Cyclin dependent protein kinases as drug targets – potential in cardiovascular diseases

Complications of atherosclerosis such as myocardial infarction and stroke are the primary cause of death in Western societies. The development of atherosclerotic lesions is a complex process, including endothelial cell dysfunction, inflammation, extracellular matrix alteration and vascular smooth muscle cell (VSMC) proliferation and migration. Various cell cycle regulatory proteins control VSMC proliferation. Protein kinases called cyclin dependent kinases (CDKs) play a major role in regulation of cell cycle progression. At specific phases of the cell cycle, CDKs pair with cyclins to become catalytically active and phosphorylate numerous substrates contributing to cell cycle progression. CDKs are also regulated by cyclin dependent kinase inhibitors, activating and inhibitory phosphorylation, proteolysis and transcription factors. This tight regulation of cell cycle is essential; thus its deregulation is connected to the development of cancer and other proliferative disorders such as atherosclerosis and restenosis as well as neurodegenerative diseases. Proteins of the cell cycle provide potential and attractive targets for drug development. Consequently, various low molecular weight CDK inhibitors have been identified and are in clinical development. Tylophorine is a phenanthroindolizidine alkaloid, which has been shown to inhibit the growth of several human cancer cell lines. It was used in Ayurvedic medicine to treat inflammatory disorders. The aim of this study was to investigate the effect of tylophorine on human umbilical vein smooth muscle cell (HUVSMC) proliferation, cell cycle progression and the expression of various cell cycle regulatory proteins in order to confirm the findings made with tylophorine in rat cells. We used several methods to determine our hypothesis, including cell proliferation assay, western blot and flow cytometric cell cycle distribution analysis. We demonstrated by cell proliferation assay that tylophorine inhibits HUVSMC proliferation dose-dependently with an IC50 value of 164 nM ± 50. Western blot analysis was used to determine the effect of tylophorine on expression of cell cycle regulatory proteins. Tylophorine downregulates cyclin D1 and p21 expression levels. The results of tylophorine’s effect on phosphorylation sites of p53 were not consistent. More sensitive methods are required in order to completely determine this effect. We used flow cytometric cell cycle analysis to investigate whether tylophorine interferes with cell cycle progression and arrests cells in a specific cell cycle phase. Tylophorine was shown to induce the accumulation of asynchronized HUVSMCs in S phase. Tylophorine has a significant effect on cell cycle, but its role as cell cycle regulator in treatment of vascular proliferative diseases and cancer requires more experiments in vitro and in vivo.

Protein Kinase C and Anaplastic Lymphoma Kinase Targeted Compounds

The protein kinases (PKs) belong to the largest single family of enzymes, phosphotransferases, which catalyze the phosphorylation of other enzymes and proteins and function primarily in signal transduction. Consequently, PKs regulate cell mechanisms such as growth, differentiation, and proliferation. Dysfunction of these cellular mechanisms may lead to cancer, a major predicament in health care. Even though there is a range of clinically available cancer-fighting drugs, increasing number of cancer cases and setbacks such as drug resistance, constantly keep cancer research active. At the commencement of this study an isophthalic acid derivative had been suggested to bind to the regulatory domain of protein kinase C (PKC). In order to investigate the biological effects and structure-activity relationships (SARs) of this new chemical entity, a library of compounds was synthesized. The best compounds induced apoptosis in human leukemia HL-60 cells and were not cytotoxic in Swiss 3T3 fibroblasts. In addition, the best apoptosis inducers were neither cytotoxic nor mutagenic. Furthermore, results from binding affinity assays of PKC isoforms revealed the pharmacophores of these isophthalic acid derivatives. The best inhibition constants of the tested compounds were measured to 210 nM for PKCα and to 530 nM for PKCδ. Among natural compounds targeting the regulatory domain of PKC, the target of bistramide A has been a matter of debate. It was initially found to activate PKCδ; however, actin was recently reported as the main target. In order to clarify and to further study the biological effects of bistramide A, the total syntheses of the natural compound and two isomers were performed. Biological assays of the compounds revealed accumulation of 4n polyploid cells as the primary mode of action and the compounds showed similar overall antiproliferative activities. However, each compound showed a distinct distribution of antimitotic effect presumably via actin binding, proapoptotic effect presumably via PKCδ, and pro-differentiation effect as evidenced by CD11b expression. Furthermore, it was shown that the antimitotic and proapoptotic effects of bistramide A were not secondary effects of actin binding but independent effects. The third aim in this study was to synthesize a library of a new class of urea-based type II inhibitors targeted at the kinase domain of anaplastic lymphoma kinase (ALK). The best compounds in this library showed IC50 values as low as 390 nM for ALK while the initial low cellular activities were successfully increased even by more than 70 times for NPM-ALK- positive BaF3 cells. More importantly, selective antiproliferative activity on ALK-positive cell lines was achieved; while the best compound affected the BaF3 and SU-DHL-1 cells with IC50 values of 0.5 and 0.8 μM, respectively, they were less toxic to the NPM-ALK-negative human leukemic cells U937 (IC50 = 3.2 μM) and BaF3 parental cells (IC50 = 5.4 μM). Furthermore, SAR studies of the synthesized compounds revealed functional groups and positions of the scaffold, which enhanced the enzymatic and cellular activities.

