostate

Prostate cancer is the most common noncutaneous malignancy and the second leading cause of cancer mortality in men. In 2004, 5237 new cases were diagnosed and altogether 25 664 men suffered from prostate cancer in Finland (Suomen Syöpärekisteri). Although extensively investigated, we still have a very rudimentary understanding of the molecular mechanisms leading to the frequent transformation of the prostate epithelium. Prostate cancer is characterized by several unique features including the multifocal origin of tumors and extreme resistance to chemotherapy, and new treatment options are therefore urgently needed. The integrity of genomic DNA is constantly challenged by genotoxic insults. Cellular responses to DNA damage involve elegant checkpoint cascades enforcing cell cycle arrest, thus facilitating damage repair, apoptosis or cellular senescence. Cellular DNA damage triggers the activation of tumor suppressor protein p53 and Wee1 kinase which act as executors of the cellular checkpoint responses. These are essential for genomic integrity, and are activated in early stages of tumorigenesis in order to function as barriers against tumor formation. Our work establishes that the primary human prostatic epithelial cells and prostatic epithelium have unexpectedly indulgent checkpoint surveillance. This is evidenced by the absence of inhibitory Tyr15 phosphorylation on Cdk2, lack of p53 response, radioresistant DNA synthesis, lack of G1/S and G2/M phase arrest, and presence of persistent gammaH2AX damage foci. We ascribe the absence of inhibitory Tyr15 phosphorylation to low levels of Wee1A, a tyrosine kinase and negative regulator of cell cycle progression. Ectopic Wee1A kinase restored Cdk2-Tyr15 phosphorylation and efficiently rescued the ionizing radiation-induced checkpoints in the human prostatic epithelial cells. As variability in the DNA damage responses has been shown to underlie susceptibility to cancer, our results imply that a suboptimal checkpoint arrest may greatly increase the accumulation of genetic lesions in the prostate epithelia. We also show that small molecules can restore p53 function in prostatic epithelial cells and may serve as a paradigm for the development of future therapeutic agents for the treatment of prostate cancer We hypothesize that the prostate has evolved to activate the damage surveillance pathways and molecules involved in these pathways only to certain stresses in extreme circumstances. In doing so, this organ inadvertently made itself vulnerable to genotoxic stress, which may have implications in malignant transformation. Recognition of the limited activity of p53 and Wee1 in the prostate could drive mechanism-based discovery of preventative and therapeutic agents.

Selenium and its effects on growth, yield and tuber quality in potato

Selenium (Se) has been demonstrated to be an essential trace element for maintenance of animal and human health. Although it has not been confirmed to be an essential micronutrient in higher plants, there is increasing evidence that Se functions as an antioxidant in plants. Selenium has been shown to exert a beneficial effect on crop growth and promotes stress tolerance at low concentrations. However, the specific physiological mechanisms that underlie the positive effects of Se in plants have not been clearly elucidated. The aims of this study were to determine the Se concentration in potato (Solanum tuberosum L.) and the effects of Se on the accumulation of carbohydrates, growth and yield in potato plants. An additional aim was to study the impact of Se on the total glycoalkaloid concentration in immature potato tubers. The distribution of Se in different biochemical Se fractions and the effect of storage on the Se concentration were studied in Se-enriched tubers. Furthermore, the effect of Se on raw darkening and translocation of Se from seed tubers to the next tuber generation was investigated. Due to the established anti-ageing properties of Se, it was of interest to study if Se affects physiological age and growth vigour of seed tubers. The Se concentrations in the upper leaves, roots, stolons and tubers of potato increased with increasing Se supplementation. The highest Se concentration was reached in young upper leaves, roots and stolons, indicating that added selenate was efficiently utilized and taken up at an early stage. During the growing period the Se concentration declined in the aerial parts, roots and stolons of potato plants whereas an intensive accumulation took place in immature and mature tubers. Selenium increased carbohydrate accumulation in the young upper leaves and in stolons, roots and tubers at maturity. This could not be explained by increased production of photoassimilates as net photosynthesis did not differ among Se treatments. The Se treated plants produced higher tuber yields than control plants, and at the highest Se concentration (0.3 mg kg-1) lower numbers of larger tubers were harvested. Increased yield of Se treated plants suggested that Se may enhance the allocation of photoassimilates for tuber growth, acting as a strong sink for both Se and for carbohydrates. Similarly as for other plant species, the positive impact of Se on the yield of potato plants could be related to its antioxidative effect in delaying senescence. The highest Se supplementation (0.9 mg kg-1) slightly decreased the glycoalkaloid concentration of immature tubers. However, at this level the Se concentration in tubers was about 20 µg g-1 DW. A 100 g consumption of potato would provide about 500 mg of Se, which exceeds the upper safe intake level of 400 µg per day for human dietary. The low Se applications (0.0035 and 0.1 mg kg-1) diminished and retarded the degree of raw darkening in tubers stored for one and eight months, which can be attributed to the antioxidative properties of Se. The storage for 1 to 12 months did not affect the Se concentrations of tubers. In the Se enriched tubers Se was allocated to the organic Se fraction, indicating that it was incorporated into organic compounds in tubers. Elevated Se concentration in the next-generation tubers produced by the Se enriched seed tubers indicated that Se could be translocated from the seed tubers to the progeny. In the seed tubers stored for 8 months, at high levels, Se had some positive effects on the growth vigour of sprouts, but Se had no consistent effect on the growth vigour of seed tubers of optimal physiological age. These results indicate that Se is a beneficial trace element in potato plants that exerts a positive effect on yield formation and improves the processing and storage quality of table potato tubers. These positive effects of Se are, however, dependent on the Se concentration and the age of the potato plant and tuber.

