Search for an Endogenous Bombesin-Like Receptor 3 (BRS-3) Ligand Using Parabiotic Mice

Bombesin-like receptor 3 (BRS-3) is an X-linked G protein-coupled receptor involved in the regulation of energy homeostasis. Brs3 null (Brs3 ^-/y) mice become obese. To date, no high affinity endogenous ligand has been identified. In an effort to detect a circulating endogenous BRS-3 ligand, we generated parabiotic pairs of mice between Brs3 ^-/y and wild type (WT) mice or between WT controls. Successful parabiosis was demonstrated by circulatory dye exchange. The Brs3 ^-/y-WT and WT-WT pairs lost similar weight immediately after surgery. After 9 weeks on a high fat diet, the Brs3 ^-/y-WT pairs weighed more than the WT-WT pairs. Within the Brs3 ^-/y-WT pairs, the Brs3 ^-/y mice had greater adiposity than the WT mice, but comparable lean and liver weights. Compared to WT mice in WT-WT pairs, Brs3 ^-/y and WT mice in Brs3 ^-/y-WT pairs each had greater lean mass, and the Brs3 ^-/y mice also had greater adiposity. These results contrast to those reported for parabiotic pairs of leptin receptor null (Lepr ^db/db) and WT mice, where high leptin levels in the Lepr ^db/db mice cause the WT parabiotic partners to lose weight. Our data demonstrate that a circulating endogenous BRS-3 ligand, if present, is not sufficient to reduce adiposity in parabiotic partners of Brs3 ^-/y mice.     

Loss of function of a lupus-associated FcgammaRIIb polymorphism through exclusion from lipid rafts

Dysfunction of receptors for IgG (FcgammaRs) has been thought to be involved in the pathogenesis of systemic lupus erythematosus (SLE). We show that a recently described SLE-associated polymorphism of FcgammaRIIb (FcgammaRIIbT(232)), encoding a single transmembrane amino acid substitution, is functionally impaired. FcgammaRIIbT(232) is unable to inhibit activatory receptors because it is excluded from sphingolipid rafts, resulting in the unopposed proinflammatory signaling thought to promote SLE.     

Sludge stabilization by composting: a Jordanian case study

Municipal sludge handling is a major problem facing wastewater treatment plants due to the high costs of treatment and disposal. This issue is of special importance in Jordan because of the critical economic situation as well as the lack of financial support for such nonprofit projects. This study investigates the possibility of solving this problem by testing a method of sludge stabilization that requires minimum initial and operating costs. The method tested here is sludge stabilization by composting which is an attempt to transform sludge into a safe, nuisance free, humus like product that can be applied safely to land and can become a source of income that would recover the costs of processing. Two types of composting systems were tested in this study, aerated static pile and windrow. Results obtained indicate that composting of dried sludge was not possible due to the extremely low moisture content; which was overcome by sludge seeding and mixing with amendment and bulking agents. This resulted in efficient stabilization and reduction of the amount of organic matter in the final compost. The experimental results obtained also indicate that both systems (aerated static pile and windrow) are efficient. The organic content of the sludge was reduced in the windrow system by 46% and in the aerated static pile by 66%. In addition, the total volatile solids had decreased in the windrow and the aerated pile by 26 and 73%, respectively. The heavy metals content of the final compost was examined and found to comply with the international standards.     

Toll-like receptor-4 regulation of hepatic Cyp3a11 metabolism in a mouse model of LPS-induced CNS inflammation

Central nervous system (CNS) infection and inflammation severely reduce the capacity of cytochrome P-450 metabolism in the liver. We developed a mouse model to examine the effects of CNS inflammation on hepatic cytochrome P-450 metabolism. FVB, C57BL/6, and C3H/HeouJ mice were given Escherichia coli LPS (2.5 microg) by intracerebroventricular (ICV) injection. The CNS inflammatory response was confirmed by the elevation of TNF-alpha and/or IL-1beta proteins in the brain. In all mouse strains, LPS produced a 60-70% loss in hepatic Cyp3a11 expression and activity compared with saline-injected controls. Adrenalectomy did not prevent the loss in Cyp3a11 expression or activity, thereby precluding the involvement of the hypothalamic-adrenal-pituitary axis. Endotoxin was detectable (1-10 ng/ml) in serum between 15 and 120 min after ICV dosing of 2.5 microg LPS. Peripheral administration of 2.5 microg LPS by intraperitoneal injection produced similar serum endotoxin levels and a similar loss (60%) in Cyp3a11 expression and activity in the liver. The loss of Cyp3a11 in response to centrally or peripherally administered LPS could not be evoked in Toll-like receptor-4 (TLR4)-mutant (C3H/HeJ) mice, indicating that TLR4 signaling pathways are directly involved in the enzyme loss. In summary, we conclude that LPS is transferred from the brain to the circulation in significant quantities in a model of CNS infection or inflammation. Subsequently, LPS that has reached the circulation stimulates a TLR4-dependent mechanism in the periphery, evoking a reduction in Cyp3a11 expression and metabolism in the liver.     