Regulation and Function of GATA Transcription Factors in Adrenocortical Tumors and Granulosa Cells

Transcription factors play a key role in tumor development, in which dysfunction of genes regulating tissue growth and differentiation is a central phenomenon. The GATA family of transcription factors consists of six members that bind to a consensus DNA sequence (A/T)GATA(A/G) in gene promoters and enhancers. The two GATA factors expressed in the adrenal cortex are GATA-4 and GATA-6. In both mice and humans, GATA-4 can be detected only during the fetal period, whereas GATA-6 expression is abundant both throughout development and in the adult. It is already established that GATA factors are important in both normal development and tumorigenesis of several endocrine organs, and expression of GATA-4 and GATA-6 is detected in adrenocortical tumors. The aim of this study was to elucidate the function of these factors in adrenocortical tumor growth. In embryonal development, the adrenocortical cells arise and differentiate from a common pool with gonadal steroidogenic cells, the urogenital ridge. As the adult adrenal cortex undergoes constant renewal, it is hypothesized that undifferentiated adrenocortical progenitor cells reside adjacent to the adrenal capsule and give rise to daughter cells that differentiate and migrate centripetally. A diverse array of hormones controls the differentiation, growth and survival of steroidogenic cells in the adrenal gland and the gonads. Factors such as luteinizing hormone and inhibins, traditionally associated with gonadal steroidogenic cells, can also influence the function of adrenocortical cells in physiological and pathophysiological states. Certain inbred strains of mice develop subcapsular adrenocortical tumors in response to gonadectomy. In this study, we found that these tumors express GATA-4, normally absent from the adult adrenal cortex, while GATA-6 expression is downregulated. Gonadal markers such as luteinizing hormone receptor, anti-Müllerian hormone and P450c17 are also expressed in the neoplastic cells, and the tumors produce gonadal hormones. The tumor cells have lost the expression of melanocortin-2 receptor and the CYP enzymes necessary for the synthesis of corticosterone and aldosterone. By way of xenograft studies utilizing NU/J nude mice, we confirmed that chronic gonadotropin elevation is sufficient to induce adrenocortical tumorigenesis in susceptible inbred strains. Collectively, these studies suggest that subcapsular adrenocortical progenitor cells can, under certain conditions, adopt a gonadal fate. We studied the molecular mechanisms involved in gene regulation in endocrine cells in order to elucidate the role of GATA factors in endocrine tissues. Ovarian granulosa cells express both GATA-4 and GATA-6, and the TGF-β signaling pathway is active in these cells. Inhibin-α is both a target gene for, and an atypical or antagonistic member of the TGF-β growth factor superfamily. In this study, we show that GATA-4 is required for TGF-β-mediated inhibin-α promoter activation in granulosa cells, and that GATA-4 physically interacts with Smad3, a TGF-β downstream protein. Apart from the regulation of steroidogenesis and other events in normal tissues, TGF-β signaling is implicated in tumors of multiple organs, including the adrenal cortex. Another signaling pathway found often to be aberrantly active in adrenocortical tumors is the Wnt pathway. As both of these pathways regulate the expression of inhibin-α, a transcriptional target for GATA-4 and GATA-6, we wanted to investigate whether GATA factors are associated with the components of these signaling cascades in human adrenocortical tumors. We found that the expression of Wnt co-receptors LRP5 and LRP6, Smad3, GATA-6 and SF-1 was diminished in adrenocortical carcinomas with poor outcome. All of these factors drive inhibin-α expression, and their expression in adrenocortical tumors correlated with that of inhibin-α. The results support a tumor suppressor role previously suggested for inhibin-α in the mouse adrenal cortex, and offer putative pathways associated with adrenocortical tumor aggressiveness. Unraveling the role of GATA factors and associated molecules in human and mouse adrenocortical tumors could ultimately contribute to the development of diagnostic tools and future therapies for these diseases.

