Spinal Muscular Atrophy (SMA) is an autosomal recessive motor neuron disease. SMA is associated with homozygous mutations in the Survival of Motor Neuron gene I (SMN1). SMN protein does not appear to exist in cells in isolation but associates with several proteins to form a large multi-protein complex. The functions of SMN complex include assembly, metabolism and transport of diverse classes of ribonucleoproteins. X- Linked Spinal Muscular Atrophy is a rare congenital disorder characterized by multiple joint contractures. It is associated with hypotonia, areflexia, chest deformities and congenital joint contractures. A candidate interval was defined for XL-SMA in Xp11.3-Xq11.2 in 1995. The purpose of this study was to refine the XL-SMA gene region and discover the XL-SMA gene. In addition to that, the gene product was investigated to delineate the genotype-phenotype correlation. My studies were focused on single nucleotide polymorphism (SNP) analysis. The candidate gene interval was refined by studying 14 SNPs in the three largest families. This analysis revealed a recombination event which allowed elimination of the NDP gene. Significantly positive LOD scores were obtained from these SNP studies. The exons and exon-intron boundaries of 12 genes were screened. No mutations were found in these genes in affected male samples. In late 2006, UBE1 (Ubiquitin activating enzyme 1) was discovered as the XL-SMA gene. UBE1 protein is responsible for the first step of ubiquitination of proteins in a cell. To investigate a possible common molecular mechanism between SMA and XL-SMA, proteins in the SMN Complex in XL-SMA patient cell lines were studied. SMN and Gemin3 protein levels were found to be consistently lower in XL-SMA patient cell lines (lymphoblasts) compared to healthy cell line. These results imply that there may be a common disease mechanism. To understand if the SMN and Gemin3 RNA levels decrease. RNA expression studies were performed. These studies confirmed that there is no difference of RNA expression of SMN and Gemin3 in XL-SMA cell lines when compared to healthy cell lines. As for UBE1, the same experimental procedure for SMN Complex proteins were repeated with antibodies to UBE1 to determine if there is any decline of UBE1 protein levels in XL-SMA patient cell lines compared to a healthy cell line. There was a decline in protein levels of UBE1 in XL-SMA patients. Two possible models are proposed for a molecular mechanism in XL-SMA: 1) UBE1 involves in degradation of a protein which downregulates SMN Complex (or a protein which stabilizes SMN Complex). When UBE1 is mutated, the protein in question is not degraded and this results in excess downregulation of SMN Complex (maybe via a pathway involving SMN-Gemin3 interaction). 2) UBE1 and UBA6 interact with the proteins of SMN Complex as they monoubiquinate them for different cellular processes. When UBE1 is mutated, UBA6 cannot compensate the deficiency of UBE1, which in turn disrupts normal cellular RNA metabolism required for motor neuron development and survival.

No description available.

Mammalian reovirus (MRV) is a prototype virus of Reoviridae family. MRV virions are composed of two concentric protein capsids that surround a genome of 10 segments of dsRNA. It has been shown that MRV can manipulate host gene expression and further induce apoptosis and cell cycle arrest in various cell lines. However, the detailed molecular mechanisms by which MRV regulates the expression of host cells are largely unknown. P53 is an important transcriptional factor which modulates the expression of more than 130 genes controlled in cell stress-response. We aimed to examine the molecular mechanisms underlying the effect of MRV infection on the expression of host genes and the possible role of p53 in the interaction of MRV and host cells. Prototype serotype 3 reovirus strain Dearing (T3D) and serotype 1 strain Lang (T1L) were used to infect different cell lines, respectively, H1299 (p53-null and p53 positive), HT1080 (p53 mutant and p53 positive) and HCT116 (p21 deficient and 14-3-3σ deficient). By comparing the virus replication curve of T1L and T3D in these cell lines, we found that MRV can replicate with a similar pattern in both p53-defective and p53-positive cell lines which indicated that p53 does not have significant impact on MRV replication in these cell lines. We further found that the level of Bcl-xL protein, which has been shown to be able to inhibit apoptosis, was increased in H1299 cell lines (both p53-null and p53 positive) infected by T3D, but decreased in the same cell lines infected by T1L. A similar change of Bcl-xL protein was not observed In HCT116 and HT1080 cell lines with MRV infection. Fifty four T1L×T3D reassortants were used to map which gene or gene combination was responsible for the changes of Bcl-xL protein. We found that the expression of Bcl-xL protein in H1299 cell line infected by MRV was majorly controlled by the S1 gene segment which encodes the σ1 cell attachment protein and the σ1s non structural protein, while minorly controlled by L3 gene segment of MRV.

