Arvostelut

H. K. Riikonen Virsiä ja niiden uudistushistoriaa Liisa Enwald ja Tuula Hökkä (toim.): Virren virtaa. Veisattu runo ennen ja nyt Merja Leppälahti Yksitoista tarinaa häpeästä Siru Kainulainen ja Viola Parente-Čapková (toim.): Häpeä vähän. Kriittisiä tutkimuksia häpeästä Eva Maria Korsisaari ”Lukijoiden yhteisö! Ei ole lukijoiden yhteisöjä.” Kuisma Korhonen: Lukijoiden yhteisö. Ystävyydestä, kansanmurhista, itkevistä kivist

E. coli Histidine Triad Nucleotide Binding Protein 1 (ecHinT) Is a Catalytic Regulator of D-Alanine Dehydrogenase (DadA) Activity In Vivo

Histidine triad nucleotide binding proteins (Hints) are highly conserved members of the histidine triad (HIT) protein superfamily. Hints comprise the most ancient branch of this superfamily and can be found in Archaea, Bacteria, and Eukaryota. Prokaryotic genomes, including a wide diversity of both Gram-negative and Gram-positive bacteria, typically have one Hint gene encoded by hinT (ycfF in E. coli). Despite their ubiquity, the foundational reason for the wide-spread conservation of Hints across all kingdoms of life remains a mystery. In this study, we used a combination of phenotypic screening and complementation analyses with wild-type and hinT knock-out Escherichia coli strains to show that catalytically active ecHinT is required in E. coli for growth on D-alanine as a sole carbon source. We demonstrate that the expression of catalytically active ecHinT is essential for the activity of the enzyme D-alanine dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a necessary component of the D-alanine catabolic pathway. Site-directed mutagenesis studies revealed that catalytically active C-terminal mutants of ecHinT are unable to activate DadA activity. In addition, we have designed and synthesized the first cell-permeable inhibitor of ecHinT and demonstrated that the wild-type E. coli treated with the inhibitor exhibited the same phenotype observed for the hinT knock-out strain. These results reveal that the catalytic activity and structure of ecHinT is essential for DadA function and therefore alanine metabolism in E. coli. Moreover, they provide the first biochemical evidence linking the catalytic activity of this ubiquitous protein to the biological function of Hints in Escherichia coli

Complementary actions of dopamine D2 receptor 1 agonist and anti-Vegf therapy on tumoral vessel normalization in a transgenic mouse model

International audienceAngiogenesis contributes in multiple ways to disease progression in tumors and reduces treatment efficiency. Molecular therapies targeting Vegf signaling combined with chemotherapy or other drugs exhibit promising results to improve efficacy of treatment. Dopamine has been recently proposed to be a novel safe anti-angiogenic drug that stabilizes abnormal blood vessels and increases therapeutic efficacy. Here, we aimed to identify a treatment to normalize tumoral vessels and restore normal blood perfusion in tumor tissue with a Vegf receptor inhibitor and/or a ligand of dopamine G protein-coupled receptor D2 (D2R). Dopamine, via its action on D2R, is an endogenous effector of the pituitary gland, and we took advantage of this system to address this question. We have used a previously described Hmga2/T mouse model developing haemorrhagic prolactin-secreting adenomas. In mutant mice, blood vessels are profoundly altered in tumors, and an aberrant arterial vascularization develops leading to the loss of dopamine supply. D2R agonist treatment blocks tumor growth, induces regression of the aberrant blood supply and normalizes blood vessels. A chronic treatment is able to restore the altered balance between pro- and anti-angiogenic factors. Remarkably, an acute treatment induces an upregulation of the stabilizing factor Angiopoietin 1. An anti-Vegf therapy is also effective to restrain tumor growth and improves vascular remodeling. Importantly, only the combination treatment suppresses intratumoral hemorrhage and restores blood vessel perfusion, suggesting that it might represent an attractive therapy targeting tumor vasculature. Similar strategies targeting other ligands of GPCRs involved in angiogenesis may identify novel therapeutic opportunities for cancer

Roles of Arrest-Defective Protein 1225 and Hypoxia-Inducible Factor 1α in Tumor Growth and Metastasis

