Skin-derived fibroblasts from long-lived species are resistant to some, but not all, lethal stresses and to the mitochondrial inhibitor rotenone

Fibroblast cell lines were developed from skin biopsies of eight species of wild-trapped rodents, one species of bat, and a group of genetically heterogeneous laboratory mice. Each cell line was tested in vitro for their resistance to six varieties of lethal stress, as well as for resistance to the nonlethal metabolic effects of the mitochondrial inhibitor rotenone and of culture at very low glucose levels. Standard linear regression of species-specific lifespan against each species mean stress resistance showed that longevity was associated with resistance to death induced by cadmium and hydrogen peroxide, as well as with resistance to rotenone inhibition. A multilevel regression method supported these associations, and suggested a similar association for resistance to heat stress. Regressions for resistance to cadmium, peroxide, heat, and rotenone remained significant after various statistical adjustments for body weight. In contrast, cells from longer-lived species did not show significantly greater resistance to ultraviolet light, paraquat, or the DNA alkylating agent methylmethanesulfonate. There was a strong correlation between species longevity and resistance to the metabolic effects of low-glucose medium among the rodent cell lines, but this test did not distinguish mice and rats from the much longer-lived little brown bat. These results are consistent with the idea that evolution of long-lived species may require development of cellular resistance to several forms of lethal injury, and provide justification for evaluation of similar properties in a much wider range of mammals and bird species.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/73523/1/j.1474-9726.2006.00255.x.pd

Mamld1 Knockdown Reduces Testosterone Production and Cyp17a1 Expression in Mouse Leydig Tumor Cells

MAMLD1 is known to be a causative gene for hypospadias. Although previous studies have indicated that MAMLD1 mutations result in hypospadias primarily because of compromised testosterone production around the critical period for fetal sex development, the underlying mechanism(s) remains to be clarified. Furthermore, although functional studies have indicated a transactivation function of MAMLD1 for the non-canonical Notch target Hes3, its relevance to testosterone production remains unknown. To examine these matters, we performed Mamld1 knockdown experiments.Mamld1 knockdown was performed with two siRNAs, using mouse Leydig tumor cells (MLTCs). Mamld1 knockdown did not influence the concentrations of pregnenolone and progesterone but significantly reduced those of 17-OH pregnenolone, 17-OH progesterone, dehydroepiandrosterone, androstenedione, and testosterone in the culture media. Furthermore, Mamld1 knockdown significantly decreased Cyp17a1 expression, but did not affect expressions of other genes involved in testosterone biosynthesis as well as in insulin-like 3 production. Hes3 expression was not significantly altered. In addition, while 47 genes were significantly up-regulated (fold change >2.0×) and 38 genes were significantly down-regulated (fold change <0.5×), none of them was known to be involved in testosterone production. Cell proliferation analysis revealed no evidence for compromised proliferation of siRNA-transfected MLTCs.The results, in conjunction with the previous data, imply that Mamld1 enhances Cyp17a1 expression primarily in Leydig cells and permit to produce a sufficient amount of testosterone for male sex development, independently of the Hes3-related non-canonical Notch signaling

Loss of Gnas Imprinting Differentially Affects REM/NREM Sleep and Cognition in Mice

It has been suggested that imprinted genes are important in the regulation of sleep. However, the fundamental question of whether genomic imprinting has a role in sleep has remained elusive up to now. In this work we show that REM and NREM sleep states are differentially modulated by the maternally expressed imprinted gene Gnas. In particular, in mice with loss of imprinting of Gnas, NREM and complex cognitive processes are enhanced while REM and REM–linked behaviors are inhibited. This is the first demonstration that a specific overexpression of an imprinted gene affects sleep states and related complex behavioral traits. Furthermore, in parallel to the Gnas overexpression, we have observed an overexpression of Ucp1 in interscapular brown adipose tissue (BAT) and a significant increase in thermoregulation that may account for the REM/NREM sleep phenotypes. We conclude that there must be significant evolutionary advantages in the monoallelic expression of Gnas for REM sleep and for the consolidation of REM–dependent memories. Conversely, biallelic expression of Gnas reinforces slow wave activity in NREM sleep, and this results in a reduction of uncertainty in temporal decision-making processes