The short term satiety peptide cholecystokinin reduces meal size and prolongs intermeal interval

Camostat mesilate (or mesylate) releases endogenous cholecystokinin (CCK) or CCK-58, the only detectable endocrine form of CCK in the rat, and reduces cumulative food intake by activating CCK₁ receptor. However, the literature lacks meal pattern analysis and an appropriate dose-response curve for this peptide. Therefore, the current study determines meal size (MS), intermeal interval (IMI) and satiety ratio (SR) by orogastric gavage of camostat (0, 12.5, 25, 50, 100, 200, 300, 400, 800 mg/kg) and compares them to those previously reported by a single dose of CCK-8 (1 nmol/kg, i.p), the most utilized form of CCK. We found that camostat (200, 300, 400 and 800 mg/kg) and CCK-8 reduced cumulative food intake and the size of the first meal, but only camostat prolonged IMI and increased SR. There was no change in the duration of the first two meals or in rated behaviors such as feeding, grooming, standing and resting in response to camostat and CCK-8, but there was more resting during the IMI in response to camostat. This study provides meal pattern analysis and an appropriate dose-response curve for camostat and CCK-8. Camostat reduces food intake by decreasing MS and prolonging IMI, whereas CCK-8 reduces food intake by reducing only meal size.     

Control of Tobacco mosaic virus Movement Protein Fate by CELL-DIVISION-CYCLE Protein48