Mammalian reovirus (MRV) is a prototype virus of Reoviridae family. MRV virions are composed of two concentric protein capsids that surround a genome of 10 segments of dsRNA. It has been shown that MRV can manipulate host gene expression and further induce apoptosis and cell cycle arrest in various cell lines. However, the detailed molecular mechanisms by which MRV regulates the expression of host cells are largely unknown. P53 is an important transcriptional factor which modulates the expression of more than 130 genes controlled in cell stress-response. We aimed to examine the molecular mechanisms underlying the effect of MRV infection on the expression of host genes and the possible role of p53 in the interaction of MRV and host cells. Prototype serotype 3 reovirus strain Dearing (T3D) and serotype 1 strain Lang (T1L) were used to infect different cell lines, respectively, H1299 (p53-null and p53 positive), HT1080 (p53 mutant and p53 positive) and HCT116 (p21 deficient and 14-3-3σ deficient). By comparing the virus replication curve of T1L and T3D in these cell lines, we found that MRV can replicate with a similar pattern in both p53-defective and p53-positive cell lines which indicated that p53 does not have significant impact on MRV replication in these cell lines. We further found that the level of Bcl-xL protein, which has been shown to be able to inhibit apoptosis, was increased in H1299 cell lines (both p53-null and p53 positive) infected by T3D, but decreased in the same cell lines infected by T1L. A similar change of Bcl-xL protein was not observed In HCT116 and HT1080 cell lines with MRV infection. Fifty four T1L×T3D reassortants were used to map which gene or gene combination was responsible for the changes of Bcl-xL protein. We found that the expression of Bcl-xL protein in H1299 cell line infected by MRV was majorly controlled by the S1 gene segment which encodes the σ1 cell attachment protein and the σ1s non structural protein, while minorly controlled by L3 gene segment of MRV.

Mammalian preimplantation embryo development is prone to high rates of early embryo demise. Two underlying causes for failed development include the effect of sub-optimal culture media and maternal lethal effect (MLE) genes. In line with the growing evidence, we hypothesize that embryo fate is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins under suboptimal culture conditions such as HTF medium and oxidative stress. Characterization of Nalp5, a MLE gene resulting in 2-cell embryo arrest, also found a significantly higher expression of pro-apoptotic proteins in knockout oocytes and embryos. With the use of two anti-apoptotic peptides, TAT-BH4 and Bax-inhibiting peptide (BIP), we attempted to improve embryo development. Our results found that neither peptide was able to improve embryo development in the Nalp5 model, or the HTF model. However, TAT-BH4 is capable of significantly improving developmental competence in embryos cultured under oxidative stress. Our findings suggest that supplementation of TAT-BH4 in embryo culture medium may offer a novel and cost-effective technique to improve embryogenesis of cultured embryos. However, further studies are still required.

Mammalian preimplantation embryo development is prone to high rates of early embryo demise. Two underlying causes for failed development include the effect of sub-optimal culture media and maternal lethal effect (MLE) genes. In line with the growing evidence, we hypothesize that embryo fate is determined by the outcome of specific intracellular interactions between pro- and anti-apoptotic proteins under suboptimal culture conditions such as HTF medium and oxidative stress. Characterization of Nalp5, a MLE gene resulting in 2-cell embryo arrest, also found a significantly higher expression of pro-apoptotic proteins in knockout oocytes and embryos. With the use of two anti-apoptotic peptides, TAT-BH4 and Bax-inhibiting peptide (BIP), we attempted to improve embryo development. Our results found that neither peptide was able to improve embryo development in the Nalp5 model, or the HTF model. However, TAT-BH4 is capable of significantly improving developmental competence in embryos cultured under oxidative stress. Our findings suggest that supplementation of TAT-BH4 in embryo culture medium may offer a novel and cost-effective technique to improve embryogenesis of cultured embryos. However, further studies are still required.