Disulphide Bridges of Phospholipase C of Chlamydomonas reinhardtii Modulates Lipid Interaction and Dimer Stability

BACKGROUND: Phospholipase C (PLC) is an enzyme that plays pivotal role in a number of signaling cascades. These are active in the plasma membrane and triggers cellular responses by catalyzing the hydrolysis of membrane phospholipids and thereby generating the secondary messengers. Phosphatidylinositol-PLC (PI-PLC) specifically interacts with phosphoinositide and/or phosphoinositol and catalyzes specific cleavage of sn-3- phosphodiester bond. Several isoforms of PLC are known to form and function as dimer but very little is known about the molecular basis of the dimerization and its importance in the lipid interaction. PRINCIPAL FINDINGS: We herein report that, the disruption of disulphide bond of a novel PI-specific PLC of C. reinhardtii (CrPLC) can modulate its interaction affinity with a set of phospholipids and also the stability of its dimer. CrPLC was found to form a mixture of higher oligomeric states with monomer and dimer as major species. Dimer adduct of CrPLC disappeared in the presence of DTT, which suggested the involvement of disulphide bond(s) in CrPLC oligomerization. Dimer-monomer equilibrium studies with the isolated fractions of CrPLC monomer and dimer supported the involvement of covalent forces in the dimerization of CrPLC. A disulphide bridge was found to be responsible for the dimerization and Cys7 seems to be involved in the formation of the disulphide bond. This crucial disulphide bond also modulated the lipid affinity of CrPLC. Oligomers of CrPLC were also captured in in vivo condition. CrPLC was mainly found to be localized in the plasma membrane of the cell. The cell surface localization of CrPLC may have significant implication in the downstream regulatory function of CrPLC. SIGNIFICANCE: This study helps in establishing the role of CrPLC (or similar proteins) in the quaternary structure of the molecule its affinities during lipid interactions

Neurite Outgrowth Mediated by Translation Elongation Factor eEF1A1: A Target for Antiplatelet Agent Cilostazol

Cilostazol, a type-3 phosphodiesterase (PDE3) inhibitor, has become widely used as an antiplatelet drug worldwide. A recent second Cilostazol Stroke Prevention Study demonstrated that cilostazol is superior to aspirin for prevention of stroke after an ischemic stroke. However, its precise mechanisms of action remain to be determined. Here, we report that cilostazol, but not the PDE3 inhibitors cilostamide and milrinone, significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Furthermore, specific inhibitors for the endoplasmic reticulum protein inositol 1,4,5-triphosphate (IP3) receptors and several common signaling pathways (PLC-γ, PI3K, Akt, p38 MAPK, and c-Jun N-terminal kinase (JNK), and the Ras/Raf/ERK/MAPK) significantly blocked the potentiation of NGF-induced neurite outgrowth by cilostazol. Using a proteomics analysis, we identified that levels of eukaryotic translation elongation factor eEF1A1 protein were significantly increased by treatment with cilostazol, but not cilostamide, in PC12 cells. Moreover, the potentiating effects of cilostazol on NGF-induced neurite outgrowth were significantly antagonized by treatment with eEF1A1 RNAi, but not the negative control of eEF1A1. These findings suggest that eEF1A1 and several common cellular signaling pathways might play a role in the mechanism of cilostazol-induced neurite outgrowth. Therefore, agents that can increase the eEF1A1 protein may have therapeutic relevance in diverse conditions with altered neurite outgrowth

N- and C-Terminal Domains of the Calcium Binding Protein EhCaBP1 of the Parasite Entamoeba histolytica Display Distinct Functions

