Eukaryotic translation initiation factor 5A (eIF-5A) is the only known cellular protein that contains the post-translationally derived amino acid, hypusine. Initially, eIF-5A was named as a translation initiation factor because of its capability to stimulate the formation of methionyl-puromycin, which mimics the first peptide bond formation during protein synthesis, under <em>in vitro</em> conditions. Subsequently, however, this proposed function of eIF-5A has been questioned because a similar effect on translation was not observed <em>in situ</em>. Moreover, eIF-5A appears not to be required for general protein synthesis. Rather, there is evidence that it facilitates the translation of specific subsets of mRNAs required for cell proliferation as well as apoptosis. <br /><br /> There are two isoforms of eIF-5A in the human genome which have designated eIF-5A1 and eIF-5A2. The objective of the present study was to gain an increased understanding of the roles of eIF-5A1 and eIF-5A2 during apoptosis and cell proliferation using human cell lines as a model system. Apoptosis was induced by treating the cells with Actinomycin D or sodium nitroprusside (SNP), which initiate programmed cell death by different mechanisms. It was observed for both normal and cancer cells that eIF-5A1 protein is up-regulated during apoptosis induced by Actinomycin D or SNP, whereas eIF-5A1 mRNA is constitutively expressed and does not change in abundance during this treatment. The up regulation of eIF-5A1 protein levels in the absence of a corresponding up-regulation in eIF-5A1 mRNA suggests that eIF-5A1 may be post-transcriptionally regulated. Moreover, eIF-5A1 protein up-regulation was stronger in normal cells than in cancer cells. By contrast, eIF-5A2 protein was below detection levels during apoptosis in both normal and cancer cells, although the corresponding transcript was detectable by semi-quantitative RT-PCR. This is attributable to inefficient translation of eIF-5A2 mRNA. <br /><br /> The effects of eIF-5A1 and eIF-5A2 on cell proliferation were examined by modulating the levels of serum in cultures of UACC-1598 cells, which are ovarian cancer cells that express high levels of both isoforms of eIF-5A. Serum starvation, which induces cell cycle arrest and ensuing apoptosis, followed by the re-addition of serum had no effect on the transcript levels of either eIF-5A1 or eIF-5A2. However, eIF-5A1 and eIF-5A2 proteins were both up-regulated within 24 hours of the initiation of serum starvation, and this coincided temporally with the onset of apoptosis as measured by TUNEL and a subsequent decline in viable cells. <br /><br /> The data indicate that eIF-5A1 and eIF-5A2 are both post-transcriptionally regulated and that they have functionally redundant roles in apoptosis.

Eukaryotic translation initiation factor 5A (eIF-5A) is the only known cellular protein that contains the post-translationally derived amino acid, hypusine. Initially, eIF-5A was named as a translation initiation factor because of its capability to stimulate the formation of methionyl-puromycin, which mimics the first peptide bond formation during protein synthesis, under <em>in vitro</em> conditions. Subsequently, however, this proposed function of eIF-5A has been questioned because a similar effect on translation was not observed <em>in situ</em>. Moreover, eIF-5A appears not to be required for general protein synthesis. Rather, there is evidence that it facilitates the translation of specific subsets of mRNAs required for cell proliferation as well as apoptosis. <br /><br /> There are two isoforms of eIF-5A in the human genome which have designated eIF-5A1 and eIF-5A2. The objective of the present study was to gain an increased understanding of the roles of eIF-5A1 and eIF-5A2 during apoptosis and cell proliferation using human cell lines as a model system. Apoptosis was induced by treating the cells with Actinomycin D or sodium nitroprusside (SNP), which initiate programmed cell death by different mechanisms. It was observed for both normal and cancer cells that eIF-5A1 protein is up-regulated during apoptosis induced by Actinomycin D or SNP, whereas eIF-5A1 mRNA is constitutively expressed and does not change in abundance during this treatment. The up regulation of eIF-5A1 protein levels in the absence of a corresponding up-regulation in eIF-5A1 mRNA suggests that eIF-5A1 may be post-transcriptionally regulated. Moreover, eIF-5A1 protein up-regulation was stronger in normal cells than in cancer cells. By contrast, eIF-5A2 protein was below detection levels during apoptosis in both normal and cancer cells, although the corresponding transcript was detectable by semi-quantitative RT-PCR. This is attributable to inefficient translation of eIF-5A2 mRNA. <br /><br /> The effects of eIF-5A1 and eIF-5A2 on cell proliferation were examined by modulating the levels of serum in cultures of UACC-1598 cells, which are ovarian cancer cells that express high levels of both isoforms of eIF-5A. Serum starvation, which induces cell cycle arrest and ensuing apoptosis, followed by the re-addition of serum had no effect on the transcript levels of either eIF-5A1 or eIF-5A2. However, eIF-5A1 and eIF-5A2 proteins were both up-regulated within 24 hours of the initiation of serum starvation, and this coincided temporally with the onset of apoptosis as measured by TUNEL and a subsequent decline in viable cells. <br /><br /> The data indicate that eIF-5A1 and eIF-5A2 are both post-transcriptionally regulated and that they have functionally redundant roles in apoptosis.