Background Vascular endothelial growth factor A (VEGFA), a critical mediator of tumor angiogenesis, is a well-characterized target of hypoxia-inducible factor 1 (HIF-1). Murine arrest-defective protein 1A (mARD1A225) acetylates HIF-1??, triggering its degradation, and thus may play a role in decreased expression of VEGFA.Methods We generated ApcMin/+/mARD1A225 transgenic mice and quantified growth of intestinal polyps. Human gastric MKN74 and murine melanoma B16F10 cells overexpressing mARD1A225 were injected into mice, and tumor growth and metastasis were measured. VEGFA expression and microvessel density in tumors were assessed using immunohistochemistry. To evaluate the role of mARD1A 225 acetylation of Lys532 in HIF-1??, we injected B16F10-mARD1A225 cell lines stably expressing mutant HIF-1??/K532R into mice and measured metastasis. All statistical tests were two-sided, and P values less than. 05 were considered statistically significant.Results ApcMin/+/mARD1A225 transgenic mice (n = 25) had statistically significantly fewer intestinal polyps than Apc Min/+ mice (n = 21) (number of intestinal polyps per mouse: Apc Min/+ mice vs ApcMin/+/mARD1A225 transgenic mice, mean = 83.4 vs 38.0 polyps, difference = 45.4 polyps, 95% confidence interval [CI] = 41.8 to 48.6; P <. 001). The growth and metastases of transplanted tumors were also statistically significantly reduced in mice injected with mARD1A225-overexpressing cells than in mice injected with control cells (P <. 01). Moreover, overexpression of mARD1A 225 decreased VEGFA expression and microvessel density in tumor xenografts (P <. 04) and ApcMin/+ intestinal polyps (P =. 001). Mutation of lysine 532 of HIF-1?? in B16F10-mARD1A225 cells prevented HIF-1?? degradation and inhibited the antimetastatic effect of mARD1A225 (P <. 001).Conclusion mARD1A225 may be a novel upstream target that blocks VEGFA expression and tumor-related angiogenesis

Requirement of TFIIH kinase subunit Mat1 for RNA Pol II C-terminal domain Ser5 phosphorylation, transcription and mRNA turnover

The relevance of serine 5 phosphorylation of RNA polymerase II carboxy-terminal domain during initiation has been difficult to determine in mammalian cells as no general in vivo Ser5 kinase has been identified. Here, we demonstrate that deletion of the TFIIH kinase subunit Mat1 in mouse fibroblasts leads to dramatically reduced Pol II Ser5 phosphorylation. This is associated with defective capping and reduced Ser2 phosphorylation, decreased Pol II progression into elongation and severely attenuated transcription detected through analysis of nascent mRNAs, establishing a general requirement for mammalian Mat1 in transcription. Surprisingly, the general defect in Pol II transcription in Mat1−/− fibroblasts is not reflected in the majority of steady-state mRNAs. This indicates widespread stabilization of mRNAs and points to the existence of a regulatory mechanism to stabilize mRNAs following transcriptional attenuation, thus revealing a potential caveat in similar studies limited to analysis of steady-state mRNAs

Endothelial Membrane Remodeling Is Obligate for Anti-Angiogenic Radiosensitization during Tumor Radiosurgery

While there is significant interest in combining anti-angiogenesis therapy with conventional anti-cancer treatment, clinical trials have as of yet yielded limited therapeutic gain, mainly because mechanisms of anti-angiogenic therapy remain to a large extent unknown. Currently, anti-angiogenic tumor therapy is conceptualized to either "normalize" dysfunctional tumor vasculature, or to prevent recruitment of circulating endothelial precursors into the tumor. An alternative biology, restricted to delivery of anti-angiogenics immediately prior to single dose radiotherapy (radiosurgery), is provided in the present study.Genetic data indicate an acute wave of ceramide-mediated endothelial apoptosis, initiated by acid sphingomyelinase (ASMase), regulates tumor stem cell response to single dose radiotherapy, obligatory for tumor cure. Here we show VEGF prevented radiation-induced ASMase activation in cultured endothelium, occurring within minutes after radiation exposure, consequently repressing apoptosis, an event reversible with exogenous C(16)-ceramide. Anti-VEGFR2 acts conversely, enhancing ceramide generation and apoptosis. In vivo, MCA/129 fibrosarcoma tumors were implanted in asmase(+/+) mice or asmase(-/-) littermates and irradiated in the presence or absence of anti-VEGFR2 DC101 or anti-VEGF G6-31 antibodies. These anti-angiogenic agents, only if delivered immediately prior to single dose radiotherapy, de-repressed radiation-induced ASMase activation, synergistically increasing the endothelial apoptotic component of tumor response and tumor cure. Anti-angiogenic radiosensitization was abrogated in tumors implanted in asmase(-/-) mice that provide apoptosis-resistant vasculature, or in wild-type littermates pre-treated with anti-ceramide antibody, indicating that ceramide is necessary for this effect.These studies show that angiogenic factors fail to suppress apoptosis if ceramide remains elevated while anti-angiogenic therapies fail without ceramide elevation, defining a ceramide rheostat that determines outcome of single dose radiotherapy. Understanding the temporal sequencing of anti-angiogenic drugs and radiation enables optimized radiosensitization and design of innovative radiosurgery clinical trials

