Intracellular calcium signals regulate growth of hepatic stellate cells via specific effects on cell cycle progression

Hepatic stellate cells (HSC) are important mediators of liver fibrosis. Hormones linked to downstream intracellular Ca²⁺ signals upregulate HSC proliferation, but the mechanisms by which this occurs are unknown. Nuclear and cytosolic Ca²⁺ signals may have distinct effects on cell proliferation, so we expressed plasmid and adenoviral constructs containing the Ca²⁺ chelator parvalbumin (PV) linked to either a nuclear localization sequence (NLS) or a nuclear export sequence (NES) to block Ca²⁺ signals in distinct compartments within LX-2 immortalized human HSC and primary rat HSC. PV-NLS and PV-NES constructs each targeted to the appropriate intracellular compartment and blocked Ca²⁺ signals only within that compartment. PV-NLS and PV-NES constructs inhibited HSC growth. Furthermore, blockade of nuclear or cytosolic Ca²⁺ signals arrested growth at the G2/mitosis (G2/M) cell-cycle interface and prevented the onset of mitosis. Blockade of nuclear or cytosolic Ca²⁺ signals downregulated phosphorylation of the G2/M checkpoint phosphatase Cdc25C. Inhibition of calmodulin kinase II (CaMK II) had identical effects on LX-2 growth and Cdc25C phosphorylation. We propose that nuclear and cytosolic Ca²⁺ are critical signals that regulate HSC growth at the G2/M checkpoint via CaMK II-mediated regulation of Cdc25C phosphorylation. These data provide a new logical target for pharmacological therapy directed against progression of liver fibrosis.     

Effect of graded levels of dietary Bacillus toyonensis and Bifidobacterium bifidum supplementation on growth,carcass traits and ileal histomorphometry and microbiota of growing quails

This experiment investigated the role of graded dietary levels of two probiotic strains (Bacillus toyonensis; BT and Bifidobacterium bifidum; BB) on the growth rate, carcass traits, physiological and histological aspects of growing Japanese quail. One thousand and three hundred sixty one-day-old un-sexed Japanese quail chicks were distributed randomly into ten groups. The 1st group served as a control and fed the basal diet without supplement while the 2nd, 3rd, 4th and 5th groups received the control diet supplemented with 0.05, 0.075, 0.10 and 0.125% BT, respectively. The 6th group fed the control diet plus 0.10% BB while the remaining groups (7th to 10th) received the basal diet incorporated with the previous levels of BT rich with 0.05% BB. Dietary supplementation of BT and/or BB increased body weight and gain; however, feed intake and feed conversion were not affected. Amylase activity was significantly elevated in 5th, 7th and 9th groups, while lipase activity was improved in all treatment groups except 3rd and 6th groups. Results obtained concluded that dietary supplementation of BT with or without BB is useful for performance, digestive enzyme activities, blood cholesterols, antioxidant status and ileal histomorphometry and microbiota of growing Japanese quail.     

SUMOylation of the hepatoma-derived growth factor negatively influences its binding to chromatin

Hepatoma-derived growth factor is a nuclear targeted mitogen containing a PWWP domain that mediates binding to DNA. To date, almost nothing is known about the molecular mechanisms of the functions of hepatoma-derived growth factor, its routes of secretion and internalization or post-translational modifications. In the present study, we show for the first time that hepatoma-derived growth factor is modified by the covalent attachment of small ubiquitin-related modifier 1 (SUMO-1), a post-translational modification with regulatory functions for an increasing number of proteins. Using a basal SUMOylation system in Escherichia coli followed by a MALDI-TOF-MS based peptide analysis, we identified the lysine residue SUMOylated located in the N-terminal part of the protein adjacent to the PWWP domain. Surprisingly, this lysine residue is not part of the consensus motif described for SUMOylation. With a series of hepatoma-derived growth factor mutants, we then confirmed that this unusual location is also used in mammalian cells and that SUMOylation of hepatoma-derived growth factor takes place in the nucleus. Finally, we demonstrate that SUMOylated hepatoma-derived growth factor is not binding to chromatin, in contrast to its unSUMOylated form. These observations potentially provide new perspectives for a better understanding of the functions of hepatoma-derived growth factor.     

Coexpression of ecto-5'-nucleotidase/CD73 with specific NTPDases differentially regulates adenosine formation in the rat liver

Ectonucleotidases modulate purinergic signaling by hydrolyzing ATP to adenosine. Here we characterized the impact of the cellular distribution of hepatic ectonucleotidases, namely nucleoside triphosphate diphosphohydrolase (NTPDase)1/CD39, NTPDase2/CD39L1, NTPDase8, and ecto-5'-nucleotidase/CD73, and of their specific biochemical properties, on the levels of P1 and P2 receptor agonists, with an emphasis on adenosine-producing CD73. Immunostaining and enzyme histochemistry showed that the distribution of CD73 (protein and AMPase activity) overlaps partially with those of NTPDase1, -2, and -8 (protein levels and ATPase and ADPase activities) in normal rat liver. CD73 is expressed in fibroblastic cells located underneath vascular endothelial cells and smooth muscle cells, which both express NTPDase1, in portal spaces in a distinct fibroblast population next to NTPDase2-positive portal fibroblasts, and in bile canaliculi, together with NTPDase8. In fibrotic rat livers, CD73 protein expression and activity are redistributed but still overlap with the NTPDases mentioned. The ability of the observed combinations of ectonucleotidases to generate adenosine over time was evaluated by reverse-phase HPLC with the recombinant rat enzymes at high "inflammatory" (500 μM) and low "physiological" (1 μM) ATP concentrations. Overall, ATP was rapidly converted to adenosine by the NTPDase1+CD73 combination, but not by the NTPDase2+CD73 combination. In the presence of NTPDase8 and CD73, ATP was sequentially dephosphorylated to the CD73 inhibitor ADP, and then to AMP, thus resulting in a delayed formation of adenosine. In conclusion, the specific cellular cocompartmentalization of CD73 with hepatic NTPDases is not redundant and may lead to the differential activation of P1 and P2 receptors, under normal and fibrotic conditions.     

Influences of total sulfur amino acids and photoperiod on growth,carcass traits,blood parameters,meat quality and cecal microbial load of broilers

The current study aimed to discuss the impact of total sulfur amino acids (TSAA) %, photoperiod, and their interaction on growth performance, carcass and blood indices of broiler chicks. A total of 300 unsexed IR broiler chicks one-week old were used in a factorial arrangement (2 × 3), including two photoperiod systems (22 L: 2 D and 16 L: 8 D) and three experimental rations having three grades of Met + Cyst (TSAA) (70%, 85% and 100% of digestible lysine in starter and finisher diets). Results revealed that the higher LBW and BWG were noticed in birds given TSAA at grades of 1.1 or 0.90 % under 22L: 2D photoperiod at five weeks of age and the whole experimental period (1–5 weeks of age), respectively. The highest live body weight (LBW (and body weight gain (BWG) were recorded in birds received 1.1% TSAA under the long photoperiod compared to the control and the other groups. Birds fed 1.3% TSAA consumed more feed than the other groups. The opposite was found in birds fed 1.1% TSAA under the short photoperiod (16L: 8D). The best feed conversion (FCR) was detected by birds fed 1.1% and 0.90% TSAA diets during the whole experimental period. All carcass traits studied were significantly influenced by TSAA levels, except for the relative weights of abdominal fat and spleen. The interaction effect on was significant on all carcass traits except spleen %. In conclusion, the addition of TSAA at level 1.1 and 0.9 % to starter and finisher diets under a long photoperiod regime improved broiler’s performance, carcass traits, and blood parameters studied.