Entamoeba histolytica, a protozoan parasite, is the causative agent of amoebiasis, and calcium signaling is thought to be involved in amoebic pathogenesis. EhCaBP1, a Ca2+ binding protein of E. histolytica, is essential for parasite growth. High resolution crystal structure of EhCaBP1 suggested an unusual arrangement of the EF-hand domains in the N-terminal part of the structure, while C-terminal part of the protein was not traced. The structure revealed a trimer with amino terminal domains of the three molecules interacting in a head-to-tail manner forming an assembled domain at the interface with EF1 and EF2 motifs of different molecules coming close to each other. In order to understand the specific roles of the two domains of EhCaBP1, the molecule was divided into two halves, and each half was separately expressed. The domains were characterized with respect to their structure, as well as specific functional features, such as ability to activate kinase and bind actin. The domains were also expressed in E. histolytica cells along with green fluorescent protein. The results suggest that the N-terminal domain retains some of the properties, such as localization in phagocytic cups and activation of kinase. Crystal structure of EhCaBP1 with Phenylalanine revealed that the assembled domains, which are similar to Calmodulin N-terminal domain, bind to Phenylalanine revealing the binding mode to the target proteins. The C-terminal domain did not show any of the activities tested. However, over-expression in amebic cells led to a dominant negative phenotype. The results suggest that the two domains of EhCaBP1 are functionally and structurally different from each other. Both the domains are required for structural stability and full range of functional diversity

Comparison of four different colorimetric and fluorometric cytotoxicity assays in a zebrafish liver cell line

Background: A broad spectrum of cytotoxicity assays is currently used in the fields of (eco)toxicology and pharmacology. To choose an appropriate assay, different parameters like test compounds, detection mechanism, specificity, and sensitivity have to be considered. Furthermore, tissue or cell line can influence test performance. For zebrafish (Danio rerio), as emerging model organism, cell lines are now increasingly used, but few studies examined cytotoxicity in these cell systems. Therefore, we compared four cytotoxicity assays in the zebrafish liver cell line, ZFL, to test four differently acting model compounds. The tests comprised two colorimetric assays (MTT assay using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, and the LDH assay detecting lactate dehydrogenase activity) and two fluorometric assays (alamarBlue® using resazurin, and CFDA-AM based on 5-carboxyfluorescein diacetate acetoxymethyl ester). Model compounds were the pharmaceutical Tamoxifen, its metabolite 4-Hydroxy-Tamoxifen, the fungicide Flusilazole and the polycyclic aromatic hydrocarbon Benzo[a]pyrene. Results: All four assays performed well in the ZFL cells and led to reproducible dose-response curves for all test compounds. Effective concentrations causing 10% or 50% loss of cell viability (EC10 and EC50 values) varied by a maximum factor of 7.0 for the EC10 values and a maximum factor of 1.8 for the EC50 values. The EC values were not statistically different between the four assays, which is due to the assessed unspecific effects of the compounds. However, most often, the MTT assay and LDH assay showed the highest and lowest EC values, respectively. Nevertheless, the LDH assay showed the highest intra- and inter-assay variabilities and the lowest signal-to-noise ratios. In contrast to MTT, the other three assays have the advantage of being non-destructive, easy to handle, and less time consuming. Furthermore, AB and CFDA-AM can be combined on the same set of cells without damaging the cells, allowing later on their use for the investigation of other endpoints. Conclusions: We recommend the alamarBlue and CFDA-AM assays for cytotoxicity assessment in ZFL cells, which can be applied either singly or combined.JRC.H.5-Rural, water and ecosystem resource

Glutamate regulation of calcium and IP3 oscillating and pulsating dynamics in astrocytes

Recent years have witnessed an increasing interest in neuron-glia communication. This interest stems from the realization that glia participates in cognitive functions and information processing and is involved in many brain disorders and neurodegenerative diseases. An important process in neuron-glia communications is astrocyte encoding of synaptic information transfer: the modulation of intracellular calcium dynamics in astrocytes in response to synaptic activity. Here, we derive and investigate a concise mathematical model for glutamate-induced astrocytic intracellular Ca2+ dynamics that captures the essential biochemical features of the regulatory pathway of inositol 1,4,5-trisphosphate (IP3). Starting from the well-known two-state Li-Rinzel model for calcium-induced-calcium release, we incorporate the regulation of the IP3 production and phosphorylation. Doing so we extended it to a three-state model (referred as the G-ChI model), that could account for Ca2+ oscillations triggered by endogenous IP3 metabolism as well as by IP3 production by external glutamate signals. Compared to previous similar models, our three-state models include a more realistic description of the IP3 production and degradation pathways, lumping together their essential nonlinearities within a concise formulation. Using bifurcation analysis and time simulations, we demonstrate the existence of new putative dynamical features. The cross-couplings between IP3 and Ca2+ pathways endows the system with self-consistent oscillator properties and favor mixed frequency-amplitude encoding modes over pure amplitude modulation ones. These and additional results of our model are in general agreement with available experimental data and may have important implications on the role of astrocytes in the synaptic transfer of information.Comment: 42 pages, 16 figures, 1 table. Figure filenames mirror figure order in the paper. Ending "S" in figure filenames stands for "Supplementary Figure". This article was selected by the Faculty of 1000 Biology: "Genevieve Dupont: Faculty of 1000 Biology, 4 Sep 2009" at http://www.f1000biology.com/article/id/1163674/evaluatio

