Project description:A fuller study of UV-induced biological effects lies beyond the scope of this paper. I will focus on the genetic interaction of cells irradiated by UV. Accordingly, in respect to genomic, the regulation of the gene expressions in cells represents how they response to the stress and repair themselves. In the past, Northern Blot has been used to detect the expression level of a few genes in one experiment. Thus, this assay is always limited to a small scope of a huge unknown network of gene expression. However, to investigate gene expression networks made of thousands of genes, a powerful tool must be implemented. As anticipated, the interactions between regulated genes in UV irradiation time-course experiment can be analyzed by cDNA microarray analysis with experimental loop design. This study provides a platform to investigate the gene networks regulated by UV irradiation. Keywords: UV, cDNA microarray, mitochondria respiratory chain, ROS, carcinogenesis Overall design: We used a loop design in this study, cDNA microarray experiment consisted of eight RNA samples, including UV-treated samples and their corresponding controls of 4 time points after UV irradiation. ***WARNING*** All samples have identical data.
Project description:BACKGROUND: Mitochondria are thought to have evolved from eubacteria-like endosymbionts; however, the origin of the mitochondrion remains a subject of debate. In this study, we investigated the phenomenon of chimerism in mitochondria to shed light on the origin of these organelles by determining which species played a role in their formation. We used the mitochondria of four distinct organisms, Reclinomonas americana, Homo sapiens, Saccharomyces cerevisiae and multichromosome Pediculus humanus, and attempted to identify the origin of each mitochondrial gene. RESULTS: Our results suggest that the origin of mitochondrial genes is not limited to the Rickettsiales and that the creation of these genes did not occur in a single event, but through multiple successive events. Some of these events are very old and were followed by events that are more recent and occurred through the addition of elements originating from current species. The points in time that the elements were added and the parental species of each gene in the mitochondrial genome are different to the individual species. These data constitute strong evidence that mitochondria do not have a single common ancestor but likely have numerous ancestors, including proto-Rickettsiales, proto-Rhizobiales and proto-Alphaproteobacteria, as well as current alphaproteobacterial species. The analysis of the multichromosome P. humanus mitochondrion supports this mechanism. CONCLUSIONS: The most plausible scenario of the origin of the mitochondrion is that ancestors of Rickettsiales and Rhizobiales merged in a proto-eukaryotic cell approximately one billion years ago. The fusion of the Rickettsiales and Rhizobiales cells was followed by gene loss, genomic rearrangements and the addition of alphaproteobacterial elements through ancient and more recent recombination events. Each gene of each of the four studied mitochondria has a different origin, while in some cases, multichromosomes may allow for enhanced gene exchange. Therefore, the tree of life is not sufficient to explain the chimeric structure of current genomes, and the theory of a single common ancestor and a top-down tree does not reflect our current state of knowledge. Mitochondrial evolution constitutes a rhizome, and it should be represented as such.
Project description:Translocation of the pro-apoptotic protein Bax from the cytosol to the mitochondria is a crucial step in DNA damage-mediated apoptosis, and is also found to be involved in mitochondrial fragmentation. Irradiation-induced cytochrome c release and apoptosis was associated with Bax activation, but not mitochondrial fragmentation. Both Bax and Drp1 translocated from the cytosol to the mitochondria in response to irradiation. However, Drp1 mitochondrial translocation and oligomerization did not require Bax, and failed to induce apoptosis in Bax deficient diffuse large B-cell lymphoma (DLBCL) cells. Using fluorescent microscopy and the intensity correlation analysis, we demonstrated that Bax and Drp1 were colocalized and the levels of colocalization were increased by UV irradiation. Using co-immuno-precipitation, we confirmed that Bax and Drp1 were binding partners. Irradiation induced a time-associated increase in the interaction between active Bax and Drp1. Knocking down Drp1 using siRNA blocked UV irradiation-mediated Bax mitochondrial translocation. In conclusion, our findings demonstrate for the first time, that Drp1 is required for Bax mitochondrial translocation, but Drp1-induced mitochondrial fragmentation alone is not sufficient to induce apoptosis in DLBCL cells.
Project description:NETosis is a unique form of neutrophil death that differs from apoptosis and necrosis. However, whether NETosis and apoptosis can occur simultaneously in the same neutrophil is unknown. In this paper, we show that increasing doses of ultraviolet (UV) irradiation increases NETosis, which is confirmed by myeloperoxidase colocalisation to neutrophil extracellular DNA. Increasing UV irradiation increases caspase 3 activation, mitochondrial reactive oxygen species (ROS) generation and p38, but not ERK, phosphorylation. Inhibition of mitochondrial ROS production and p38 activation, but not NADPH oxidase (NOX) activity, suppresses UV-induced NETosis, indicating that UV induces NOX-independent NETosis. Like classical NOX-dependent and -independent NETosis, UV-induced NETosis requires transcriptional firing for chromatin decondensation. Cell death-specific inhibitor studies indicate that UV-mediated NETosis is not apoptosis, necrosis or necroptosis. Collectively, these studies indicate that increasing doses of UV irradiation induce both apoptosis and NETosis simultaneously, but the ultimate outcome is the induction of a novel form of NOX-independent NETosis, or "ApoNETosis".
