Mineralocorticoid receptor is involved in the regulation of genes responsible for hepatic glucose production

The mineralocorticoid receptor (MR) is expressed in kidney and plays a central role in the control of sodium, homeostatic fluid, and blood pressure. It has also been implicated in other functions in cardiovascular system, central nervous system, and adipose tissue. This study revealed a novel role of MR in the gene regulation related to hepatic glucose production. RNAi-mediated MR silencing led to a decrease in the expression of glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase 1, the enzymes known to be involved in glucose production in liver. The MR-specific antagonists also down-regulated the expression of G6Pase, while the specific agonist enhanced G6Pase expression. These observations, for the first time, revealed a novel role for MR and its ligands in the regulation of de novo glucose synthesis in hepatocytes. It also suggests the potential of liver-specific MR modulation for the treatment of hyperglycemia.     

Growth Conditions in In Vitro Culture Can Induce Oxidative Stress in <Emphasis Type="Italic">Mammillaria gracilis</Emphasis> Tissues

In vitro propagated plants of Mammillaria gracilis Pfeiff. (Cactaceae) develop calli without any exogenous growth regulators. This habituated tissue spontaneously regenerates morphologically normal as well as hyperhydric shoots. In this study, a possible involvement of activated oxygen metabolism in habituation and hyperhydricity in in vitro propagated plants of Mammillaria gracilis Pfeiff. (Cactaceae) was investigated. Significantly higher malondialdehyde (MDA) and carbonyl contents as well as hydrogen peroxide (H₂O₂) production were observed in habituated callus (HC), hyperhydric regenerated shoots (HS), and tumors (TT) in comparison to normal regenerated shoots (NS). Lipoxygenase (LOX) activity showed a similar trend, with a clear increase in activity in HC and HS. The activities of antioxidative enzymes, namely, peroxidase (POX), ascorbate peroxidase (APX), and catalase (CAT), were also higher in HC, HS, and TT, whereas an increase in superoxide dismutase (SOD) activity was observed in HC and HS. The majority of antioxidative isoenzymes were common to all cactus tissues, although a few tissue-specific bands were noticed. Significant decreases in phenylalanine ammonia lyase (PAL) activity, total phenolic content, and lignification were found in HS, HC, and TT in comparison to NS. Our results showed the appearance of a prominent oxidative stress in HC, HS, and TT as well as a strong induction of the antioxidant system indicating that activated oxygen metabolism could be involved in habituation and hyperhydricity and linked to the loss of tissue organization in M. gracilis. B. Balen and M. Tkalec contributed equally to this work.     

Divergent β-hairpins determine double-strand versus single-strand substrate recognition of human AlkB-homologues 2 and 3

Human AlkB homologues ABH2 and ABH3 repair 1-methyladenine and 3-methylcytosine in DNA/RNA by oxidative demethylation. The enzymes have similar overall folds and active sites, but are functionally divergent. ABH2 efficiently demethylates both single- and double-stranded (ds) DNA, whereas ABH3 has a strong preference for single-stranded DNA and RNA. We find that divergent F1 β-hairpins in proximity of the active sites of ABH2 and ABH3 are central for substrate specificities. Swapping F1 hairpins between the enzymes resulted in hybrid proteins resembling the donor proteins. Surprisingly, mutation of the intercalating residue F102 had little effect on activity, while the double mutant V101A/F102A was catalytically impaired. These residues form part of an important hydrophobic network only present in ABH2. In this functionally important network, F124 stacks with the flipped out base while L157 apparently functions as a buffer stop to position the lesion in the catalytic pocket for repair. F1 in ABH3 contains charged and polar residues preventing use of dsDNA substrate. Thus, E123 in ABH3 corresponds to F102 in ABH2 and the E123F-variant gained capacity to repair dsDNA with no loss in single strand repair capacity. In conclusion, divergent sequences outside of the active site determine substrate specificities of ABH2 and ABH3.     

Trastuzumab Mediated T-Cell Response against HER-2/Neu Overexpressing Esophageal Adenocarcinoma Depends on Intact Antigen Processing Machinery

Background

Esophageal adenocarcinoma (EAC) is a highly aggressive disease with poor prognosis, which frequently exhibits HER-2 gene amplification. Trastuzumab, the humanized antibody against HER-2, has potent growth inhibitory effects on HER-2 overexpressing cancers. One effect of trastuzumab is that it causes HER-2 receptor internalization and degradation, enhancing presentation of HER-2 epitopes on MHC-Class I molecules. This enhances the ability of HER-2 specific cytotoxic T lymphocytes (CTLs) to recognize and kill cancer cells. Novel strategies targeting the HER-2 receptor either directly by trastuzumab and/or indirectly by inducing a CTL response against HER-2 epitopes with, for instance, DC immunotherapy and consequently combining these strategies might prove to be very effective.