The role of cyclic guanosine monophosphate (cGMP) dependent protein kinase (PKG) in necrotic and apoptotic pathways of many cell types is well established; however its role in the ischemic preconditioning (IPC) of cardiomyocytes is not clearly defined. In the current study, we assessed the hypothesis that PKG protects against cell death following ischemidreperfusion injury in myocytes subjected to IPC. Freshly isolated adult rat ventricular myocytes were subjected to IPC by incubating in ischemic buffer for 30 minutes (min) followed by incubation in normal medium for 30 min. Prolonged simulated ischemia (SI) was created by incubating myocytes in the ischemic buffer for 90 min and reoxygenation (RO) for 120 min in the normal medium. Necrosis was determined by trypan blue exclusion and apoptosis was assessed by TUNEL assay. IPC reduced necrosis as shown by significant decrease in trypan blue positive cells as compared to virgin non-preconditioned myocytes subjected to SI and RO alone (p<.01). Similarly, the number of TUNEL positive myocytes following SI and 18 hrs of RO were significantly reduced in the IPC group. Treatment with PKG inhibitor, KT5832 (2pM) completely abolished the protection against necrosis by IPC. However, KT5832 failed to abolish the protective affect of IPC against apoptosis. Furthermore, myocytes infected with an adenoviral construct of PKG-la (1 x 1 o4 particles/cell) significantly reduced the number of trypan blue and TUNEL positive cells. These results suggest that the PKG signaling pathway plays an essential role in the preconditioning of myocytes against necrosis following SI / RO injury. Furthermore, while the overexpression of PKG protects myocytes against necrosis, as well as apoptosis, IPC may not induce a sufficient level of PKG during 18 hours of RO to induce protection against apoptosis.

Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.

Spermatogenesis is a complex process of male germ cell development from diploid spermatogonia to haploid fertile spermatozoa. Apoptosis plays a vital role in limiting cell numbers and eliminating defective germ cells. This requires novel gene products, and precise and well-coordinated programmes of gene expression. It is therefore possible that a disruption of transcription factor function would significantly impact germ cell development. The present work was undertaken to use Staput separation followed by culture of purified germ cells of rodent testis since mammalian spermatogenesis cannot yet be recreated in vitro. Specificity of separation was assessed using immunocytochemistry to identify spermatogonia, spermatocytes and spermatids. The genotoxins H2O2, doxorubicin, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, 6-mercaptopurine, 5-bromodeoxyuridine, methyl methanesulphonate and ethyl methanesulphonate were investigated. Cells were cultured and treated with different concentrations for each agent. DNA damage and apoptosis were measured by Comet and TUNEL assay respectively. Up-regulation of expression of the transcription factors Tbpl1, FHL5 and Gtf2a1l that are important post-meiotically, were examined using RT- PCR and qPCR. Protein production was evaluated using Western blotting. Tbpl1, FHL5 and Gtf2a1l were cloned in-frame into the inducible expression vector pET/100-TOPO. The recombinant clones were induced and successful expression of the proteins in E. coli was confirmed by SDS-PAGE and Western blotting. The recombinant clones obtained were used to demonstrate genotoxin induced impairment of gene expression. Thus, Staput-isolated rodent testicular germ cells seem to be a suitable model to study genotoxicity in vitro yielding result comparable to those reported in vivo. Furthermore, the work shows that genotoxins can impair gene expression.

