The reticulons: guardians of the structure and function of the endoplasmic reticulum

The endoplasmic reticulum (ER) consists of the nuclear envelope and a peripheral network of tubules and membrane sheets. The tubules are shaped by a specific class of curvature stabilizing proteins, the reticulons and DP1; however it is still unclear how the sheets are assembled. The ER is the cellular compartment responsible for secretory and membrane protein synthesis. The reducing conditions of ER lead to the intra/inter-chain formation of new disulphide bonds into polypeptides during protein folding assessed by enzymatic or spontaneous reactions. Moreover, ER represents the main intracellular calcium storage site and it plays an important role in calcium signaling that impacts many cellular processes. Accordingly, the maintenance of ER function represents an essential condition for the cell, and ER morphology constitutes an important prerogative of it. Furthermore, it is well known that ER undergoes prominent shape transitions during events such as cell division and differentiation. Thus, maintaining the correct ER structure is an essential feature for cellular physiology. Now, it is known that proper ER-associated proteins play a fundamental role in ER tubules formation. Among these ER-shaping proteins are the reticulons (RTN), which are acquiring a relevant position. In fact, beyond the structural role of reticulons, in very recent years new and deeper functional implications of these proteins are emerging in relation to their involvement in several cellular processes.     

The insulin receptor substrate 1 (IRS1) in intestinal epithelial differentiation and in colorectal cancer

Colorectal cancer (CRC) is associated with lifestyle factors that affect insulin/IGF signaling, of which the insulin receptor substrate 1 (IRS1) is a key transducer. We investigated expression, localization and pathologic correlations of IRS1 in cancer-uninvolved colonic epithelium, primary CRCs with paired liver metastases and in vitro polarizing Caco2 and HT29 cells. IRS1 mRNA and protein resulted higher, relative to paired mucosa, in adenomas of familial adenomatous polyposis patients and in CRCs that overexpressed c-MYC, ß-catenin, InsRß, and IGF1R. Analysis of IRS1 immunostaining in 24 cases of primary CRC with paired colonic epithelium and hepatic metastasis showed that staining intensity was significantly higher in metastases relative to both primary CRC (P<0.01) and colonic epithelium (P<0.01). Primary and metastatic CRCs, compared to colonic epithelium, contained significantly higher numbers of IRS1-positive cells (P = 0.013 and P = 0.014, respectively). Pathologic correlations in 163 primary CRCs revealed that diffuse IRS1 staining was associated with tumors combining differentiated phenotype and aggressive markers (high Ki67, p53, and ß-catenin). In Caco 2 IRS1 and InsR were maximally expressed after polarization, while IGF1R was highest in pre-polarized cells. No nuclear IRS1 was detected, while, with polarization, phosphorylated IRS1 (pIRS1) shifted from the lateral to the apical plasma membrane and was expressed in surface cells only. In HT29, that carry mutations constitutively activating survival signaling, IRS1 and IGF1R decreased with polarization, while pIRS1 localized in nuclear spots throughout the course. Overall, these data provide evidence that IRS1 is modulated according to CRC differentiation, and support a role of IRS1 in CRC progression and liver metastatization.     

Bovine fetal microchimerism in normal and embryo transfer pregnancies and its implications for biotechnology applications in cattle

Fetal cells and DNA have been detected in the maternal circulation during and after pregnancy in a few mammalian species. The incidence of similar microchimerism in cattle could have repercussion for the application of modern biotechnologies such as the transfer of transgenic embryos. To determine if feto-maternal leakage can occur in pregnant cows, we have analyzed maternal blood samples for the presence of fetal DNA during gestation and post-partum periods. Y chromosome-specific DNA was detected in up to 73% of blood samples from naturally mated heifers carrying conventional bull calves and a transgene-specific sequence in up to 50% of recipient cows carrying transgenic fetuses. These findings document for the first time that transplacental leakage of fetal DNA into the maternal circulation can occur in cattle despite the epitheliochorial placenta of ruminants, with potential implications for the utilization of recipient cows in the food chain.     

