Jianjun Hu1,2* Shaorong Gao1*
Abstract: ZC3H14 proteins bind poly (A) tail of RNAs through the CCCH (5) zinc finger domain, shuttle between the nucleus and the cytoplasm, and play roles in nuclear exporting and proper polyadenylation of mRNA transcripts. However, molecular and cellular mechanisms of mammalian ZC3H14 remain largely unknown. Here we identified the mouse Zc3h14 gene and its encoded Zc3h14 protein. We found that Mouse Zc3h14 gene had at least two transcript isoforms, both were widely expressed in various tissues. Mouse ZC3H14 protein had one N-terminus Q-rich domain and one CCCH (5) zinc fingers domain. Both the two isoforms localized to the nucleus, and intriguingly, formed multiple nuclear speckles. We mapped the core nuclear localization signal to 292-RKRK sequences. Serial mutation analysis showed that the CCCH (5) zinc fingers domain was necessary but not sufficient for nuclear speckle formation. Over-expression of CCCH(5) domain as a dominant negative mutant, caused dramatic cell cycle delay, accompanied with multiple morphological abnormalities, such as long processes growing-out, multinucleation, and nuclear fragmentation. Further studies demonstrated that the CCCH (5) domain induced nuclear foci formation of the DNA damage marker g-H2AX. RT-PCR analysis showed that a group of DNA damage/repair genes, such as Cdk2, Ddb1, Mdm2, p21, p53, Rad50, as well as two RNA binding proteins, Pabpc1 and Rbm10, were dramatically up-regulated. Immunoprecipitation and mass spectrometry analysis further showed that the CCCH (5) domain could bind with RNA binding protein RBM10 in an RNA-dependent manner, and the CCCH (5) domain could also immunoprecipitate a group of chromatin stability/DNA damage-related proteins. In brief, our data for the first time demonstrated that the RNA binding protein, ZC3H14, is associated with genome/chromatin stability and DNA damage/repair.
Nicholas Card, William S. Garver and Robert A. Orlando*
Abstract: Obesity often leads to increased systemic inflammation which is now thought to play a causative role in the development of atherosclerotic disease and insulin resistance. This inflammatory response originates within large adipose tissue depots and is initiated by classically activated macrophages that infiltrate the tissue from the circulation. The large number of macrophages resides in obese adipose tissue lead to significant increases in interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα) secretion; achieving levels sufficient to elevate circulating plasma concentrations. These cytokines activate potent signals to initiate lipolysis, to release free fatty acids from triacylglycerol stores and contribute to hyperlipidemia in obese individuals. Obese adipose tissue responds to normal β-adrenergic and glucagon stimuli to recover from negative energy balance by inducing lipolysis. However, it is not clear what quantitative influence additional lipolytic stimulation by IL-6 and TNFα has on normal β-adrenergic activity. Although, β-adrenergic and cytokine signaling activate separate pathways for lipolytic activation, it is undefined if the effects of multiple signaling events on lipolysis are additive or coincident. To clarify this issue, we measured lipolytic activity in 3T3-L1-derived adipocytes stimulated by a β-adrenergic agonist (isoproterenol), IL-6 or TNFα individually and in combinations as co- and tri-stimulation. Treatment of adipocytes with isoproterenol and either IL-6 or TNFα as co-stimulants increased lipolytic activation by approximately the sum of the individual ligands suggesting contributions from two independent pathways. Co-stimulation with IL-6 and TNFα provided slightly more than an additive response indicating signaling contributions from independent and common pathways. Tri-stimulation resulted in the largest level of lipolytic activation with a value approximate to adding isoproterenol stimulation to a combined treatment of IL-6 and TNFα. The additive nature of cytokine signaling to β-adrenergic activity suggests its therapeutic inhibition will prevent excessive lipolysis, yet minimally interfere with maintaining normal responses to varying energy demands.
John Adeolu Falode1*, Okonji, Raphael Emuebie2 and Komolafe O O3
Abstract: The cyanide detoxifying enzymes (rhodanese and mercaptopyruvate sulphurtransferase [MST]) were estimated in different tissues of three fishes (Tilapia zillii, Hepstus odea and Sarothodelon galileaus) from Igun River in Ilesa, South-Western region of Nigeria. The enzyme activities were carried out by measuring the amount of thiocyanate produced by the two enzymes using specific substrates in each case. The results also showed that both enzymes have a statistically high specific activity in Hepstus odea in all the tissues, followed by Sarothodelon galileaus and Tilapia zillii. Moreover, the tissue with the highest protein concentration is the gut, then the gills and the flesh, although the differences in their protein concentrations were statistically insignificant. The gut also showed the highest MST specific activity statistically, afterwards the gills and the flesh. Conversely, the rhodanese specific activity was however revealed to be high in the flesh than other tissues, but not statistically significantly different from the other tissues. The study showed the activities of two cyanide detoxifying enzymes (rhodanese and mercaptopyruvate sulphurtransferase) in the different fishes indicating the existence of a strong cyanide detoxifying mechanisms
Janine M. Preble, Hiroshi Kondo, Sidney Levitsky, James D. McCully*
Abstract: Transplant of mitochondria for cardioprotection has been shown to be efficacious in vitro and in vivo in animal models. The transition from animal to human models requires that effective quality control be used in the isolation and verification of mitochondrial viability and function. Herein, we describe standard procedures that can be used to isolate mitochondria and assess their quality and function. These procedures include hemocytometry, Coulter Counting, EM, fluorescent staining, respiration analysis and ATP assays. These quality control parameters for mitochondria transplant in cardiac and other tissue will ensure a high yield of isolated mitochondria from suitable tissue sources, and will ensure that the isolated mitochondria are viable, respiration competent and free from contamination.
The mitochondrial outer membrane (MOM) is the interface between mitochondria and other cellular compartments. Signaling cascades organized on the MOM convey signaling to and from mitochondria in order to coordinate appropriate responses to stimuli. This type of signal transduction ultimately influences mitochondrial physiology by retrograde or an anteriograde mechanisms. Scaffold and adaptor proteins positioned on the MOM are responsible for organizing signaling components (kinases, phosphatases, etc.) in close proximity with one another in order to facilitate communication between mitochondria and the rest of the cell [1,2].