Enhanced photodynamic effect of cobalt(III) dipyridophenazine complex on thyrotropin receptor expressing HEK293 cells

Ternary cobalt(III) complexes CoL(B)] (1-3) of a trianionic tetradentate phenolate-based ligand (L) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyridoquinoxaline (dpq in 2) and dipyridophenazine (dppz in 3) are synthesized, characterized from X-ray crystallographic, analytical and spectral techniques, and their utility in photodynamic therapy (PDT) of thyroid diseases caused by TSH receptor dysfunction is probed. The complexes display a visible spectral band within the PDT spectral window at similar to 690 nm. Photodynamic potential was estimated through DNA cleavage activity of the dpq and dppz complexes in UV-A light of 365 nm and red light of 676 nm. The reactions proceed via the hydroxyl radical pathway. The complexes retain their DNA photocleavage activity in red light under anaerobic conditions, a situation normally prevails in hypoxic tumor core. Investigation into the photocytotoxic potential of these complexes showed that the dppz complex 3 is approximately 4-fold more active in the HEK293 cells expressing human thyrotropin receptor (HEK293-hTSHR) than in the parental cell line and has an insignificant effect on an unrelated human cervical carcinoma cell line (HeLa). Photoexcitation of complex 3 in HEK293-hTSHR cells leads to damage hTSHR as evidenced from the decrease in cAMP formation both in absence and presence of hTSH and decrease in the TSHR immunofluorescence with a concomitant cytoplasmic translocation of the membrane protein, cadherin. The involvement of hTSHR is evidenced from the ability of complex 3 to bind to the extracellular domain of hTSHR (hTSHR-ECD) with a K-d value of 81 nM and from the photocleavage of hTSHR-ECD.

Molecular genetics of Usher syndrome -inherited deafness and blindness

Usher syndrome (USH) is an inherited blindness and deafness disorder with variable vestibular dysfunction. The syndrome is divided into three subtypes according to the progression and severity of clinical symptoms. The gene mutated in Usher syndrome type 3 (USH3), clarin 1 (CLRN1), was identified in Finland in 2001 and two mutations were identified in Finnish patients at that time. Prior to this thesis study, the two CLRN1 gene mutations were the only USH mutations identified in Finnish USH patients. To further clarify the Finnish USH mutation spectrum, all nine USH genes were studied. Seven mutations were identified: one was a previously known mutation in CLRN1, four were novel mutations in myosin VIIa (MYO7A) and two were a novel and a previously known mutation in usherin (USH2A). Another aim of this thesis research was to further study the structure and function of the CLRN1 gene, and to clarify the effects of mutations on protein function. The search for new splice variants resulted in the identification of eight novel splice variants in addition to the three splice variants that were already known prior to this study. Studies of the possible promoter regions for these splice variants showed the most active region included the 1000 bases upstream of the translation start site in the first exon of the main three exon splice variant. The 232 aa CLRN1 protein encoded by the main (three-exon) splice variant was transported to the plasma membrane when expressed in cultured cells. Western blot studies suggested that CLRN1 forms dimers and multimers. The CLRN1 mutant proteins studied were retained in the endoplasmic reticulum (ER) and some of the USH3 mutations caused CLRN1 to be unstable. During this study, two novel CLRN1 sequence alterations were identified and their pathogenicity was studied with cell culture protein expression. Previous studies with mice had shown that Clrn1 is expressed in mouse cochlear hair cells and spiral ganglion cells, but the expression profile in mouse retina remained unknown. The Clrn1 knockout mice display cochlear cell disruption/death, but do not have a retinal phenotype. The zebrafish, Danio rerio, clrn1 was found to be expressed in hair cells associated with hearing and balance. Clrn1 expression was also found in the inner nuclear layer (INL), photoreceptor layer and retinal pigment epithelium layer (RPE) of the zebrafish retina. When Clrn1 production was knocked down with injected morpholino oligonucleotides (MO) targeting Clrn1 translation or correct splicing, the zebrafish larvae showed symptoms similar to USH3 patients. These larvae had balance/hearing problems and reduced response to visual stimuli. The knowledge this thesis research has provided about the mutations in USH genes and the Finnish USH mutation spectrum are important in USH patient diagnostics. The extended information about the structure and function of CLRN1 is a step further in exploring USH3 pathogenesis caused by mutated CLRN1 as well as a step in finding a cure for the disease.