Yes / Anthracyclines such as doxorubicin (Dox), widely used to treat various types of tumours, may result in induced testicular toxicity and oxidative stress. The present investigation was designed to determine whether exposure of isolated and purified mouse germ cells to Dox induces DNA damage in the form of strand breaks (presumably) resulting in apoptosis and to investigate the relative sensitivity of specific cell types. DNA damage was assessed using the Comet assay and the presence of apoptosis was determined by TUNEL assay. Isolated mouse germ cells were treated with different concentrations (0.05, 0.5 and 1 mM, respectively) of Dox, and fixed 1 h after treatment. The incidences of both DNA damage shown by single cell gel-electrophoresis and of apoptosis increased significantly in each specific cell type in a concentration-dependent manner. The DNA damage and apoptosis incidences gradually increased with concentration from 0.05 to 1 mM with Dox. Our results indicate that apoptosis plays a vital role in the induction of germ cell phase-specific toxicity caused by Dox with pre-meiotically and meiotically dividing spermatogonia and spermatocytes respectively as highly susceptible target cells. / Higher Education Funding Council for England (HEFCE)

Concerns about climate changes and scarcity of fossil fuels are rising. Hence wood is becoming an attractive source of renewable energy and raw material and these new dimensions have prompted increasing interest in wood formation in trees, in both the scientific community and wider public. In this thesis, the focus is on a key process in wood development: programmed cell death (PCD) in the development of xylem elements. Since secondary cell wall formation is dependent, inter alia, upon the life time of xylem elements, the qualitative features of wood will be affected by PCD in xylem, about which there is little information. This thesis focuses on the anatomical, morphological and transcriptional features of PCD during xylem development in both the stem of hybrid aspen, Populus tremula (L.) x tremuloides (Michx.) and the hypocotyl of the herbaceous model system Arabidopsis thaliana (L. Heynh.). In Populus, the progressive removal of organelles from the cytoplasm before the time of death (vacuolar bursts) and the slowness of the cell death process, illustrated by DNA fragmentation assays (such as TUNEL and Comet assays), have been ascertained in the xylem fibres by microscopic analyses. Furthermore, candidate genes for the regulation of fibre cell death were identified either from a Populus EST library obtained from woody tissues undergoing fibre cell death or from microarray experiments in Populus stem, and further assessed in an in silico comparative transcriptomic analysis of Arabidopsis thaliana. These candidate genes were either putative novel regulators of fibre cell death or members of previously described families of cell death-related genes, such as autophagy-related genes. The induction of the latter and the previous microscopic observations suggest the importance of autophagy in the degradation of the cytoplasmic contents specifically in the xylem fibres. Vacuolar bursts in the vessels were the only previously described triggers of PCD in the xylem, which induce the very rapid degradation of the nuclei and surrounding cytoplasmic contents, therefore unravelling a unique previously unrecorded type of PCD in the xylem fibres, principally involving autophagy. Arabidopsis is an attractive alternative model plant for exploring some aspects of wood formation, such as the characterisation of negative regulators of PCD. Therefore, the anatomy of Arabidopsis hypocotyls was also investigated and the ACAULIS5 (ACL5) gene, encoding an enzyme involved in polyamine biosynthesis, was identified as a key regulator of xylem specification, specifically in the vessel elements, though its negative effect on the cell death process. Taken together, PCD in xylem development seems to be a highly specific process, involving unique cell death morphology and molecular machinery. In addition, the technical challenges posed by the complexity of the woody tissues examined highlighted the need for specific methods for assessing PCD and related phenomena in wood. / Oron för klimatförändringar och brist på fossila bränslen har ökat påtagligt under de senaste åren. De enorma möjligheter som skogsråvaran erbjuder som alternativ källa för förnyelsebar energi och råmaterial har väckt ett stort intresse också för den biologiska processen bakom vedbildning i träd. Denna avhandling fokuserar på en viktig process i vedbildning: programmerad celldöd (PCD) i xylemet. Xylemcellernas livstid påverkar bildningen av sekundära cellväggar, vilket i sin tur påverkar vedens kvalitativa egenskaperna, så som veddensitet. Trots dess betydelse för viktiga egenskaper hos vedråvaran existerar fortfarande väldigt lite information om xylem PCD på cellulär eller molekylär nivå. I den här avhandlingen belyses de anatomiska, morfologiska och genetiska aspekterna av PCD i xylemutveckling i både stam av hybridasp, Populus tremula (L.) x tremuloides (Michx.) och hypokotyl av det örtartade modellsystemet Arabidopsis thaliana (L. Heynh.). Xylemet i både Populus och Arabidopsis består av två olika celltyper; de vattentransporterade kärlen och de stödjande fibrerna. Det är känt att celldöd i kärlen pågår mycket snabbt efter att den centrala vakuolen brister och de hydrolytiska enzymer släpps in i cytoplasman. I den här avhandlingen ligger fokus på fibrerna i Populus xylemet. Med hjälp av mikroskopianalyser av cellmorfologin (elektronmikroskopi) och DNA-fragmentering i cellkärnan (TUNEL- och Comet-analyser) kunde vi konstatera att till skillnad från kärlen så uppvisar fibrerna en långsam och progressiv nedbrytning av organellerna och cellkärnans DNA före vakuolbristning. Dessutom har kandidatgener för reglering av fibercelldöd identifierats antingen från ett Populus EST bibliotek från vedartade vävnader som genomgår fibercelldöd eller från mikroarray experiment i Populus stam. Dessa kandidatgener är antingen potentiella nya regulatorer av fibercelldöd eller medlemmar av tidigare beskrivna familjer av celldödsrelaterade gener. Bland de sistnämnda finns autofagi-relaterade gener, vilket stöder funktionen av autofagi i samband med autolys av cellinnehållet i xylemfibrerna. Dessa studier pekar därför på en typ av PCD som har inte tidigare beskrivits för xylemet. Arabidopsis är ett alternativt växtmodellsystem för studier av vissa aspekter av vedbildningen, såsom karakteriseringen av negativa regulatorer av PCD. Därför har också hypokotylanatomin analyserats, och ACAULIS5 (ACL5) genen, som kodar för ett enzym i biosyntesen av polyaminer, har visats vara en viktig regulator av xylemspecifikation genom dess negativa effekt på kärlens celldöd. Sammantaget visar denna avhandling att PCD i xylemutvecklingen verkar involvera unika morfologiska och molekylära mekanismer. Vi visar dessutom att komplexiteten hos de vedartade vävnaderna leder till ett behov av bättre anpassade verktyg för att djupare kunna bedöma PCD och liknande fenomen i veden. / Även med namnet Moreau-Courtois, Charleen L. samt Moreau, Charleen.

