MitoTimer

Fluorescent Timer, or DsRed1-E5, is a mutant of the red fluorescent protein, dsRed, in which fluorescence shifts over time from green to red as the protein matures. This molecular clock gives temporal and spatial information on protein turnover. To visualize mitochondrial turnover, we targeted Timer to the mitochondrial matrix with a mitochondrial-targeting sequence (coined “MitoTimer”) and cloned it into a tetracycline-inducible promoter construct to regulate its expression. Here we report characterization of this novel fluorescent reporter for mitochondrial dynamics. Tet-On HEK 293 cells were transfected with pTRE-tight-MitoTimer and production was induced with doxycycline (Dox). Mitochondrial distribution was demonstrated by fluorescence microscopy and verified by subcellular fractionation and western blot analysis. Dox addition for as little as 1 h was sufficient to induce MitoTimer expression within 4 h, with persistence in the mitochondrial fraction for up to 6 d. The color-specific conformation of MitoTimer was stable after fixation with 4% paraformaldehyde. Ratiometric analysis of MitoTimer revealed a time-dependent transition from green to red over 48 h and was amenable to analysis by fluorescence microscopy and flow cytometry of whole cells or isolated mitochondria. A second Dox administration 48 h after the initial induction resulted in a second round of expression of green MitoTimer. The extent of new protein incorporation during a second pulse was increased by administration of a mitochondrial uncoupler or simvastatin, both of which trigger mitophagy and biogenesis. MitoTimer is a novel fluorescent reporter protein that can reveal new insights into mitochondrial dynamics within cells. Coupled with organelle flow cytometry, it offers new opportunities to investigate mitochondrial subpopulations by biochemical or proteomic methods.     

The public health and safety consequences of sleep disorders

Sleep deprivation and medical disorders of sleep are common in today's society and have significant public health implications. In this article, we address 3 specific issues related to the public health and safety consequences of sleep disorders. First, we review data that has linked sleep restriction to a variety of adverse physiologic and long-term health outcomes including all-cause mortality, diabetes, and cardiovascular disease. Second, we will review recent data that has demonstrated that therapy for obstructive sleep apnea (the most common respiratory disorder of sleep) is an extremely efficient use of healthcare resources (in terms of dollars spent per quality adjusted life year gained), and compares favorably with other commonly funded medical therapies. Finally, we will review data that illustrate the potential adverse patient and occupational safety impacts of the extreme work schedules of housestaff (physicians in training).     

Identification and Characterization of Thioredoxin H-Type Gene Family in Triticum turgidum ssp. durum in Response to Natural and Environmental Factor-Induced Oxidative Stress

Plant Molecular Biology Reporter - Key message: Thioredoxin h-type isoforms are tissue-specific, differentially expressed in germinating seeds and under salinity stress and highly regulated by...     

A Bayesian network approach incorporating imputation of missing data enables exploratory analysis of complex causal biological relationships

Bayesian networks can be used to identify possible causal relationships between variables based on their conditional dependencies and independencies, which can be particularly useful in complex biological scenarios with many measured variables. Here we propose two improvements to an existing method for Bayesian network analysis, designed to increase the power to detect potential causal relationships between variables (including potentially a mixture of both discrete and continuous variables). Our first improvement relates to the treatment of missing data. When there is missing data, the standard approach is to remove every individual with any missing data before performing analysis. This can be wasteful and undesirable when there are many individuals with missing data, perhaps with only one or a few variables missing. This motivates the use of imputation. We present a new imputation method that uses a version of nearest neighbour imputation, whereby missing data from one individual is replaced with data from another individual, their nearest neighbour. For each individual with missing data, the subsets of variables to be used to select the nearest neighbour are chosen by sampling without replacement the complete data and estimating a best fit Bayesian network. We show that this approach leads to marked improvements in the recall and precision of directed edges in the final network identified, and we illustrate the approach through application to data from a recent study investigating the causal relationship between methylation and gene expression in early inflammatory arthritis patients. We also describe a second improvement in the form of a pseudo-Bayesian approach for upweighting certain network edges, which can be useful when there is prior evidence concerning their directions.     