Poststroke survival and ischemic stroke recurrence : the cerebral small-vessel disease perspective

The aim of the present study was to investigate the influence of different manifestations of cerebral SVD on poststroke survival and ischemic stroke recurrence in long-term follow-up. The core imaging features of small-vessel disease (SVD) are confluent and extensive white matter changes (WMC) and lacunar infarcts. These are associated with minor motor deficits but a major negative influence on cognition, mood, and functioning in daily life, resulting from small-vessel lesions in the fronto-subcortical brain network. These sub-studies were conducted as part of the Helsinki Stroke Aging Memory (SAM) study. The SAM cohort consisted of 486 consecutive patients aged 55 to 85 years who were admitted to Helsinki University Central Hospital with acute ischemic stroke. The study included comprehensive clinical, neuropsychological, psychiatric and radiological assessment three months poststroke. The patients were followed up up for 12 years using extensive national registers. The effect of different manifestations of cerebral SVD on poststroke survival and stroke recurrence was analyzed controlling for factors such as age, education, and cardiovascular risk factors. Poststroke dementia and cognitive impairment relate to poor long-term survival. In particular, deficits in executive functions as well as visuospatial and constructional abilities predict poor outcome. The predictive value of cognitive deficits is further underlined by the finding that depression-executive dysfunction syndrome (DES), but not depression in itself, is associated with poor poststroke survival. Delirium is not independently associated with increased risk for long-term poststroke mortality, although it is associated with poststroke dementia. Furthermore, acute index stroke attributable to SVD is associated with poorer long-term survival and a higher risk for cardiac death than other stroke subtypes. Severe WMC, a surrogate of SVD, is independently related to an increased risk of stroke recurrence at five years. In summary, cognitive poststroke outcomes reflecting changes in the executive network brain, and the presence of cerebral SVD are important determinants of poststroke mortality and ischemic stroke recurrence, regardless of whether SVD is the cause of the index stroke or a condition concurrent to some other etiology.

Pain measurement and management in elderly patients : Clinical studies in long term hospital care and after cardiac surgery

The proportion of patients over 75 years of age, receiving all different types of healthcare, is constantly increasing. The elderly undergo surgery and anaesthetic procedures more often than middle-aged patients. Poor pain management in the elderly is still an issue. Although the elderly consumes the greatest proportion of prescribed medicines in Western Europe, most clinical pharmacological studies have been performed in healthy volunteers or middle-aged patients. The aim of this study was to investigate pain measurement and management in cognitively impaired patients in long term hospital care and in cognitively normal elderly patients after cardiac surgery. This thesis incorporated 366 patients, including 86 home-dwelling or hospitalized elderly with chronic pain and 280 patients undergoing cardiac surgery with acute pain. The mean age of patients was 77 (SD ± 8) years and approximately 8400 pain measurements were performed with four pain scales: Verbal Rating Scale (VRS), the Visual Analogue Scale (VAS), the Red Wedge Scale (RWS), and the Facial Pain Scale (FPS). Cognitive function, depression, functional ability in daily life, postoperative sedation and postoperative confusion were assessed with MMSE, GDS, Barthel Index, RASS, and CAM-ICU, respectively. The effects and plasma concentrations of fentanyl and oxycodone were measured in elderly (≥ 75 years) and middle-aged patients (≤ 60 years) and the opioid-sparing effect of pregabalin was studied after cardiac surgery. The VRS pain scores after movement correlated with the Barthel Index. The VRS was most successful in the groups of demented patients (MMSE 17-23, 11-16 and ≤ 10) and in elderly patients on the first day after cardiac surgery. The elderly had a higher plasma concentration of fentanyl at the end of surgery than younger patients. The plasma concentrations of oxycodone were comparable between the groups. Pain intensity on the VRS was lower and the sedation scores were higher in the elderly. Total oxycodone consumption during five postoperative days was reduced by 48% and the CAM-ICU scores were higher on the first postoperative day in the pregabalin group. The incidence of postoperative pain during movement was lower in the pregabalin group three months after surgery. This investigation demonstrates that chronic pain did not seem to impair daily activities in home-dwelling Finnish elderly. The VRS appeared to be applicable for elderly patients with clear cognitive dysfunction (MMSE ≤17) and it was the most feasible pain scale for the early postoperative period after cardiac surgery. After cardiac surgery, plasma concentrations of fentanyl in elderly were elevated, although oxycodone concentrations were at similar level compared to middle-aged patients. The elderly had less pain and were more sedated after doses of oxycodone. Therefore, particular attention must be given to individual dosing of the opioids in elderly surgical patients, who often need a smaller amount for adequate analgesia than middle-aged patients. The administration of pregabalin reduced postoperative oxycodone consumption after cardiac surgery. Pregabalin-treated patients had less confusion, and additionally to less postoperative pain on the first postoperative day and during movement at three months post-surgery. Pregabalin might be a new alternative as analgesic for acute postoperative and chronic pain management in the elderly. Its clinical role and safety remains to be verified in large-scale randomized and controlled studies. In the future, many clinical trials in the older category of patients will be needed to facilitate improvements in health care methods.