Methodology/Principal Findings

In this study we report that trastuzumab has potent growth inhibitory effects on two HER-2 overexpressing EAC cell lines OE33 and OE19. However, we found that trastuzumab and HER-2 specific CTLs act synergistically in inducing tumor lysis in OE33 but not in OE19. We discovered that in OE19 this deficient response is due to a down-regulation of the Transporter Associated with Antigen Processing-2 (TAP-2). TAP-2 is an important member of the Antigen Processing Machinery (APM), and is one of the essential elements for loading antigens on MHC class I molecules. Importantly, we demonstrated that by inducing re-expression of TAP-2 in OE19 with INF-γ treatment or by incubating the cells with INF-γ producing CTLs, the specific anti HER-2 CTL tumor lysis response and synergistic effect with trastuzumab can be restored.

Conclusion

An inefficient response of HER-2 overexpressing EAC to trastuzumab and/or DC immunotherapy can be due to a down-regulated TAP-2 expression and thus a deficient APM. Future studies combining trastuzumab with IFN-γ and/or immune-therapies inducing potent anti HER-2 CTL responses could lead to an effective combinatorial strategy for successful treatment of HER-2 overexpressing but APM defective cancers.     

Identification and characterization of a critical region in the glycogen synthase from Escherichia coli

The cysteine-specific reagent 5,5'-dithiobis(2-nitrobenzoic acid) inactivates the Escherichia coli glycogen synthase (Holmes, E., and Preiss, J. (1982) Arch. Biochem. Biophys. 216, 736-740). To find the responsible residue, all cysteines, Cys(7), Cys(379), and Cys(408), were substituted combinatorially by Ser. 5,5'-Dithiobis(2-nitrobenzoic acid) modified and inactivated the enzyme if and only if Cys(379) was present and it was prevented by the substrate ADP-glucose (ADP-Glc). Mutations C379S and C379A increased the S(0.5) for ADP-Glc 40- and 77-fold, whereas the specific activity was decreased 5.8- and 4.3-fold, respectively. Studies of inhibition by glucose 1-phosphate and AMP indicated that Cys(379) was involved in the interaction of the enzyme with the phosphoglucose moiety of ADP-Glc. Other mutations, C379T, C379D, and C379L, indicated that this site is intolerant for bulkier side chains. Because Cys(379) is in a conserved region, other residues were scanned by mutagenesis. Replacement of Glu(377) by Ala and Gln decreased V(max) more than 10,000-fold without affecting the apparent affinity for ADP-Glc and glycogen binding. Mutation of Glu(377) by Asp decreased V(max) only 57-fold indicating that the negative charge of Glu(377) is essential for catalysis. The activity of the mutation E377C, on an enzyme form without other Cys, was chemically restored by carboxymethylation. Other conserved residues in the region, Ser(374) and Gln(383), were analyzed by mutagenesis but found not essential. Comparison with the crystal structure of other glycosyltransferases suggests that this conserved region is a loop that is part of the active site. The results of this work indicate that this region is critical for catalysis and substrate binding.     

Saprolegnia oliviae sp. nov. isolated from an Argentine river (Tierra del Fuego Province,Argentina)

Saprolegnia oliviae sp. nov. is described from litter (floating dead twigs, leaves and roots) in the Olivia River, Ushuaia Department, Tierra del Fuego Province (Argentina). The new species is illustrated and compared with other species of the genus. Distinguishing characteristics of S. oliviae are the production of smooth oogonia (with some lateral or terminal projections) and the absence of antheridial branches on the majority of the oogonia, but when present, they are mostly diclinous, at times oogonia are supplied with androgynous and monoclinous antheridial branches. The oogonial stalks are predominantly short and straight or long and bent, curved or many times coiled; oospores are distinctive subcentric, (1-) 15-50 (-70) per oogonium. Morphological details of the new species and its comparison with other described species are discussed here.     

