Substantial Metabolic Activity of Human Brown Adipose Tissue during Warm Conditions and Cold-Induced Lipolysis of Local Triglycerides

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Brain ATP Depletion Induced by Acute Ammonia Intoxication in Rats Is Mediated by Activation of the NMDA Receptor and Na+, K+-ATPase

Abstract: Injection of large doses of ammonia into rats leads to depletion of brain ATP. However, the molecular mechanism leading to ATP depletion is not clear. The aim of the present work was to assess whether ammonium-induced depletion of ATP is mediated by activation of the NMDA receptor. It is shown that injection of MK-801, an antagonist of the NMDA receptor, prevented ammonia-induced ATP depletion but did not prevent changes in glutamine, glutamate, glycogen, glucose, and ketone bodies. Ammonia injection increased Na⁺,K⁺-ATPase activity by 76%. This increase was also prevented by previous injection of MK-801. The molecular mechanism leading to activation of the ATPase was further studied. Na⁺,K⁺-ATPase activity in samples from ammonia-injected rats was normalized by "in vitro" incubation with phorbol 12-myristate 13-acetate, an activator of protein kinase C. The results obtained suggest that ammonia-induced ATP depletion is mediated by activation of the NMDA receptor, which results in decreased protein kinase C-mediated phosphorylation of Na⁺,K⁺-ATPase and, therefore, increased activity of the ATPase and increased consumption of ATP.     

Draft Genome Sequence of the Hydrocarbon-Degrading and Emulsan-Producing Strain Acinetobacter venetianus RAG-1T

We report the draft genome sequence of Acinetobacter venetianus strain RAG-1(T), which is able to degrade hydrocarbons and to synthesize a powerful biosurfactant (emulsan) that can be employed for oil removal and as an adjuvant for vaccine delivery. The genome sequence of A. venetianus RAG-1(T) might be useful for bioremediation and/or clinical purposes.     

Dexamethasone modulates interleukin-12 production by inducing monocyte chemoattractant protein-1 in human dendritic cells

Glucocorticoids have long been used as first-line immunosuppressants, although their precise mechanism of action has not been fully elucidated yet. This study evaluated the gene and protein expression of monocyte chemoattractant protein-1 (MCP-1), and its relationship with interleukin-12 and interleukin-10 synthesis, in human monocyte-derived dendritic cells exposed to dexamethasone. Dendritic cells were differentiated in the presence or in the absence of dexamethasone and then activated by IFN-gamma+soluble CD40 ligand; the gene and protein expression of target cytokines was measured by real-time PCR and ELISA, respectively. Our results showed that dexamethasone-primed mature dendritic cells expressed low levels of interleukin-12, and, at the opposite, high levels of interleukin-10 and MCP-1. Transfection experiments confirmed the ability of dexamethasone to activate MCP-1 gene promoter. Dexamethasone increased also MCP-2, but not MCP-3 synthesis, and the gene expression of CC chemokine receptor-2 by mature dendritic cells. The addition of anti-MCP-1 blocking antibody depressed MCP-1 release, and increased interleukin-12 production in dexamethasone-treated dendritic cells, thus demonstrating that interleukin-12 downregulation is largely dependent on MCP-1 overexpression. Our findings suggest that the induction of MCP expression in human dendritic cells by dexamethasone, and the amplification of cell response via the upregulation of the chemokine cognate receptor, may be critical to inhibit type 1 T-helper-biased immune response and, possibly, to favor type 2 T-helper-skewed response.     

Interaction of KAI1 on tumor cells with DARC on vascular endothelium leads to metastasis suppression

CD82, also known as KAI1, was recently identified as a prostate cancer metastasis suppressor gene on human chromosome 11p1.2 (ref. 1). The product of CD82 is KAI1, a 40- to 75-kDa tetraspanin cell-surface protein also known as the leukocyte cell-surface marker CD82 (refs. 1,2). Downregulation of KAI1 has been found to be clinically associated with metastatic progression in a variety of cancers, whereas overexpression of CD82 specifically suppresses tumor metastasis in various animal models. To define the mechanism of action of KAI1, we used a yeast two-hybrid screen and identified an endothelial cell-surface protein, DARC (also known as gp-Fy), as an interacting partner of KAI1. Our results indicate that the cancer cells expressing KAI1 attach to vascular endothelial cells through direct interaction between KAI1 and DARC, and that this interaction leads to inhibition of tumor cell proliferation and induction of senescence by modulating the expression of TBX2 and p21. Furthermore, the metastasis-suppression activity of KAI1 was significantly compromised in DARC knockout mice, whereas KAI1 completely abrogated pulmonary metastasis in wild-type and heterozygous littermates. These results provide direct evidence that DARC is essential for the function of CD82 as a suppressor of metastasis.     