Myoblast Sheet Transplantation for Treatment of Heart Failure after Myocardial Infarction

Heart failure is a common, severe, and progressive condition associated with high mortality and morbidity. Because of population-aging in the coming decades, heart failure is estimated to reach epidemic proportions. Current medical and surgical treatments have reduced mortality, but the prognosis for patients has remained poor. Transplantation of skeletal myoblasts has raised hope of regenerating the failing heart and compensating for lost cardiac contractile tissue. In the present work, we studied epicardial transplantation of tissue-engineered myoblast sheets for treatment of heart failure. We employed a rat model of myocardial infarction-induced acute and chronic heart failure by left anterior descending coronary artery ligation. We then transplanted myoblast sheets genetically modified to resist cell death after transplantation by expressing antiapoptotic gene bcl2. In addition, we evaluated the regenerative capacity of myoblast sheets expressing the cardioprotective cytokine hepatocyte growth factor in a rat chronic heart failure model. Furthermore, we utilized in vitro cardiomyocyte and endothelial cell culture models as well as microarray gene expression analysis to elucidate molecular mechanisms mediating the therapeutic effects of myoblast sheet transplantation. Our results demonstrate that Bcl2-expression prolonged myoblast sheet survival in rat hearts after transplantation and induced secretion of cardioprotective, proangiogenic cytokines. After acute myocardial infarction, these sheets attenuated left ventricular dysfunction and myocardial damage, and they induced therapeutic angiogenesis. In the chronic heart failure model, inhibition of graft apoptosis by Bcl-2 improved cardiac function, supported survival of cardiomyocytes in the infarcted area, and induced angiogenesis in a vascular endothelial growth factor receptor 1- and 2-dependent mechanism. Hepatocyte growth factor-secreting myoblast sheets further enhanced the angiogenic efficacy of myoblast sheet therapy. Moreover, myoblast-secreted paracrine factors protected cardiomyocytes against oxidative stress in an epidermal growth factor receptor- and c-Met dependent manner. This protection was associated with induction of antioxidative genes and activation of the unfolded protein response. Our results provide evidence that inhibiting myoblast sheet apoptosis can enhance the sheets efficacy for treating heart failure after acute and chronic myocardial infarction. Furthermore, we show that myoblast sheets can serve as vehicles for delivery of growth factors, and induce therapeutic angiogenesis in the chronically ischemic heart. Finally, myoblasts induce, in a paracine manner, a cardiomyocyte-protective response against oxidative stress. Our study elucidates novel mechanisms of myoblast transplantation therapy, and suggests effective means to improve this therapy for the benefit of the heart failure patient.

Suolistopatogeenisten Escherichia coli -bakteerien esiintyvyys ripulipotilailla ja raa’oissa lihatuotteissa Burkina Fasossa sekä enterohemorraagisten E. coli -kantojen karakterisointi