Characterization of new maize chloroplastic copper/zinc superoxide dismutase isoforms by high resolution native two-dimensional polyacrylamide gel electrophoresis. Identification of chilling responsive chloroplastic superoxide dismutase isoforms

The response of superoxide dismutases (SOD, EC1.15.1.1) to chilling-induced oxidative stress in differentially sensitive maize genotypes ( Zea mays L) was examined. A native 2D-PAGE system that resolves the maize leaf SOD isoforms has been developed. The chloroplastic SOD activity was resolved into four Cu/Zn SOD isoforms designated SOD_1a→d with pI values of 3.9, 4.0, 4.5 and 5.6, respectively. These SODs are located in the stroma and display a higher resistance to hydrogen peroxide inactivation than the cytosol Cu/ZnSODs. They operate as 32 kDa homodimers and have an AT motif at the NH₂-terminal, which characterizes the chloroplastic SODs of most species. A light chilling treatment resulted in a rapid increase in the activity of SOD_1a and SOD_1b. Because this increase was observed in the presence of the protein synthesis inhibitor cycloheximide, it is suggested that short-term regulation of chloroplastic SODs occurs at a post-translational level.     

Solid-phase Synthesis of ω-Agatoxin IVA,a P-type Calcium Channel Blocker

ω-Agatoxin IVA, isolated from the venom of funnel web spider Agelenopsis aperta, blocks potently and selectively P-type calcium channels. This toxin, composed of 48 amino acids and containing 8 cysteine residues, was synthesized by the solid-phase procedure. The Cys residues were protected by acetamidomethyl (Acm) groups which were removed by mercuric acetate. During treatment with mercuric acetate, a by-product was detected, involving modification of tryptophan residues by the Acm groups. This side reaction can be completely prevented by addition of an excess of tryptophan in the reaction medium during Acm deprotection. The resulting peptide was submitted to an oxidative refolding, in different conditions, in order to determine the most favourable protocol. After formation of the four disulphide bonds, the toxin was purified by successive preparative HPLC, on two different supports, and fully characterized by analytical HPLC, capillary electrophoresis, amino acid analysis, mass spectrometry and Edman degradation. It was found to block the P-type calcium channel with a similar biological potency as described for the natural product.     

State-Dependency Effects on TMS: A Look at Motive Phosphene Behavior

Transcranial magnetic stimulation (TMS) is a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.^1,2 Within the field of TMS, the term state dependency refers to the initial, baseline condition of the particular neural region targeted for stimulation. As can be inferred, the effects of TMS can (and do) vary according to this primary susceptibility and responsiveness of the targeted cortical area.^3,4,5In this experiment, we will examine this concept of state dependency through the elicitation and subjective experience of motive phosphenes. Phosphenes are visually perceived flashes of small lights triggered by electromagnetic pulses to the visual cortex. These small lights can assume varied characteristics depending upon which type of visual cortex is being stimulated. In this particular study, we will be targeting motive phosphenes as elicited through the stimulation of V1/V2 and the V5/MT+ complex visual regions.⁶Download video file.^{(78M, mov)}     

Regulation of neuronal nitric-oxide synthase activity by somatostatin analogs following SST5 somatostatin receptor activation

Somatostatin receptor SST5 is an inhibitory G protein-coupled receptor that exerts a strong cytostatic effect on various cell types. We reported previously that the SST5 anti-proliferative effect results in the inhibition of mitogen-induced increases in intracellular cGMP levels and MAPK activity. This study was conducted to define the early molecular events accountable for the SST5-mediated anti-proliferative effect. Here, we demonstrate that, in Chinese hamster ovary cells expressing SST5 (CHO/SST5 cells), somatostatin inhibited cell proliferation induced by nitric oxide donors and overexpression of the neuronal nitric-oxide synthase (nNOS) protein isoform. Accordingly, nNOS activity and dimerization were strongly inhibited following SST5 activation by the somatostatin analog RC-160. In CHO/SST5 cells, nNOS was dynamically recruited by the SST5 receptor and phosphorylated at tyrosyl residues following RC-160 treatment. RC-160 induced SST5-p60(src) kinase complex formation and subsequent p60(src) kinase activation. Coexpression of an inactive p60(src) kinase mutant with SST5 blocked RC-160-induced nNOS phosphorylation and inactivation and prevented the SST5-mediated anti-proliferative effect. In CHO/SST5 cells, p60(src) kinase associated with nNOS to induce its inactivation by phosphorylation at tyrosyl residues following RC-160 treatment. Using recombinant proteins, we demonstrated that such phosphorylation prevented nNOS homodimerization. Next, surface plasmon resonance and mutation analysis revealed that p60(src) directly associated with nNOS phosphorylated Tyr604. SST5-mediated inhibition of nNOS activity was demonstrated to be essential to the RC-160 anti-proliferative effect on pancreatic endocrine tumor-derived cells. We therefore identified nNOS as a new p60(src) kinase substrate essential for SST5-mediated anti-proliferative action.