A mathematical model of mitochondrial swelling

<p>Abstract</p> <p>Background</p> <p>The <it>permeabilization </it>of mitochondrial membranes is a decisive event in apoptosis or necrosis culminating in cell death. One fundamental mechanism by which such permeabilization events occur is the calcium-induced mitochondrial permeability transition. Upon Ca²⁺-uptake into mitochondria an increase in inner membrane permeability occurs by a yet unclear mechanism. This leads to a net water influx in the mitochondrial matrix, mitochondrial swelling, and finally the rupture of the outer membrane. Although already described more than thirty years ago, many unsolved questions surround this important biological phenomenon. Importantly, theoretical modeling of the mitochondrial permeability transition has only started recently and the existing mathematical models fail to characterize the swelling process throughout the whole time range.</p> <p>Results</p> <p>We propose here a new mathematical approach to the mitochondrial permeability transition introducing a specific delay equation and resulting in an optimized representation of mitochondrial swelling. Our new model is in accordance with the experimentally determined course of volume increase throughout the whole swelling process, including its initial lag phase as well as its termination. From this new model biological consequences can be deduced, such as the confirmation of a positive feedback of mitochondrial swelling which linearly depends on the Ca²⁺-concentration, or a negative exponential dependence of the average swelling time on the Ca²⁺-concentration. Finally, our model can show an initial shrinking phase of mitochondria, which is often observed experimentally before the actual swelling starts.</p> <p>Conclusions</p> <p>We present a model of the mitochondrial swelling kinetics. This model may be adapted and extended to diverse other inducing/inhibiting conditions or to mitochondria from other biological sources and thus may benefit a better understanding of the mitochondrial permeability transition.</p

INT reduction is a valid proxy for eukaryotic plankton respiration despite the inherent toxicity of INT and differences in cell wall structure

The reduction of 2-para (iodophenyl)-3(nitrophenyl)-5(phenyl) tetrazolium chloride (INT) is increasingly being used as an indirect method to measure plankton respiration. Its greater sensitivity and shorter incubation time compared to the standard method of measuring the decrease in dissolved oxygen concentration, allows the determination of total and size-fractionated plankton respiration with higher precision and temporal resolution. However, there are still concerns as to the method’s applicability due to the toxicity of INT and the potential differential effect of plankton cell wall composition on the diffusion of INT into the cell, and therefore on the rate of INT reduction. Working with cultures of 5 marine plankton (Thalassiosira pseudonana CCMP1080/5, Emiliania huxleyi RCC1217, Pleurochrysis carterae PLY-406, Scrippsiella sp. RCC1720 and Oxyrrhis marina CCMP1133/5) which have different cell wall compositions (silica frustule, presence/absence of calcite and cellulose plates), we demonstrate that INT does not have a toxic effect on oxygen consumption at short incubation times. There was no difference in the oxygen consumption of a culture to which INT had been added and that of a replicate culture without INT, for periods of time ranging from 1 to 7 hours. For four of the cultures (T. pseudonana CCMP1080/5, P. carterae PLY-406, E. huxleyi RCC1217, and O. marina CCMP1133/5) the log of the rates of dissolved oxygen consumption were linearly related to the log of the rates of INT reduction, and there was no significant difference between the regression lines for each culture (ANCOVA test, F = 1.696, df = 3, p = 0.18). Thus, INT reduction is not affected by the structure of the plankton cell wall and a single INT reduction to oxygen consumption conversion equation is appropriate for this range of eukaryotic plankton. These results further support the use of the INT technique as a valid proxy for marine plankton respiration