Suolistopatogeeniset Escherichia coli -bakteerit eli ripulikolit aiheuttavat ihmisellä suolistoinfektioita. Kuten normaalimikrobiston E. coli -bakteerit, ne esiintyvät ihmisen lisäksi muiden nisäkkäiden, etenkin märehtijöiden, ja lintujen suolistossa. Lisäksi ne voivat esiintyä maaperässä ja vesistöissä. Ihminen voi saada tartunnan eläinperäisten elintarvikkeiden välityksellä tai juomalla eläinten tai ihmisen ulosteilla saastunutta vettä. Ripulikolit voidaan jakaa ainakin viiteen ryhmään perustuen niiden erilaisiin virulenssiominaisuuksiin: enteropatogeeninen E. coli (EPEC), enterotoksigeeninen E. coli (ETEC), enterohemorraaginen E. coli (EHEC), enteroinvasiivinen E. coli (EIEC) ja enteroaggregatiivinen E. coli (EAEC). EPEC aiheuttaa etenkin kehitysmaissa pikkulapsille ripulia. ETEC aiheuttaa turistiripulia ja vastasyntyneiden ripulia kehitysmaissa. EHEC aiheuttaa ripulia tai veriripulia, joka voi varsinkin pienillä lapsilla johtaa hemolyyttis-ureemiseen oireyhtymään (HUS) ja munuaisten vaurioitumiseen. EIEC aiheuttaa Shigellan kaltaista ripulia, joka voi olla veristä. EAEC on yhdistetty lähinnä pitkittyneisiin ripuleihin. Tutkimuksessa selvitettiin suolistopatogeenisten E. coli -bakteerien esiintyvyyttä Burkina Fasossa, josta ei ole saatavilla aikaisempaa tietoa ripulikolien esiintymisestä ihmisissä ja elintarvikkeissa. Ulostenäytteitä otettiin ripulia sairastavilta alle viisivuotiailta lapsilta maaseudulta kahdesta kylästä, Boromosta ja Gourcysta, ja maan pääkaupungista Ouagadougousta (110 näytettä). Lihanäytteitä (kanaa, nautaa, lammasta ja naudan suolta, jota käytetään ihmisravinnoksi) otettiin Ouagadougoun toreilla myytävistä kypsentämättömistä lihoista (120 näytettä). Näytteistä saadut bakteerisekaviljelmät tutkittiin monialukkeisella PCR-menetelmällä, joka tunnistaa viiden ripulikoliryhmän virulenssigeenejä. Lisäksi lihanäytteistä eristettiin 20 EHEC-kantaa shigatoksiinin stx-geenin havaitsemiseen perustuvalla pesäkehybridisaatiolla ja PCR-seulonnalla, ja karakterisoitiin mahdollisten virulenssiominaisuuksien selvittämiseksi. Tutkimus osoitti, että ripulikolien aiheuttamat suolistoinfektiot ovat yleisiä ripulia sairastavilla pikkulapsilla Burkina Fasossa. Ulostenäytteistä 59 % oli positiivisia. Useimmiten lapsilla esiintyi EAEC- (32 %) ETEC- (31 %) ja EPEC-patoryhmiä (20 %). EIEC- (2 %) ja EHEC-patoryhmiä (1 %) esiintyi vähän. Myös useamman patoryhmän sekainfektiot olivat yleisiä (24 %). Eri paikkakuntien välillä oli tilastollisesti merkitseviä eroja ripulikolien esiintymisessä. Gourcyssa ripulikoleja esiintyi useammin kuin Ouagadougoussa ja Boromossa. Tutkimuksessa kävi ilmi, että Ouagadougoun toreilla myytävissä lihoissa on paljon ripulikoleja. Lihanäytteistä 43 % oli positiivisia. Yleisimmin lihoissa esiintyi EHEC (28 %), EPEC (20 %), ETEC (8 %) ja EAEC (5 %). EIEC-ryhmää ei havaittu lihoissa. Myös useamman patoryhmän sekakontaminaatioita löytyi (17 %) lihoista. Ripulikolien esiintyvyydessä eri lihojen välillä ei ollut tilastollisesti merkitseviä eroja, kun tarkasteltiin kaikkia patoryhmiä yhdessä. Eri patoryhmien esiintyvyyttä tarkasteltaessa EHEC-patoryhmää ei esiintynyt ollenkaan kanassa ja ero oli tilastollisesti merkitsevä muihin lihoihin verrattuna. Lihoista eristetyt 20 EHEC-kantaa kuuluivat 14 eri serotyyppiin, joista osa on aikaisemmin eristetty suolistoinfektioihin ja HUSoireyhtymään sairastuneilta ihmisiltä. Kaikki kannat olivat stx1-positiivisia ja puolella oli lisäksi stx2-geeni, jota pidetään shigatoksiinin virulentimpana muotona. Kahdelta EHEC-kannalta löytyi myös ETECpatoryhmän lämpöstabiilin enterotoksiini Ia:n geeni eli kannat olivat kahden patoryhmän välimuotoa ja osoitus geenien siirtymisestä eri patoryhmien välillä. Vaikka nuorimmat näytteen antaneet lapsipotilaat tuskin söivät lihaa, sen voidaan ajatella silti olevan edustava näyte lasten elinympäristöstä, sillä lasten ruoka valmistetaan usein samoissa oloissa, joissa raakaa lihaa käsitellään. Saastunut liha voi siten olla pikkulasten ripulikoli-infektioiden aiheuttaja.

