Benzamide riboside induces apoptosis independent of Cdc25A expression in human ovarian carcinoma N.1 cells.

One of the mechanisms of action of a new oncolytic agent, benzamide riboside (BR) is by inhibiting inosine 5'-monophosphate dehydrogenase (IMPDH) which catalyzes the formation of xanthine 5'-monophosphate from inosine 5'-monophosphate and nicotinamide adenine dinucleotide, thereby restricting the biosynthesis of guanylates. In the present study BR (10 - 20 microM) induced apoptosis in a human ovarian carcinoma N.1 cell line (a monoclonal derivative of its heterogenous parent line HOC-7). This was ascertained by DNA fragmentation, TUNEL assay, [poly(ADP)ribose polymerase]-cleavage and alteration in cell morphology. Apoptosis was accompanied by sustained c-Myc expression, concurrent down-regulation of cdc25A mRNA and protein, and by inhibition of Cdk2 activity. Both Cdk2 and cdc25A are G1 phase specific genes and Cdk2 is the target of Cdc25A. These studies demonstrate that BR exhibits dual mechanisms of action, first by inhibiting IMPDH, and second by inducing apoptosis, which is associated with repression of components of the cell cycle that are downstream of constitutive c-Myc expression.     

Bipolar‐associated miR‐499‐5p controls neuroplasticity by downregulating the Cav1.2 subunit CACNB2

Bipolar disorder (BD) is a chronic mood disorder characterized by manic and depressive episodes. Dysregulation of neuroplasticity and calcium homeostasis are frequently observed in BD patients, but the underlying molecular mechanisms are largely unknown. Here, we show that miR‐499‐5p regulates dendritogenesis and cognitive function by downregulating the BD risk gene CACNB2. miR‐499‐5p expression is increased in peripheral blood of BD patients, as well as in the hippocampus of rats which underwent juvenile social isolation. In rat hippocampal neurons, miR‐499‐5p impairs dendritogenesis and reduces surface expression and activity of the L‐type calcium channel Cav1.2. We further identified CACNB2, which encodes a regulatory β‐subunit of Cav1.2, as a direct functional target of miR‐499‐5p in neurons. miR‐499‐5p overexpression in the hippocampus in vivo induces short‐term memory impairments selectively in rats haploinsufficient for the Cav1.2 pore forming subunit Cacna1c. In humans, miR‐499‐5p expression is negatively associated with gray matter volumes of the left superior temporal gyrus, a region implicated in auditory and emotional processing. We propose that stress‐induced miR‐499‐5p overexpression contributes to dendritic impairments, deregulated calcium homeostasis, and neurocognitive dysfunction in BD.     

Unique Responses of Differentiating Neuronal Growth Cones to Inhibitory Cues Presented by Oligodendrocytes

During central nervous system development, neurons differentiate distinct axonal and dendritic processes whose outgrowth is influenced by environmental cues. Given the known intrinsic differences between axons and dendrites and that little is known about the response of dendrites to inhibitory cues, we tested the hypothesis that outgrowth of differentiating axons and dendrites of hippocampal neurons is differentially influenced by inhibitory environmental cues. A sensitive growth cone behavior assay was used to assess responses of differentiating axonal and dendritic growth cones to oligodendrocytes and oligodendrocyte- derived, myelin-associated glycoprotein (MAG). We report that >90% of axonal growth cones collapsed after contact with oligodendrocytes. None of the encounters between differentiating, MAP-2 positive dendritic growth cones and oligodendrocytes resulted in growth cone collapse. The insensitivity of differentiating dendritic growth cones appears to be acquired since they develop from minor processes whose growth cones are inhibited (nearly 70% collapse) by contact with oligodendrocytes. Recombinant MAG(rMAG)-coated beads caused collapse of 72% of axonal growth cones but only 29% of differentiating dendritic growth cones. Unlike their response to contact with oligodendrocytes, few growth cones of minor processes were inhibited by rMAG-coated beads (20% collapsed). These results reveal the capability of differentiating growth cones of the same neuron to partition the complex molecular terrain they navigate by generating unique responses to particular inhibitory environmental cues.     

Bistable Perception Modeled as Competing Stochastic Integrations at Two Levels

We propose a novel explanation for bistable perception, namely, the collective dynamics of multiple neural populations that are individually meta-stable. Distributed representations of sensory input and of perceptual state build gradually through noise-driven transitions in these populations, until the competition between alternative representations is resolved by a threshold mechanism. The perpetual repetition of this collective race to threshold renders perception bistable. This collective dynamics – which is largely uncoupled from the time-scales that govern individual populations or neurons – explains many hitherto puzzling observations about bistable perception: the wide range of mean alternation rates exhibited by bistable phenomena, the consistent variability of successive dominance periods, and the stabilizing effect of past perceptual states. It also predicts a number of previously unsuspected relationships between observable quantities characterizing bistable perception. We conclude that bistable perception reflects the collective nature of neural decision making rather than properties of individual populations or neurons.     