Cellular responses to environmental contaminants in amoebic cells of the slime mould Dictyostelium discoideum

Amoebic Dictyostelium discoideum cells were employed in a bioassay to evaluate stress responses after exposures to the polyaromatic hydrocarbon benzo[a]pyrene (B[a]P) and two heavy metals (copper and mercury). Furthermore, we developed a recombinant cell line expressing a labile Green Fluorescent Protein (GFP) variant expressed under the control of an actin promoter to monitor stress-related protein degradation. Finally, cell viability was monitored to discriminate lethal exposure concentrations. The results demonstrated that exposure to sub-micromolar concentrations of mercury rendered significant changes in all studied physiological parameters, whereas B[a]P became toxic at low micromolar, and copper at high micromolar concentrations. Exposure to 0.5 microM mercury significantly reduced lysosomal membrane stability (LMS), endocytosis rate, GFP expression, and further resulted in the elevation of cytosolic free Ca(2+) ([Ca(2+)](i)). LMS in mercury-treated cells that had been pre-incubated with a specific Ca(2+)-dependent phospholipase A2 blocking agent was however not affected by the exposure, indicating that the toxic action of mercury is linked to the activation of phospholipase A2 via a Ca(2+)-signaling pathway. Exposure to 20 microM B[a]P significantly reduced LMS, endocytosis rate, and GFP expression, however without affecting [Ca(2+)](i), suggesting a calcium-independent route of toxicity for this compound. None of the physiological parameters were significantly affected by copper exposure at concentrations <400 microM, demonstrating a high resistance to this metal. Our results further showed that neither cell growth nor viability was affected by concentrations altering the studied physiological parameters. LMS, endocytosis rate, and [Ca(2+)](I), therefore, appear sensitive biomarkers of pollutant-related stress in amoebic cells.     

Phylogenetic analysis informed by geological history supports multiple, sequential invasions of the Mediterranean Basin by the angiosperm family Araceae

Despite the remarkable species richness of the Mediterranean flora and its well-known geological history, few studies have investigated its temporal and spatial origins. Most importantly, the relative contribution of geological processes and long-distance dispersal to the composition of contemporary Mediterranean biotas remains largely unknown. We used phylogenetic analyses of sequences from six chloroplast DNA markers, Bayesian dating methods, and ancestral area reconstructions, in combination with paleogeographic, paleoclimatic, and ecological evidence, to elucidate the time frame and biogeographic events associated with the diversification of Araceae in the Mediterranean Basin. We focused on the origin of four species, Ambrosina bassii, Biarum dispar, Helicodiceros muscivorus, Arum pictum, subendemic or endemic to Corsica, Sardinia, and the Balearic Archipelago. The results support two main invasions of the Mediterranean Basin by the Araceae, one from an area connecting North America and Eurasia in the Late Cretaceous and one from the Anatolian microplate in western Asia during the Late Eocene, thus confirming the proposed heterogeneous origins of the Mediterranean flora. The subendemic Ambrosina bassii and Biarum dispar likely diverged sympatrically from their widespread Mediterranean sister clades in the Early-Middle Eocene and Early-Middle Miocene, respectively. Combined evidence corroborates a relictual origin for the endemic Helicodiceros muscivorus and Arum pictum, the former apparently representing the first documented case of vicariance driven by the initial splitting of the Hercynian belt in the Early Oligocene. A recurrent theme emerging from our analyses is that land connections and interruptions, caused by repeated cycles of marine transgressions-regressions between the Tethys and Paratethys, favored geodispersalist expansion of biotic ranges from western Asia into the western Mediterranean Basin and subsequent allopatric speciation at different points in time from the Late Eocene to the Late Oligocene.     

Mutant PrP is delayed in its exit from the endoplasmic reticulum,but neither wild-type nor mutant PrP undergoes retrotranslocation prior to proteasomal degradation

The cellular mechanisms by which prions cause neurological dysfunction are poorly understood. To address this issue, we have been using cultured cells to analyze the localization, biosynthesis, and metabolism of PrP molecules carrying mutations associated with familial prion diseases. We report here that mutant PrP molecules are delayed in their maturation to an endoglycosidase H-resistant form after biosynthetic labeling, suggesting that they are impaired in their exit from the endoplasmic reticulum (ER). However, we find that proteasome inhibitors have no effect on the maturation or turnover of either mutant or wild-type PrP molecules. Thus, in contrast to recent studies from other laboratories, our work indicates that PrP is not subject to retrotranslocation from the ER into the cytoplasm prior to degradation by the proteasome. We find that in transfected cells, but not in cultured neurons, proteasome inhibitors cause accumulation of an unglycosylated, signal peptide-bearing form of PrP on the cytoplasmic face of the ER membrane. Thus, under conditions of elevated expression, a small fraction of PrP chains is not translocated into the ER lumen during synthesis, and is rapidly degraded in the cytoplasm by the proteasome. Finally, we report a previously unappreciated artifact caused by treatment of cells with proteasome inhibitors: an increase in PrP mRNA level and synthetic rate when the protein is expressed from a vector containing a viral promoter. We suggest that this phenomenon may explain some of the dramatic effects of proteasome inhibitors observed in other studies. Our results clarify the role of the proteasome in the cell biology of PrP, and suggest reasonable hypotheses for the molecular pathology of inherited prion diseases.