Biomarkers in acute respiratory failure

Acute respiratory failure (ARF) is the most common type of organ failure leading to the need for intensive care. It is often secondary to acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS). ARF, and especially ALI and ARDS, cause increased morbidity, and mortality rates remain high (up to 40%). These disorders are characterised by inflammatory reaction and tissue damage. In some cases, inflammation continues and leads to an overwhelming repair process with ongoing fibrosis, accompanied by organ dysfunction and eventually a loss of function. Measuring the magnitude of the inflammation, and the repair process, would theoretically offer information concerning outcome. Early identification of patients whose disease process is likely to proceed unfavourably, would help clinicians to optimise their treatment. The aim of this study was to evaluate the epidemiology of ARF, its treatment, and outcome in Finland, with special interest in biomarkers, and their value in the prediction of mortality. Altogether, 958 adult patients treated with ventilatory support were prospectively included in this study during an eight week period in 2007 in 25 intensive care units. Plasma aminoterminal pro-brain natriuretic peptide (NT-pro-BNP) was assessed in 602 patients, and plasma cell-free DNA in 580 patients, to evaluate their prognostic value in ARF. Markers of collagen metabolism were studied in longitudinal serum samples in 68 patients in order to evaluate their evolution in ARF and the association to multiple organ dysfunction (MOD). Ventilatory support was used in 39% of all ICU patients. The estimated incidence of ARF was 149.5/100 000 per year. Median tidal volumes used were higher than recommended. Overall mortality at 90 days was 31%. Plasma NT-pro-BNP and cell-free DNA were highly increased in the majority of patients. Both markers were independent predictors of 90-day mortality, but their discriminative power was at most moderate when used separately. The mortality was highest in those patients, in whom both biomarkers were over their separate cut-off values. Thus, combined use of these biomarkers may increase their clinical value in the mortality prediction. The markers of collagen metabolism changed significantly over time in surviving patients. None of these markers did associate with MOD in these patients.

Computation of restoration of ligand response in the random kinetics of a prostate cancer cell signaling pathway

Mutation and/or dysfunction of signaling proteins in the mitogen activated protein kinase (MAPK) signal transduction pathway are frequently observed in various kinds of human cancer. Consistent with this fact, in the present study, we experimentally observe that the epidermal growth factor (EGF) induced activation profile of MAP kinase signaling is not straightforward dose-dependent in the PC3 prostate cancer cells. To find out what parameters and reactions in the pathway are involved in this departure from the normal dose-dependency, a model-based pathway analysis is performed. The pathway is mathematically modeled with 28 rate equations yielding those many ordinary differential equations (ODE) with kinetic rate constants that have been reported to take random values in the existing literature. This has led to us treating the ODE model of the pathways kinetics as a random differential equations (RDE) system in which the parameters are random variables. We show that our RDE model captures the uncertainty in the kinetic rate constants as seen in the behavior of the experimental data and more importantly, upon simulation, exhibits the abnormal EGF dose-dependency of the activation profile of MAP kinase signaling in PC3 prostate cancer cells. The most likely set of values of the kinetic rate constants obtained from fitting the RDE model into the experimental data is then used in a direct transcription based dynamic optimization method for computing the changes needed in these kinetic rate constant values for the restoration of the normal EGF dose response. The last computation identifies the parameters, i.e., the kinetic rate constants in the RDE model, that are the most sensitive to the change in the EGF dose response behavior in the PC3 prostate cancer cells. The reactions in which these most sensitive parameters participate emerge as candidate drug targets on the signaling pathway. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

A Study of Long-range Correlations in Schizophrenia EEG using Detrended Fluctuation Analysis

In this paper, we have studied electroencephalogram (EEG) activity of schizophrenia patients, in resting eyes closed condition, with detrended fluctuation analysis (DFA). The DFA gives information about scaling and long-range correlations in time series. We computed DFA exponents from 30 scalp locations of 18 male neuroleptic-naIve, recent-onset schizophrenia (NRS) subjects and 15 healthy male control subjects. Our results have shown two scaling regions in all the scalp locations in all the subjects, with different slopes, corresponding to two scaling exponents. No significant differences between the groups were found with first scaling exponent (short-range). However, the second scaling exponent (long-range) were significantly lower in control subjects at all scalp locations (p<0.05, Kruskal-Wallis test). These findings suggest that the long-range scaling behavior of EEG is sensitive to schizophrenia, and this may provide an additional insight into the brain dysfunction in schizophrenia.