Project description:UV irradiation and other stress-activated signals activate the Jun N-terminal kinase (JNK, SAPK) pathway. The induction of JNK activity results in the activation of proto-oncogene c-Jun and activator protein-1 (AP-1) transcriptional activity. Data presented here show that UV mediated the activation of JNK correlated with UV-induced apoptosis and that overexpression of a dominant negative JNK blocked UV-induced apoptosis. However, the molecular events that lead to JNK activation in response to UV treatment are not clear. In this report, we provide evidence that a Fas receptor binding protein, Daxx, mediates UV-induced JNK activation and apoptosis. A dominant negative Daxx, coding for the C-terminal region (112 amino acids) of Daxx, was constructed and used in the experiments. Our data show that overexpression of the dominant negative Daxx partially inhibits UV-induced JNK phosphorylation in 293 cells. Inhibition of JNK phosphorylation resulted in the inhibition of c-Jun activation upon UV irradiation. Our data also show that the inhibition of JNK activation by dominant negative Daxx correlates with the reduced rate of apoptotic death of 293 cells after UV irradiation. Surprisingly, overexpression of wild-type Daxx also inhibited UV-induced apoptosis, suggesting that Daxx competes for Fas receptor binding sites with other proapoptotic factors such as FADD. In addition, overexpression of a dominant negative mutant of FADD did not affect UV-induced JNK activation but does inhibit UV-induced apoptosis. These results suggest that UV-induced JNK activation is not sufficient but required for induction of apoptosis.
Project description:UV irradiation elicits acute inflammation in the skin by increasing proinflammatory cytokine production in keratinocytes. However, the downstream protein target(s) that link UV radiation to the activation of signaling pathways responsible for cytokine expression have not been fully elucidated. In this study, we report a novel role of transglutaminase 2 (TG2), a member of the TG enzyme family whose activities are critical for cornified envelope formation, in mediating UV-induced inflammation. Our results showed that TG2-deficient mice exhibited reduced inflammatory responses to UV irradiation, including reduced erythema, edema, dilation of blood vessels, inflammatory cell infiltration, and levels of inflammatory cytokines. Using primary mouse keratinocytes and HaCaT cells, we found that UV irradiation-induced cytokine production by activating TG2, but not by upregulating TG2 expression, and that ER calcium release triggered by the UV-induced activation of phospholipase C was required for TG2 activation. Moreover, TG2 activity enhanced p65 phosphorylation, leading to an increase in NF-?B transcriptional activity. These results indicate that TG2 is a critical mediator of cytokine expression in the UV-induced inflammatory response of keratinocytes, and suggest that TG2 inhibition might be useful for preventing UV-related skin disorders, such as photoaging and skin cancer caused by chronic UV exposure.
Project description:Ultraviolet (UV) B radiation is a dangerous environmental stressor, which can lead to photoaging, inflammation, immune suppression and tumour formation. A recent report has shown the transcriptional activation of several skin-specific genes including matrix metalloproteases (MMPs) in response to UV irradiation. Here, we use a novel human keratinocyte model, HKerE6SFM, to demonstrate that UVB activates the transcription of most members of the 11q22.3 MMP gene cluster including MMP13, MMP12, MMP3, MMP1 and MMP10. Curiously, the expression of the well-characterized UVB-inducible MMP9, which is located outside of the cluster, remains unchanged. In accordance with the increased expression of the MMP gene cluster upon UVB irradiation, RNA polymerase II showed increased occupancy at their promoters following UVB irradiation. The results also demonstrate increased acetylated histone H3K9 levels at the promoters of the MMP13, MMP12, MMP3, MMP1 and MMP10 genes. These findings suggest a coordinated transcriptional activation of genes in the MMP cluster at 11q22.3 and that acetylation of histone H3 at lysine 9 has an important role in the UVB-dependent enhancement of transcription of MMP genes in this region.
Project description:Several species of Sulfolobales have been used as model organisms in the study of response mechanisms to ultraviolet (UV) irradiation in hyperthermophilic crenarchaea. To date, the transcriptional responses of genes involved in the initiation of DNA replication, transcriptional regulation, protein phosphorylation, and hypothetical function have been observed in Sulfolobales species after UV irradiation. However, due to the absence of knockout experiments, the functions of these genes under in situ UV irradiation have not yet been demonstrated. In the present study, we constructed five gene knockout strains (cdc6-2, tfb3, rio1, and two genes encoding the hypothetical proteins, Saci_0951 and Saci_1302) of Sulfolobus acidocaldarius and examined their sensitivities to UV irradiation. The knockout strains exhibited significant sensitivities to UV-B irradiation, indicating that the five UV-regulated genes play an important role in responses to UV irradiation in vivo. Furthermore, ?cdc6-2, ?rio1, ?Saci_0951, and ?tfb3 were sensitive to a wide variety of helix-distorting DNA lesions, including UV-induced DNA damage, an intra-strand crosslink, and bulky adducts. These results reveal that cdc6-2, tfb3, rio1, and Saci_0951 are play more important roles in broad responses to helix-distorting DNA damage than in specific responses to UV irradiation.