Stress and developmental responses in Arabidopsis thaliana: regulation through the transcription factor-interacting protein RCD1

Plants constantly face adverse environmental conditions, such as drought or extreme temperatures that threaten their survival. They demonstrate astonishing metabolic flexibility in overcoming these challenges and one of the key responses to stresses is changes in gene expression leading to alterations in cellular functions. This is brought about by an intricate network of transcription factors and associated regulatory proteins. Protein-protein interactions and post-translational modifications are important steps in this control system along with carefully regulated degradation of signaling proteins. This work concentrates on the RADICAL-INDUCED CELL DEATH1 (RCD1) protein which is an important regulator of abiotic stress-related and developmental responses in Arabidopsis thaliana. Plants lacking this protein function display pleiotropic phenotypes including sensitivity to apoplastic reactive oxygen species (ROS) and salt, ultraviolet B (UV-B) and paraquat tolerance, early flowering and senescence. Additionally, the mutant plants overproduce nitric oxide, have alterations in their responses to several plant hormones and perturbations in gene expression profiles. The RCD1 gene is transcriptionally unresponsive to environmental signals and the regulation of the protein function is likely to happen post-translationally. RCD1 belongs to a small protein family and, together with its closest homolog SRO1, contains three distinguishable domains: In the N-terminus, there is a WWE domain followed by a poly(ADP-ribose) polymerase-like domain which, despite sequence conservation, does not seem to be functional. The C-terminus of RCD1 contains a novel domain called RST. It is present in RCD1-like proteins throughout the plant kingdom and is able to mediate physical interactions with multiple transcription factors. In conclusion, RCD1 is a key point of signal integration that links ROS-mediated cues to transcriptional regulation by yet unidentified means, which are likely to include post-translational mechanisms. The identification of RCD1-interacting transcription factors, most of whose functions are still unknown, opens new avenues for studies on plant stress as well as developmental responses.

European aspen and hybrid aspen under changing environment : Leaf traits, growth and litter decomposition

Understanding the responses of species and ecosystems to human-induced global environmental change has become a high research priority. The main aim of this thesis was to investigate how certain environmental factors that relate to global change affect European aspen (Populus tremula), a keystone species in boreal forests, and hybrid aspen (P. tremula × P. tremuloides), cultivated in commercial plantations. The main points under consideration were the acclimatization potential of aspen through changes in leaf morphology, as well as effects on growth, leaf litter chemistry and decomposition. The thesis is based on two experiments, in which young aspen (< 1 year) were exposed either to an atmospheric pollutant [elevated ozone (O3)] or variable resource availability [water, nitrogen (N)]; and two field studies, in which mature trees (> 8 years) were growing in environments exposed to multiple environmental stress factors (roadside and urban environments). The field studies included litter decomposition experiments. The results show that young aspen, especially the native European aspen, was sensitive to O3 in terms of visible leaf injuries. Elevated O3 resulted in reduced biomass allocation to roots and accelerated leaf senescence, suggesting negative effects on growth in the long term. Water and N availability modified the frost hardening of young aspen: High N supply, especially when combined with drought, postponed the development of frost hardiness, which in turn may predispose trees to early autumn frosts. This effect was more pronounced in European aspen. The field studies showed that mature aspen acclimatized to roadside and urban environments by producing more xeromorphic leaves. Leaf morphology was also observed to vary in response to interannual climatic variation, which further indicates the ability of aspen for phenotypic plasticity. Intraspecific variation was found in several of the traits measured, although intraspecific differences in response to the abiotic factors examined were generally small throughout the studies. However, some differences between clones were found in sensitivity to O3 and the roadside environment. Aspen leaf litter decomposition was retarded in the roadside environment, but only initially. By contrast, decomposition was found to be faster in the urban than the rural environment throughout the study. The higher quality of urban litter (higher in N, lower in lignin and phenolics), as well as higher temperature, N deposition and humus pH at the urban site were factors likely to promote decay. The phenotypic plasticity combined with intraspecific variation found in the studies imply that aspen has potential for withstanding environmental changes, although some global change factors, such as rising O3 levels, may adversely affect its performance. The results also suggest that the multiple environmental changes taking place in urban areas which correspond closely with the main drivers of global change can modify ecosystem functioning by promoting litter decomposition, mediated partly by alterations in leaf litter quality.