Subclinical autonomic neuropathy in alcoholics: Decreased heart rate variability

Cardiac autonomic neuropathy is known to occur in alcoholics but the extent of its subclinical form is not usually recognized, Heart Rate Variability (HRV) analysis can detect subclinical autonomic neuropathy. In this study the HRV parameters were compared in 20 neurologically asymptomatic alcoholics, 20 age-matched normals and 16 depressives. All were males, ECG was recorded in a quiet room for four minutes in supine position. Time and Frequency domain parameters of HRV were computed by a researcher blind to clinical details. Alcoholics had significantly smaller Coefficient of Variation of R-R intervals (CVR-R) on time domain analysis and smaller HF band (0.15-0.5 Hz) power on spectral analysis. The decreased Heart Rate Variability indicates cardiac autonomic dysfunction.

Reach task-associated excitatory overdrive of motor cortical neurons following infusion with ALS-CSF

Converging evidence from transgenic animal models of amyotrophic lateral sclerosis (ALS) and human studies suggest alterations in excitability of the motor neurons in ALS. Specifically, in studies on human subjects with ALS the motor cortex was reported to be hyperexcitable. The present study was designed to test the hypothesis that infusion of cerebrospinal fluid from patients with sporadic ALS (ALS-CSF) into the rat brain ventricle can induce hyperexcitability and structural changes in the motor cortex leading to motor dysfunction. A robust model of sporadic ALS was developed experimentally by infusing ALS-CSF into the rat ventricle. The effects of ALS-CSF at the single neuron level were examined by recording extracellular single unit activity from the motor cortex while rats were performing a reach to grasp task. We observed an increase in the firing rate of the neurons of the motor cortex in rats infused with ALS-CSF compared to control groups. This was associated with impairment in a specific component of reach with alterations in the morphological characteristics of the motor cortex. It is likely that the increased cortical excitability observed in the present study could be the result of changes in the intrinsic properties of motor cortical neurons, a dysfunctional inhibitory mechanism and/or an underlying structural change culminating in a behavioral deficit.

The Presence of Multiple Cellular Defects Associated with a Novel G50E Iron-Sulfur Cluster Scaffold Protein ( ISCU) Mutation Leads to Development of Mitochondrial Myopathy

Background: Muscle-specific deficiency of iron-sulfur (Fe-S) cluster scaffold protein (ISCU) leads to myopathy. Results: Cells carrying the myopathy-associated G50E ISCU mutation demonstrate impaired Fe-S cluster biogenesis and mitochondrial dysfunction. Conclusion: Reduced mitochondrial respiration as a result of diminished Fe-S cluster synthesis results in muscle weakness in myopathy patients. Significance: The molecular mechanism behind disease progression should provide invaluable information to combat ISCU myopathy. Iron-sulfur (Fe-S) clusters are versatile cofactors involved in regulating multiple physiological activities, including energy generation through cellular respiration. Initially, the Fe-S clusters are assembled on a conserved scaffold protein, iron-sulfur cluster scaffold protein (ISCU), in coordination with iron and sulfur donor proteins in human mitochondria. Loss of ISCU function leads to myopathy, characterized by muscle wasting and cardiac hypertrophy. In addition to the homozygous ISCU mutation (g.7044GC), compound heterozygous patients with severe myopathy have been identified to carry the c.149GA missense mutation converting the glycine 50 residue to glutamate. However, the physiological defects and molecular mechanism associated with G50E mutation have not been elucidated. In this report, we uncover mechanistic insights concerning how the G50E ISCU mutation in humans leads to the development of severe ISCU myopathy, using a human cell line and yeast as the model systems. The biochemical results highlight that the G50E mutation results in compromised interaction with the sulfur donor NFS1 and the J-protein HSCB, thus impairing the rate of Fe-S cluster synthesis. As a result, electron transport chain complexes show significant reduction in their redox properties, leading to loss of cellular respiration. Furthermore, the G50E mutant mitochondria display enhancement in iron level and reactive oxygen species, thereby causing oxidative stress leading to impairment in the mitochondrial functions. Thus, our findings provide compelling evidence that the respiration defect due to impaired biogenesis of Fe-S clusters in myopathy patients leads to manifestation of complex clinical symptoms.