Expression of delta-like ligand 4 (Dll4) and markers of hypoxia in colon cancer

BACKGROUND: Delta-like ligand 4 (Dll4) is a Notch ligand that is upregulated by hypoxia and vascular endothelial growth factor-A (VEGF-A) and is reported to have a role in tumor angiogenesis. Evidence from xenograft studies suggests that inhibiting Dll4-Notch signalling may overcome resistance to anti-VEGF therapy. The aim of this study was to characterise the expression of Dll4 in colon cancer and to assess whether it is associated with markers of hypoxia and prognosis. METHOD: In all, 177 colon cancers were represented in tissue microarrays. Immunohistochemistry was performed using validated antibodies against Dll4, VEGF, hypoxia-inducible factor (HIF)-1alpha, HIF-2alpha, prolyl hydroxylase (PHD)1, PHD2, PHD3 and carbonic anhydrase 9 (CA9). RESULTS: The expression of Dll4 was observed preferentially in the endothelium of 71% (125 out of 175) of colon cancers, but not in the endothelium adjacent to normal mucosa (none out of 107, P<0.0001). The expression of VEGF was significantly associated with HIF-2alpha (P<0.0001) and Dll4 (P=0.010). Only HIF-2alpha had a significant multivariate prognostic effect (hazard ratio 1.61, 95% confidence interval 1.01-2.57). Delta-like ligand 4 was also expressed by neoplastic cells, particularly neoplastic goblet cells. CONCLUSION: Endothelial expression of Dll4 is not a prognostic factor, but is significantly associated with VEGF. Assessing endothelial Dll4 expression may be critical in predicting response to anti-VEGF therapies

Studies on PKCI, a putative CDK7 interacting growth inhibitor

Cells in tissues have only three serious options in life; they can grow and divide, remain static, or die by apoptosis. Upon growth factor stimulation a cell enters the so called cell cycle which will eventually lead to the division of the cell. Cell cycle can be divided into four phases; G1, S, G2 and M. The current model of the cell cycle control holds that the transitions between different cell cycle states are regulated by cyclin dependent kinases (CDK) with their activator subunits, the cyclins. CDK regulation can be separated into four distinct mechanisms, one of which being phosphorylation on the so called T-loop leading to complete activation. This phosphorylating activity is mediated by apparently a single enzymatic activity termed the CDK activating kinase, CAK. CAK activity was originally isolated as a biochemical purification extract and the enzyme was surprisingly noticed to be structurally related to CDKs. Since a novel cyclin was identified to be associated to it, the enzyme exercising CAK activity was named CDK7 and the cyclin was designated cyclin H. An entirely new perspective on CDK7 function was opened when CDK7 was identified as a subunit of transcription factor IIH (TFIIH) and shown to phosphorylate the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). CDK7 has also been suggested to be involved in irradiation sensitivity pathways and nucleotide excision repair functions. To elucidate the intriguing in vivo role of CDK7, proteins interacting with CDK7 were screened for using the yeast two-hybrid method as part of previous studies of the laboratory. The results showed that 15 out of 144 (10,4%) positive clones were identified to encode a peptide sequence of a protein previously known as the inhibitor/interactor of protein kinase C (PKCI). These yeast colonies had an unexpected phenotype; contradictory to a dark blue color of the colonies, indicating strong interaction, the size of the PKCI colonies was small compared to others, indicating a possible growth inhibition effect. Several DNA open reading frames (ORF) coding for proteins related to human PKCI have been identified in a broad range of species representing mammalian, plant, fungal and bacterial kingdoms, all these forming a HIT (conserved triad of histidines) protein family. Another human member, part of this now super family, named FHIT (fragile triad of histidines) was identified with a dinucleoside 5’,5’’’-P1,P3-triphosphate hydrolase activity. These molecules; substrates of FHIT and related enzymes have been proposed to have various intracellular functions, including signalling stress responses. The aim of this study was to extend the investigation of the interaction between CDK7 and PKCI observed in yeast two-hybrid by means of several genetic and biochemical approaches to determine if this observed interaction and growth phenotype has any physiological significance. Investigations included performing yeast two-hybrid screening for PKCI, developing yeast three-hybrid system and carrying out growth rate assays for yeast liquid cultures. These studies also included performing biochemical purifications of over-expressed proteins, immunoprecipitations, western blot analysis and kinase activity assays. Protein extracts originated from transformed yeast cells, transfected mammalian cells or from in vitro transcription and translation reactions. On basis of growth rate assays it can be concluded that PKCI has an inhibitory growth effect in yeast. The preliminary finding of a specific PKCI-CDK7 interaction in yeast two-hybrid, however could not be conclusively verified by the other methods that were used in this study. Studies of PKCI characterisation also included examination of the subcellular localisation of PKCI in mammalian cells by immunofluorescence labelling of HA-PKCI. Results showed PKCI to localize both in the nucleus and in the cytoplasm. Also, studies to elucidate the function of PKCI were performed; whether it possesses enzymatic activity related to that of FHIT. By NMR spectroscopy using bacterially produced GST-PKCI, hydrolase activity towards ADP was indeed observed. Future studies will include elucidation of possible links between growth inhibition and hydrolase activity, in the form of stress signalling functions. The main focus of our future studies will be the generation of mice with targeted PKCI alleles offering powerful means to reveal the function of PKCI through observing phenotypes and through detailed analysis of these mice harbouring wild type, hypomorphic or null alleles

