Hepatic steatosis by dietary-conjugated linoleic acid is accompanied by accumulation of diacylglycerol and increased membrane-associated protein kinase C ε in mice

Scope : Conjugated linoleic acid reduces weight gain and adipose mass while inducing liver enlargement, hepatic steatosis, and insulin resistance in mice. The objective of this study was to determine if hepatic steatosis induced by conjugated linoleic acid would predict for hepatic diacylglycerol accumulation, increased membrane‐associated protein kinase C ε, and hyperglycemia. Methods and results : Six‐wk‐old C57Bl/6 male mice were fed a high‐saturated fat diet for 3 wk and were then randomized to high‐saturated fat diet with or without conjugated linoleic acid (1.5% wt). Following a 6‐wk intervention, hepatic triacylglycerol, diacylglycerol, membrane‐associated protein kinase C ε, and gluconeogenic gene expression were determined. Fasting glucose was determined at baseline and at the end of the study. Conjugated linoleic acid increased hepatic triacylglycerol and diacylglycerol concentration in association with increased membrane‐associated protein kinase C ε. Diacylglycerol concentration proved to be a better predictor than triacylglycerol concentration for the change from baseline in fasting glucose concentration and final insulin concentration. The increase in diacylglycerol concentration was also associated with increased hepatic gluconeogenic gene expression in conjugated linoleic acid‐treated animals. Conclusion : Our data suggest that conjugated linoleic acid can initiate the pathophysiology responsible for hepatic insulin resistance. Additional studies are needed to determine if the accumulation of hepatic diacylglycerol by conjugated linoleic acid leads to hepatic insulin resistance.     

Structural interpretation of site-directed mutagenesis and specificity of the catalytic subunit of protein kinase CK2 using comparative modelling

The catalytic subunit of protein kinase casein kinase 2 (CK2),which has specificity for both ATP and GTP, shows significantamino acid sequence similarity to the cyclin-dependent kinase2 (CDK2). We constructed site-directed mutants of CK2 and useda three-dimensional model to investigate the basis for the dualspecificity. Introduction of Phe and Gly at positions 50 and51, in order to restore the pattern of the glycine-rich motif,did not seriously affect the specificity for ATP or GTP. Weshow that the dual specificity probably originates from theloop situated around the position His115 to Asp120 (HVNNTD).The insertion of a residue in this loop in CK2 subunits, comparedwith CDK2 and other kinases, might orient the backbone to interactwith the base A and G; this insertion is conserved in all knownCK2. The mutant N118, the design of which was based on the modelling,showed reduced affinity for GTP as predicted from the model.Other mutants were intended to probe the integrity of the catalyticloop, alter the polarity of a buried residue and explore theimportance of the carboxy terminus. Introduction of Arg to replaceAsn189, which is mapped on the activation loop, results in amutant with decreased k_cat, possibly as a result of disruptionof the interaction between this residue and basic residues inthe vicinity. Truncation at position 331 eliminates the last60 residues of the subunit and this mutant has a reduced catalyticefficiency compared with the wild-type. Catalytic efficiencyis restored in the truncation mutant by the replacement of apotentially buried Glu at position 252 by Lys, probably owingto a higher stability resulting from the formation of a saltbridge between Lys252 and Asp208.     

Synthesis,Cytotoxicity, and Insight into the Mode of Action of Re(CO)3 Thymidine Complexes

Nucleoside analogues are extensively used in the treatment of cancer and viral diseases. The antiproliferative properties of organorhenium(I) complexes, however, have been scarcely explored to date. Herein we present the syntheses, characterization, and in vitro evaluation of Re^I(CO)₃ core complexes of thymidine and uridine. For the binding of the Re^I(CO)₃ core, a tridentate dipicolylamine metal chelate was introduced at positions C5′, C2′, N3, and C5 with spacers of various lengths. The corresponding organometallic thymidine complexes were fully characterized by IR and NMR spectroscopy and mass spectrometry. Their cytotoxicity was assessed against the A549 lung carcinoma cell line. Toxicity is dependent on the site and mode of conjugation as well as on the nature and the length of the tether. Moderate toxicity was observed for conjugates carrying the rhenium moiety at position C5′ or N3 (IC₅₀=124–160 μM ). No toxicity was observed for complexes modified at C2′ or C5. Complex 53 , with a dodecylene spacer at C5′, exhibits remarkable toxicity and is more potent than cisplatin, with an IC₅₀ value of 6.0 μM . To the best of our knowledge, this is the first report of the antiproliferative properties of [M(CO)₃]⁺¹–nucleoside conjugates. In competitive inhibition experiments with A549 cell lysates and purified recombinant human thymidine kinase 1 (hTK‐1), enzyme inhibition was observed for complexes modified at either N3 or C5′, but our results suggest that the toxicity cannot be attributed solely to interaction with hTK‐1.     

Dysregulation of PAK1 Is Associated with DNA Damage and Is of Prognostic Importance in Primary Esophageal Small Cell Carcinoma

Primary esophageal small cell carcinoma (PESCC) is a rare, but fatal subtype of esophageal carcinoma. No effective therapeutic regimen for it. P21-activated kinase 1 (PAK1) is known to function as an integrator and an indispensable node of major growth factor signaling and the molecular therapy targeting PAK1 has been clinical in pipeline. We thus set to examine the expression and clinical impact of PAK1 in PESCC. The expression of PAK1 was detected in a semi-quantitative manner by performing immunohistochemistry. PAK1 was overexpressed in 22 of 34 PESCC tumors, but in only 2 of 18 adjacent non-cancerous tissues. Overexpression of PAK1 was significantly associated with tumor location (p = 0.011), lymph node metastasis (p = 0.026) and patient survival (p = 0.032). We also investigated the association of PAK1 with DNA damage, a driven cause for malignancy progression. γH2AX, a DNA damage marker, was detectable in 18 of 24 (75.0%) cases, and PAK1 expression was associated with γH2AX (p = 0.027). Together, PAK1 is important in metastasis and progression of PESCC. The contribution of PAK1 to clinical outcomes may be involved in its regulating DNA damage pathway. Further studies are worth determining the potentials of PAK1 as prognostic indicator and therapeutic target for PESCC.