Diabetic cardiomyopathy and post-infarct ventricular remodelling : Effects of levosimendan in a rodent model of type II diabetes

Type 2 diabetes is a risk factor for the development of cardiovascular disease. Recently, the term diabetic cardiomyopathy has been proposed to describe the changes in the heart that occur in response to chronic hyperglycemia and insulin resistance. Ventricular remodelling in diabetic cardiomyopathy includes left ventricular hypertrophy, increased interstitial fibrosis, apoptosis and diastolic dysfunction. Mechanisms behind these changes are increased oxidative stress and renin-angiotensin system activation. The diabetic Goto-Kakizaki rat is a non-obese model of type 2 diabetes that exhibits defective insulin signalling. Recently two interconnected stress response pathways have been discovered that link insulin signalling, longevity, apoptosis and cardiomyocyte hypertrophy. The insulin-receptor PI3K/Ak pathway inhibits proapoptotic FOXO3a in response to insulin signalling and the nuclear Sirt1 deacetylase inhibits proapoptotic p53 and modulates FOXO3a in favour of survival and growth. --- Levosimendan is a calcium sensitizing agent used for the management of acute decompensated heart failure. Levosimendan acts as a positive inotrope by sensitizing cardiac troponin C to calcium and exerts vasodilation by opening mitochondrial and sarcolemmal ATP-sensitive potassium channels. Levosimendan has been described to have beneficial effects in ventricular remodelling after myocardial infarction. The aims of the study were to characterize whether diabetic cardiomyopathy associates with cardiac dysfunction, cardiomyocyte apoptosis, hypertrophy and fibrosis in spontaneously diabetic Goto-Kakizaki (GK) rats, which were used to model type 2 diabetes. Protein expression and activation of the Akt FOXO3a and Sirt1 p53 pathways were examined in the development of ventricular remodelling in GK rats with and without myocardial infarction (MI). The third and fourth studies examined the effects of levosimendan on ventricular remodelling and gene expression in post-MI GK rats. The results demonstrated that diabetic GK rats develop both modest hypertension and features similar to diabetic cardiomyopathy including cardiac dysfunction, LV hypertrophy and fibrosis and increased apoptotic signalling. MI induced a sustained increase in cardiomyocyte apoptosis in GK rats together with aggravated LV hypertrophy and fibrosis. The GK rat myocardium exhibited decreased Akt- FOXO3a phosphorylation and increased nuclear translocation of FOXO3a and overproduction of the Sirt1 protein. Treatment with levosimendan decreased cardiomyocyte apoptosis, senescence and LV hypertrophy and altered the gene expression profile in GK rat myocardium. The findings indicate that impaired cardioprotection via Akt FOXO3a and p38 MAPK is associated with increased apoptosis, whereas Sirt1 functions in counteracting apoptosis and the development of LV hypertrophy in the GK rat myocardium. Overall, levosimendan treatment protects against post-MI ventricular remodelling and alters the gene expression profile in the GK rat myocardium.

Merkel cell carcinoma : Epidemiological study with special reference to polyomavirus and vascular factors in pathogenesis