Cells of the retinal pigment epithelium (RPE) are generated prenatally and generally survive the lifetime of the individual without undergoing proliferation or replacement. Therefore, the mechanisms promoting individual RPE cell survival and longevity in vivo may be distinct from, or a limited subset of, the mechanisms known to promote survival in proliferative cells in culture. To identify specific factors that sustain cell viability independent of effects on cell division, we studied RPE cells in low-density suspension culture, in which cell proliferation is inhibited. Single cells from Xenopus laevis eyes were plated onto a non-adhesive surface in protein-free medium, then assayed for survival using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell viability in these cultures was essentially undiminished over the initial 2 days. However, by approximately 1 week in culture, only an average of 53% of the cells remained alive. Plating cells on a fibronectin-coated substratum significantly enhanced survival, such that the number of cells alive at 1 week was 80-90% of the initial level. Essentially identical results were obtained with laminin- or collagen IV-coated substrata, or with insulin (5μg ml-1) in the medium. The absence of cell division in these cultures was confirmed by cell counting and BrdU incorporation experiments. Interestingly, in suspension cultures derived from monolayers previously established on microporous membrane filters, cells lost viability much faster (average of 80% dead at 3 days), and showed a relatively greater response to extracellular matrix proteins (five-fold increase in cell survival at 3 days). Enhanced RPE survival in response to fibronectin required spreading of the cell on a substratum, rather than mere adherence, as there was a high correlation between the percentage of spread cells and the percentage that were MTT-positive (r = 0·940). Cell spreading apparently enhanced survival by preventing the initiation of programmed cell death: unattached non-viable cells in culture exhibited morphological features expected of apoptosis, as well as positive staining by the TUNEL reaction. These studies demonstrate that, of several factors shown to maintain or increase cell number in proliferating cultures, some have their effect, at least in part, by promoting the survival of individual cells. The increased susceptibility of subcultured RPE to cell death has implications for clinical transplantation applications that may require manipulation of RPE in vitro.