Like many other viruses, Tobacco mosaic virus replicates in association with the endoplasmic reticulum (ER) and exploits this membrane network for intercellular spread through plasmodesmata (PD), a process depending on virus-encoded movement protein (MP). The movement process involves interactions of MP with the ER and the cytoskeleton as well as its targeting to PD. Later in the infection cycle, the MP further accumulates and localizes to ER-associated inclusions, the viral factories, and along microtubules before it is finally degraded. Although these patterns of MP accumulation have been described in great detail, the underlying mechanisms that control MP fate and function during infection are not known. Here, we identify CELL-DIVISION-CYCLE protein48 (CDC48), a conserved chaperone controlling protein fate in yeast (Saccharomyces cerevisiae) and animal cells by extracting protein substrates from membranes or complexes, as a cellular factor regulating MP accumulation patterns in plant cells. We demonstrate that Arabidopsis (Arabidopsis thaliana) CDC48 is induced upon infection, interacts with MP in ER inclusions dependent on the MP N terminus, and promotes degradation of the protein. We further provide evidence that CDC48 extracts MP from ER inclusions to the cytosol, where it subsequently accumulates on and stabilizes microtubules. We show that virus movement is impaired upon overexpression of CDC48, suggesting that CDC48 further functions in controlling virus movement by removal of MP from the ER transport pathway and by promoting interference of MP with microtubule dynamics. CDC48 acts also in response to other proteins expressed in the ER, thus suggesting a general role of CDC48 in ER membrane maintenance upon ER stress.Plant viruses are obligate intracellular pathogens that replicate in association with host membranes (Laliberté and Sanfaçon, 2010) and subvert host intra- and intercellular trafficking pathways to achieve cell-to-cell and systemic spread (Harries and Ding, 2011; Niehl and Heinlein, 2011). In the case of the well-studied Tobacco mosaic virus (TMV), viral replication factories form on membranes of the endoplasmic reticulum (ER; Heinlein et al., 1995, 1998). As the plant ER is continuous between cells through plasmodesmata (PD; Ding et al., 1992), this membrane network provides a direct pathway for the spread of replicated virus from the replication sites in infected cells into the ER network of noninfected cells. The spread of plant viruses depends on virus-encoded movement proteins (MPs; Deom et al., 1987; Lucas, 2006). The MP of TMV facilitates the cell-to-cell passage of the infectious particle by forming a ribonucleoprotein complex with the viral RNA (Citovsky et al., 1990) and by increasing the size exclusion limit of PD (Wolf et al., 1989).During the course of infection, as well as when ectopically expressed, the MP associates with PD, the ER/actin network, and microtubules (Heinlein et al., 1995, 1998; Reichel and Beachy, 1998; Wright et al., 2007; Sambade et al., 2008; Hofmann et al., 2009; Boutant et al., 2010; Peña and Heinlein, 2012; Supplemental Fig. S1). Shortly after infection of a new cell, the MP localizes to small, mobile, ER-associated particles proposed to play a role in PD targeting of the viral RNA (Boyko et al., 2007; Sambade et al., 2008). Similar small, mobile MP particles are observed early upon ectopic expression of the protein. These particles colocalize with RNA and undergo stop-and-go movements in association with the ER (Sambade et al., 2008). The particle movements pause at microtubule proximal sites and their detachment requires microtubule polymerization (Sambade et al., 2008). These observations suggest that the interaction with the microtubule system plays a critical role in the maturation and ER-mediated delivery of infectious viral RNA particles to PD during early infection stages. Consistently, tobacco (Nicotiana tabacum) mutants with reduced microtubule dynamics exhibit reduced TMV movement (Ouko et al., 2010). Following virus movement, the previously infected cell further accumulates MP at the ER, a process that coincides with the formation of large ER inclusions that contain viral replicase and viral RNA in addition to MP and likely function as virus factories (Heinlein et al., 1998; Más and Beachy, 1999). In mature form, these inclusions may represent the so-called viroplasms or X-bodies described in the classical literature (Bawden and Sheffield, 1939; Esau and Cronshaw, 1967; Hills et al., 1987). Their formation is associated with rearrangements of the ER membrane and likely mediated by the accumulated MP since the inclusions diminish and reconstitute a native ER structure when MP becomes degraded by the 26S proteasome (Reichel and Beachy, 1998, 2000). Transfected cells accumulate MP in similar inclusions as those formed during infection, indicating that accumulated MP is indeed necessary and sufficient to form inclusions in association with the ER (Reichel and Beachy, 1998; Supplemental Fig. S1). Following accumulation of MP in virus factories, the infected cells accumulate the MP also along microtubules (Heinlein et al., 1998). The accumulation of MP in virus factories and on microtubules in cells behind the leading front of infection is dispensable for virus movement (Heinlein et al., 1998; Boyko et al., 2000a). At these late infection stages, the virus factories may enable the virus to produce high virion titers (Laliberté and Sanfaçon, 2010; Tilsner et al., 2012), and the subsequent accumulation along microtubules may play a role in withdrawing MP from the cell-to-cell communication pathway (Curin et al., 2007) and in stockpiling MP prior to degradation (Padgett et al., 1996; Gillespie et al., 2002).The molecular mechanisms that guide the MP to the ER and subsequently to microtubules during infection are not known. The MP is a hydrophobic protein that behaves like a membrane-integral or tightly membrane-associated protein in differential fractionation experiments and contains two predicted transmembrane domains (Reichel and Beachy, 1998; Brill et al., 2000, 2004) involved in ER association (Fujiki et al., 2006). The association with microtubules depends on MP amino acids 1 to 213 required for MP function (Kahn et al., 1998; Boyko et al., 2000b,Boyko et al., 2000c, 2002; Kotlizky et al., 2001). Moreover, certain amino acid exchange mutations known to affect the function of MP in virus movement in a temperature-sensitive manner also affect the ability of MP to interact with microtubules (Boyko et al., 2007,Boyko et al., 2000b). Interestingly, these mutations cluster together in a short domain of 25 amino acids showing a structural similarity with the M-loop of tubulin involved in tubulin-tubulin interactions (Boyko et al., 2000b; Waigmann et al., 2007). Importantly, this M-loop similarity domain overlaps with the predicted transmembrane domain (Brill et al., 2000, 2004) thus suggesting that the association of MP with membranes or microtubules is an alternative event that may depend on specific posttranslational modifications or specific folds of MP. However, although the different subcellular localizations of MPs during the course of infection indicate directional transport of MP from the ER to microtubules and may indicate different folds and functions of the protein when associated with these different subcellular components, the mechanism that controls the subcellular localization and, thus, the fate and function of MP is not known.Here, we identify CELL-DIVISION-CYCLE protein48 (CDC48), named p97/VCP (Valosin-containing protein) in mammals and Cdc48p in yeast (Saccharomyces cerevisiae), as a cellular factor regulating MP subcellular accumulation patterns. CDC48 functions are well characterized in mammalian and yeast systems but remain poorly investigated in plants. Yeast and mammalian CDC48s are essential, conserved chaperones involved in diverse cellular processes by controlling protein fate through extraction of substrates from membranes or complexes (Tsai et al., 2002; Meusser et al., 2005; Römisch, 2005; Rumpf and Jentsch, 2006; Schrader et al., 2009; Eisele et al., 2010; Meyer et al., 2012; Yamanaka et al., 2012). We show that virus infection leads to the induction of Arabidopsis (Arabidopsis thaliana) CDC48 isoforms and demonstrate a function of CDC48 in ER maintenance upon ER stress conditions. We further demonstrate that CDC48 interacts with MP and that CDC48 activity is required for MP degradation. Interaction of CDC48 with MP depends on the MP N terminus, which is required for degradation of the protein, for PD localization and microtubule accumulation of MP, and for function of MP in cell-to-cell transport of the viral RNA. Overexpressed CDC48 shifts MP subcellular localization from ER inclusions to microtubules, suggesting that CDC48 extracts the MP from ER-associated inclusions, where it accumulates in midstages of infection, to the cytosol, where it accumulates along microtubules during late infection stages. Moreover, overexpression of active, but not inactive, CDC48 inhibits virus movement. Our data demonstrate that a CDC48-dependent pathway leading to the clearance of ER-associated protein inclusions exists in plants, that plant viral MPs are substrates for this pathway, and that this pathway determines viral protein fate during infection. We suggest that CDC48-mediated extraction of MP from the ER is part of a plant defense response to remove MP from the ER, the compartment the virus uses for replication and movement.