Resolution of mitochondrial and chloroplast membrane protein complexes from green leaves of potato on blue-native polyacrylamide gels

Purification of mitochondria and mitochondrial protein complexes from green tissues is often severely impaired by the presence of chloroplasts and their proteins. Here we present a method which allows analysis of respiratory protein complexes from potato leaves. The procedure includes the preparation of an organellar fraction specifically enriched in mitochondria and the separation of organellar protein complexes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). For the first time mitochondrial and chloroplast protein complexes have been resolved simultaneously in a native gel. BN-PAGE allowed the separation of eleven bands, including the mitochondrial NADH-dehydrogenase, the bc1 complex and the mitochondrial F1-ATP synthase as well as the chloroplast F1-ATP synthase, the cytochrome b6f complex, the two photosystems and the light harvesting complex. The resolution of the protein complexes in the first dimension was good enough to allow identification of all subunits of individual complexes in the second dimension under denaturing conditions. Thus, BN-PAGE offers an opportunity to analyze mitochondrial and chloroplast protein complexes from a single preparation from very small amounts of tissue. The implications of our findings, for studies on protein expression and turnover in different tissues and developmental stages, are discussed.     

The mitochondrial complexome of Arabidopsis thaliana

Mitochondria are central to cellular metabolism and energy conversion. In plants they also enable photosynthesis through additional components and functional flexibility. A majority of those processes relies on the assembly of individual proteins to larger protein complexes, some of which operate as large molecular machines. There has been a strong interest in the makeup and function of mitochondrial protein complexes and protein–protein interactions in plants, but the experimental approaches used typically suffer from selectivity or bias. Here, we present a complexome profiling analysis for leaf mitochondria of the model plant Arabidopsis thaliana for the systematic characterization of protein assemblies. Purified organelle extracts were separated by 1D Blue native (BN) PAGE, a resulting gel lane was dissected into 70 slices (complexome fractions) and proteins in each slice were identified by label free quantitative shot‐gun proteomics. Overall, 1359 unique proteins were identified, which were, on average, present in 17 complexome fractions each. Quantitative profiles of proteins along the BN gel lane were aligned by similarity, allowing us to visualize protein assemblies. The data allow re‐annotating the subunit compositions of OXPHOS complexes, identifying assembly intermediates of OXPHOS complexes and assemblies of alternative respiratory oxidoreductases. Several protein complexes were discovered that have not yet been reported in plants, such as a 530 kDa Tat complex, 460 and 1000 kDa SAM complexes, a calcium ion uniporter complex (150 kDa) and several PPR protein complexes. We have set up a tailored online resource ( https://complexomemap.de/at_mito_leaves ) to deposit the data and to allow straightforward access and custom data analyses.     

Level of ribosomal RNA required for stimulation from quiescence increases during cellular aging in vitro of mammalian fibroblasts

We have investigated the relation between cell size in terms of cellular ribosomal RNA (rRNA) content and proliferation of diploid human and rat embryo fibroblasts during their aging in vitro. During phase III of the proliferative lifespan in vitro, cellular rRNA content increases by a factor of nearly 3. For very different regimes of stimulation of quiescent cells, a strict correlation was observed, between the proportion of cells stimulated and cellular rRNA content, resembling a steep threshold curve. During aging in vitro, these characteristic curves exhibit an essentially parallel shift to higher values of cellular rRNA content (to higher 'thresholds'). Upon establishment as a permanent cell line, the relation between proliferation stimulation and cellular rRNA ceases to change with further subculturing. It is suggested that the essence of transformation of fibroblasts with a myc-type of oncogenes is a reduction and stabilizing of the critical rRNA content required for proliferation.     

Acetic acid increases stability of silage under aerobic conditions

The effects of various compounds on the aerobic stability of silages were evaluated. It has been observed that inoculation of whole-crop maize with homofermentative lactic acid bacteria leads to silages which have low stability against aerobic deterioration, while inoculation with heterofermentative lactic acid bacteria, such as Lactobacillus brevis or Lactobacillus buchneri, increases stability. Acetic acid has been proven to be the sole substance responsible for the increased aerobic stability, and this acid acts as an inhibitor of spoilage organisms. Therefore, stability increases exponentially with acetic acid concentration. Only butyric acid has a similar effect. Other compounds, like lactic acid, 1,2-propanediol, and 1-propanol, have been shown to have no effect, while fructose and mannitol reduce stability.     

Chemiluminescence in peripheral blood mononuclear cells of solid tumor cancer patients

Summary Levels of chemiluminescence were measured in peripheral blood mononuclear cells (PBMC) from normal subjects and from solid tumor cancer patients. Patients with advanced malignant disease were found to have significantly elevated baseline chemiluminescence activity in their resting PBMC as compared to normal subjects or to cancer patients with, at most, minimum residual disease. Patients with either advanced disease or minimum residual disease, however, were found to exhibit significantly elevated activation of chemiluminescence by treatment of cells with phorbol myristic acetate (PMA). Treatment of surgically resected stage I lung cancer patients with Freund's complete adjuvant alone or emulsified with extracted lung cancer antigens was found to elevate chemiluminescence levels in patient PBMC. Serum from those vaccinated patients was found to elevate chemiluminescence levels of resting PBMC from normal subjects. That serum activity did net correlate with levels of immune complexes measurable in the Clq or Raji cell assay.