Merkel cell carcinoma (MCC) is a rare cutaneous malignancy that occurs predominantly on sun exposed skin areas. A new polyomavirus (MCPyV) was identified in MCC tumor tissues in 2008 suggesting that a viral infection might be an etiological factor. A typical MCC is a rapidly growing painless purple nodule. In its early stage it can be misjudged by its appearance as a cyst or abscess. Recurrences are common and approximately half of the patients will develop lymph node metastases and third of the patents will have distant metastases. It affects mostly elderly persons at an average age of 70 at the time of diagnosis. MCC was first described in 1972 and the first MCC patient in Finland was identified in 1983. MCC has been poorly recognized, but increased awareness and better diagnostic accuracy has increased the incidence since the early years. In this study, all cases with a notation of MCC during 1979 2008 were obtained from the Finnish Cancer Registry. Based on this data, the incidence is 0.11 for men and 0.12 for women. It is similar than that of other Nordic countries, but lower than in the USA. For clinical series, the files of patients diagnosed with MCC during 1983 2004 were reviewed, and the tissue samples were re-evaluated, if available (n=181). Third of the patients were men, and the most common site of the primary tumor was the head and neck (53%). The majority of the patients (86%) presented with a clinically node-negative (Stage I or II) disease, but the disease recurred in 38% of them. The treatment schemes were heterogeneous. No additional benefit from a wide margin (≥2 cm) was found compared to a margin of 0.1-1.9 cm, but intralesional excision was more often associated with local recurrence. None of the patients with Stage I-II disease who had received postoperative radiotherapy had local recurrence during the follow-up period. The 5-year relative survival ratio for Stage I disease was 68%, for Stage II 67%, for Stage III 16%, and for Stage IV 0%. The relative excess risk of death was significantly lower among women than among men. Some of these tissue samples were further analyzed for vascular invasion (n=126) by immunohistochemistry using vascular endothelial markers CD-31 and D2-40. Vascular invasion was seen in 93% of the samples and it was observed already in very small, <5mm tumors. The tissue samples were also analyzed for the presence of MCPyV by using a polymerase chain reaction (PCR) and quantitative PCR. MCPyV DNA was present in 80% of 114 samples studied. The patients with virus-positive tumors had better overall survival than patients with virus-negative tumors. Immunohistochemical analyses were performed for the expression of VEGFR-2 (n=21) and endostatin (n=19), but they had no prognostic value. Our results support the concept of treating MCC with margin-negative excision and radiotherapy to the tumor bed to reduce local recurrence. The finding of a high frequency of lymphovascular invasion reduces its value as a prognostic factor, but emphasizes the role of sentinel node biopsy even in very small primary MCC.

Diversitet och tillväxtfrämjande egenskaper hos Stenotrophomonas-endofyter isolerade från Calliandra calothyrsus Meisn. rotknölar

The bacterial genus Stenotrophomonas comprises 12 species. They are widely found throughout the environment and particularly S. maltophilia, S. rhizophila and S. pavanii are closely associated with plants. Strains of the most common Stenotrophomonas species, S. maltophilia, promote plant growth and health, degrade natural and man-made pollutants and produce biomolecules of biotechnological and economical value. Many S. maltophilia –strains are also multidrug resistant and can act as opportunistic human pathogens. During an INCO-project (1998-2002) rhizobia were collected from root nodules of the tropical leguminous tree Calliandra calothyrsus Meisn. from several countries in Central America, Africa and New Caledonia. The strains were identified by the N2-group (Helsinki university) and some strains turned out to be members of the genus Stenotrophomonas. Several Stenotrophomonas strains induced white tumor- or nodule-like structures on Calliandra?s roots in plant experiments. The strains could, besides from root nodules, also be isolated from surface sterilized roots and stems. The purpose of my work was to investigate if the Stenotrophomonas strains i) belong to a new Stenotrophomonas species, ii) have the same origin, iii) if there are other differences than colony morphology between phase variations of the same strain, iv) have plant growth-promoting (PGP) activity or other advantageous effects on plants, and v) like rhizobia have ability to induce root nodule formation. The genetic diversity and clustering of the Stenotrophomonas strains were analyzed with AFLP fingerprinting to get indications about their geographical origin. Differences in enzymatic properties and ability to use different carbon and energy sources were tested between the two phases of each strain with commercial API tests for bacterial identification. The ability to infect root hairs and induce root nodule formation was investigated both using plant tests with the host plant Calliandra and PCR amplification of nodA and nodC genes for nodulation. The PGP activity of the strains was tested in vitro mainly with plate methods. The impact on growth, nitrogen content and nodulation in vivo was investigated through greenhouse experiments with the legumes Phaseolus vulgaris and Galega orientalis. Both the genetic and phenotypic diversity among the Stenotrophomonas strains was small, which proposes that they have the same origin. The strains brought about changes on the root hairs of Calliandra and they also increased the amount of root hairs. However, no root nodules were detected. The strains produced IAA, protease and lipase in vitro. They also showed plant a growth-promoting effect on G. orientalis, both alone and together with R. galegae HAMBI 540, and also activated nodulation among efficient rhizobia on P. vulgaris in greenhouse. It requires further research to get a better picture about the mechanisms behind the positive effects. The results in this thesis, however, confirm earlier studies concerning Stenotrophomonas positive impact on plants.