Acidosis improves uptake of antigens and MHC class I-restricted presentation by dendritic cells

It is widely appreciated that inflammatory responses in peripheral tissues are usually associated to the development of acidic microenvironments. Despite this, there are few studies aimed to analyze the effect of extracellular pH on immune cell functions. We analyzed the impact of acidosis on the behavior of dendritic cells (DCs) derived from murine bone marrow. We found that extracellular acidosis (pH 6.5) markedly stimulated the uptake of FITC-OVA, FITC-dextran, and HRP by DCs. In fact, to reach similar levels of endocytosis, DCs cultured at pH 7.3 required concentrations of Ag in the extracellular medium almost 10-fold higher compared with DCs cultured at pH 6.5. Not only the endocytic capacity of DCs was up-regulated by extracellular acidosis, but also the expression of CD11c, MHC class II, CD40, and CD86 as well as the acquisition of extracellular Ags by DCs for MHC class I-restricted presentation. Importantly, DCs pulsed with Ag under acidosis showed an improved efficacy to induce both specific CD8(+) CTLs and specific Ab responses in vivo. Our results suggest that extracellular acidosis improves the Ag-presenting capacity of DCs.     

Modulation of NF-kappa B activity and apoptosis in chronic lymphocytic leukemia B cells

Chronic lymphocytic leukemia (CLL) is an indolent malignancy of CD5+ B lymphocytes. CLL cells express CD40, a key regulator of B cell proliferation, differentiation, and survival. In nonmalignant B cells, CD40 ligation results in nuclear translocation and activation of NF-kappaB proteins. Based on observations that in some CLL cases, the tumor cells express both CD40 and its ligand, CD154 (CD40 ligand), we proposed a model for CLL pathogenesis due to CD40 ligation within the tumor. To evaluate this issue, we used freshly isolated CLL B cells to examine constitutive and inducible NF-kappaB activity by electrophoretic mobility shift assay. We consistently observed high levels of nuclear NF-kappaB-binding activity in unstimulated CLL B cells relative to that detected in nonmalignant human B cells. In each case examined, CD40 ligation further augmented NF-kappaB activity and prolonged CLL cell survival in vitro. The principle NF-kappaB proteins in stimulated CLL cells appear to be quite similar to those in nonmalignant human B cells and include p50, p65, and c-Rel. In a CD154-positive case, blocking CD154 engagement by mAb to CD154 resulted in inhibition of NF-kappaB activity in the CLL cells. The addition of anti-CD154 mAb resulted in accelerated CLL cell death to a similar degree as was observed in cells exposed to dexamethasone. These data indicate that CD40 engagement has a profound influence on NF-kappaB activity and survival in CLL B cells, and are consistent with a role for CD154-expressing T and B cells in CLL pathogenesis. The data support the development of novel therapies based on blocking the CD154-CD40 interaction in CLL.     

Reduction of infarct size and postischemic inflammation from ATL-146e, a highly selective adenosine A2A receptor agonist, in reperfused canine myocardium

Adenosine and adenosine A(2A) receptor agonists have been shown to limit myocardial infarct size when given at vasodilatory doses during reperfusion. This beneficial effect is thought to be due, in part, to stimulation of adenosine A(2A) receptors on inflammatory cells. The specific aims of this study were to determine whether the anti-inflammatory and cardioprotective properties of a novel adenosine A(2A) receptor agonist, ATL-146e (ATL), alone or in combination with the phosphodiesterase IV inhibitor rolipram would occur using very low, nonvasodilating doses. In a canine model of reperfused myocardial infarction, low-dose ATL given alone reduced infarct size by 45% (P < 0.05 vs. control). When ATL was combined with a very low dose of rolipram (0.001 microg.kg(-1).min(-1)), a marked reduction in P-selectin expression and neutrophil infiltration (51% lower; P < 0.001 vs. control) was seen and the infarct size reduction (58% lower; P < 0.01 vs. control) was greater than observed with ATL (45% lower; P < 0.05) or rolipram (33% lower; P < 0.05) alone. In conclusion, a low, nonvasodilating dose of ATL, a highly selective adenosine A(2A) receptor agonist, reduced infarct size after reperfusion. Furthermore, combining ATL and the phosphodiesterase IV inhibitor rolipram reduced infarct size even more than either agent alone. Such combination therapy may be beneficial clinically by potentiating cardioprotection after coronary reperfusion at doses far below those producing vasodilatation or side effects.