The anthracycline quinone, doxorubicin (Adriamycin) is an antineoplastic agent that has substantial therapeutic activity against a broad variety of human cancers. Unfortunately, the use of this agent is limited by its cardiac toxicity, which is associated with free radical formation leading to apoptotic cell death. The goal of this work is to improve our understanding about doxorubicin induced cardiomyopathy and to identify compounds to limit doxorubicin induced cardiomyopathy. The knowledge gained here will have a generalized impact on all cardiac diseases involving oxidative stress and apoptosis. We show that doxorubicin induced apoptosis in primary neonatal rat cardiomyocytes can be attenuated by progesterone (PG). The anti-apoptotic action of PG was blocked by a progesterone receptor antagonist, Mifepristone (MF), indicating a progesterone receptor dependent pathyway. Affymetrix gene analyses found that PG treated cardiomyocytes increased the expression of 180 genes. Among the genes upregulated is NAD(P)H: Quinone Oxidoreductase-1 (NQO1) gene. NQO1 is a flavo-enzyme that can catalyze a two-electron reduction of Dox to a more stable hydroquinone, thereby acting as a defense mechanism against oxidative stress. The induction of NQO1 mRNA and NQO1 activity in cardiomyocytes was observed in a dose and time-dependent manner with PG treatment and was blocked by MF. Induction of NQO1 by b-naphoflavone, an inducer of NQO1, resulted in a decrease in caspase-3 activity. However, inhibition of NQO1 by dicoumarol did not attenuate the cytoprotective effect of PG. This data indicates that although induction of NQO1 can decrease Dox induced apoptosis, this is not the primary mechanism of cytoprotection induced by PG. Microarray analyses revealed that PG induced an increase of Bcl-XL mRNA. Inhibiting the expression of Bcl-XL using siRNA reduced the anti-apoptotic effect of PG, suggesting that Bcl-XL is a key player in PG induced cytoprotection. Western blot analyses indicated that PG induced the expression of Bcl-XL in a dose and time dependent manner consistent with the protective effect of PG. Induction of Bcl-XL by PG was blocked by cyclohexamide, but was not blocked by Actinomycin D indicating that a transcriptionally independent mechanism is responsible for the induction of Bcl-XL by PG. The activity of a bcl-x 3'UTR reporter was induced by PG and blocked by MF. These data suggest that PG may induce stabilization of the Bcl-X mRNA. We further explored the mechanism of PG induced Bcl-XL gene expression by comparing the effect of PG to two other steroids: corticosterone (CT) and retinoic acid (RA). Both CT and RA attenuate Dox induced apoptosis in cardiomyocytes. CT, but not RA or PG induced the activity of a GRE reporter plasmid. Analysis of the 5' region of the Bcl-XL promoter indicated that RA and CT, but not PG induced the activity of the 0.9kb region of the Bcl-XL promoter. The induction of the 0.9kb reporter plasmid by CT was glucocorticoid receptor dependent, since it was inhibited by MF. The Bcl-XL promoter does not contain any glucocorticoid or retinoid response elements, but does have AP-1 and NFkB response elements. CT, but not RA or PG induced the activity of an AP-1 reporter plasmid. RA, but not CT or PG induced the activity of an NFkB reporter plasmid. The induction of the 0.9kb Bcl-XL reporter plasmid by CT was blocked by expression of a dominant negative c-jun, TAM67 as well SB202190 indicating a nongenomic effect of CT in activating the Bcl-XL promoter through a p38 MAPK mediated AP-1 mechanism. Therefore although all three types of nuclear receptor ligands induce bcl-xL expression, the effect of CT is mediated by transcriptional activation by AP-1 signaling while NF-kB transcription factor appears to be involved in RA indced bcl-xL transcription.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22688号 / 医博第4632号 / 新制||医||1045(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 武藤 学, 教授 小川 誠司, 教授 溝脇 尚志 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Apoptosis is the process by which organisms eliminate excess, damaged or hazardous cells in a controlled manner. This process is controlled by the Bcl-2 family of proteins. Bcl-2 and Bcl-XL are anti-apoptotic paralogues that can replace CED-9, the sole homologue in C. elegans. It has therefore been assumed that Bcl-2 and Bcl-XL are replaceable and functionally identical. However, evidence in some mammalian cells indicates that this may not be the case. The purpose of this project was to exhaustively compare the anti-apoptotic activities of Bcl-2 and Bcl-XL in one cell type. As Bcl-2 and Bcl-XL have been found to localize to the ER and the outer mitochondrial membrane, we also determined whether subcellular location affects the function of these proteins differently. MCF-7 breast cancer cells were stably transfected with Bcl-2 and Bcl-XL alternatively targeted to the ER or mitochondria, and exposed to various doses of doxorubicin; PARP cleavage was measured using quantitative Western blotting as an indication of apoptosis to obtain EC₅₀ values in the different cell lines. The levels of both Bcl-2 and Bcl-XL affected anti-apoptotic activity; specific degradation of both proteins was noted at higher doses of doxorubicin. Nevertheless, the results indicated clearly that there was a difference between Bcl-2 and Bcl-XL. Using EC₅₀ values Bcl-XL mutants were at least 8 times more protective than Bcl-2 mutants. Furthermore, most of the cleavage products of PARP in Bcl-XL expressing clones were due to non-caspase-7 proteases, a pattern not seen with Bcl-2. Bcl-2 and Bcl-XL localized to mitochondria were most effective, while cytosolic and ER localized Bcl-XL were less effective, and Bcl-2 at these sites did not inhibit apoptosis. / Thesis / Master of Science (MSc)