Tule, rakkaani! : Naisen ja miehen välisestä etiikasta kirjallisuuden rakkauskuvauksissa

Tutkin väitöskirjassani naisen ja miehen välistä rakkautta ja naisen ja miehen välisen rakkauden kuvauksia kirjallisuudessa ranskalaisen filosofin Luce Irigarayn samuuden järjestyksen kritiikin ja sukupuolieron etiikan valossa. Osoitan ensinnäkin, kuinka Platonin Pidoissa, Percy Bysshe Shelleyn Epipsychidionissa (1821) ja Barbara Cartlandin Liekehtivässä lumessa (1975) kuvataan rakkautta kahden rakastavaisen yhteensulautumiseksi - tutkin millaisia haltuunottamisen ja sulauttamisen muotoja rakastavaisten välillä näissä teoksissa tulee esille. Toiseksi osoitan, kuinka Laulujen laulussa, Dian kreivittären 1100-luvulla kirjoittamassa lyriikassa ja Guillevicin runokokoelmasta Trouées (1981) löytyvässä sikermässä Ylistyslaulu kuvataan rakkautta kahden toisiinsa palautumattoman rakastavaisen väliseksi suhteeksi - syvennyn näistä teoksista löytyviin kuvauksiin toista ihmettelevästä kohtaamisesta, toisen huomioonottavasta puhuttelemisesta ja toista hyväilevästä koskettamisesta. Tutkimukseni keskiössä on siis rakastavaisten välinen etiikka eli kysymykset siitä, kuinka rakastavaisten välinen suhde voisi toteutua ilman että kumpikaan pyrkisi haltuunottamaan toista. Objektivoivan analyysin sijaan tutkimusmenetelmänäni on rakkaudellinen vuoropuhelu niin valitsemieni kaunokirjallisten teosten kuin Irigarayn filosofiankin kanssa. Seuraan tässä Irigarayn ja toisen ranskalaisen ajattelijan Hélène Cixous'n työskentelytapoja - heidän mukaansa pysähtyvä ja kuulosteleva asenne on pyrkivää ja tarttuvaa otetta hedelmällisempi niin inhimillistä toista kuin kirjallistakin toista lähestyttäessä, sillä sen avulla toinen voi säilyttää liikkuvuutensa, arvaamattomuutensa ja vapautensa. Työni tulokset ovat johtopäätösten sijaan avauksia. Jatkan tutkimuksessani Irigarayn työtä ulottamalla hänen samuuden järjestyksen kritiikkinsä ja sukupuolieron etiikan hahmottelunsa kirjallisuudentutkimuksen alueelle. Irigarayta seuraten ajattelen uudelleen naisen ja miehen, henkisen ja ruumiillisen, aistimellisen ja transsendentaalisen välisiä suhteita. Tehtävänämme ei ole muodostaa rakkauden teoriaa tai rakastavaisten välisen etiikan ohjelmaa, vaan tutkia mitä etiikka merkitsee ja voisi merkitä rakkauden alueella