Antioxidant activity of oligomeric acylphloroglucinols from Myrtus communis L

The use of myrtle (Myrtus communis L.) as a culinary spice and as a flavoring agent for alcoholic beverages is widespread in the Mediterranean area, and especially in Sardinia. Myrtle contains unique oligomeric non-prenylated acylphloroglucinols, whose antioxidant activity was investigated in various systems. Both semimyrtucommulone (1) and myrtucommulone A (2) showed powerful antioxidant properties, protecting linoleic acid against free radical attack in simple in vitro systems, inhibiting its autoxidation and its FeCl3- and EDTA-mediated oxidation. While both compounds lacked pro-oxidant activity, semimyrtucommulone was more powerful than myrtucommulone A, and was further evaluated in rat liver homogenates for activity against lipid peroxidation induced by ferric-nitrilotriacetate, and in cell cultures for cytotoxicity and the inhibition of TBH- or FeCl3-induced oxidation. The results of these studies established semimyrtucommulone as a novel dietary antioxidant lead.     

Intracellular Ionic Variations in the Apoptotic Death of L Cells by Inhibitors of Cell Cycle Progression

Treatment with VP-16 (1-50 μM) or excess thymidine (5 mM) caused a block of L cells at different steps in their progression through the replicative cycle. The arrest was followed by an asynchronous process of cell death that conformed to criteria for apoptosis. Careful monitoring of this process in the whole cell population by flow cytometry showed a virtual absence of necrosis, an increase in side light scattering, followed by the occurrence of a population with subdiploid DNA fluorescence as well as reduced forward and side light seattering. The development of apoptosis required sufficient time and adequate ion gradients in the cells. By the combined use of flow cytometry and fluorescence microscopy data were obtained suggesting that (i) intracellular free Ca²⁺ and pH and/or their drug-induced alterations had to be adequately controlled for the apoptotic process to evolve; (ii) mitochondria were compromised earlier than the plasma membrane or lysosomes; and (iii) K⁺ extrusion possibly played a role in the final loss of cell volume. Interfering with the control of ion gradients and/or their changes in drug-treated cells resulted in cell death by necrosis.     

PKR activity is required for acute leukemic cell maintenance and growth: A role for PKR‐mediated phosphatase activity to regulate GSK‐3 phosphorylation

Recent reports demonstrate that PKR is constitutively active in a variety of tumors and is required for tumor maintenance and growth. Here we report acute leukemia cell lines contain elevated levels of p‐T451 PKR and PKR activity as compared to normal controls. Inhibition of PKR with a specific inhibitor, as well as overexpression of a dominant‐negative PKR, inhibited cell proliferation and induced cell death. Interestingly, PKR inhibition using the specific inhibitor resulted in a time‐dependent augmentation of AKT S473 and GSK‐3α S21 phosphorylation, which was confirmed in patient samples. Increased phosphorylation of AKT and GSK‐3α was not dependent on PI3K activity. PKR inhibition augmented levels of p‐S473 AKT and p‐S21/9 GSK‐3α/β in the presence of the PI3K inhibitor, LY294002, but was unable to augment GSK‐3α or β phosphorylation in the presence of the AKT inhibitor, A443654. Pre‐treatment with the PKR inhibitor blocked the ability of A443654 and LY294002 to promote phosphorylation of eIF2α, indicating the mechanism leading to AKT phosphorylation and activation did not require eIF2α phosphorylation. The effects of PKR inhibition on AKT and GSK‐3 phosphorylation were found to be, in part, PP2A‐dependent. These data indicate that, in acute leukemia cell lines, constitutive basal activity of PKR is required for leukemic cell homeostasis and growth and functions as a negative regulator of AKT, thereby increasing the pool of potentially active GSK‐3. J. Cell. Physiol. 221: 232–241, 2009. © 2009 Wiley‐Liss, Inc     

IL-15 augments antitumoral activity of an ErbB2/HER2 cancer vaccine targeted to professional antigen-presenting cells

Targeted delivery of tumor-associated antigens to professional antigen-presenting cells (APC) is being explored as a strategy to enhance the antitumoral activity of cancer vaccines. Here, we generated a cell-based system for continuous in vivo production of a CTLA-4-ErbB2 fusion protein as a therapeutic vaccine. The chimeric CTLA-4-ErbB2 molecule contains the extracellular domain of CTLA-4 for specific targeting to costimulatory B7 molecules on the surface of APC, genetically fused to residues 1-222 of human ErbB2 (HER2) as an antigenic determinant. In wild-type BALB/c mice, inoculation of syngeneic epithelial cells continuously secreting the CTLA-4-ErbB2 fusion vaccine in the vicinity of subcutaneously growing ErbB2-expressing renal cell carcinomas resulted in the rejection of established tumors, accompanied by the induction of ErbB2-specific antibodies and cytotoxic T cells. In contrast, treatment with CTLA-4-ErbB2 vaccine-secreting producer cells alone was insufficient to induce tumor rejection in ErbB2-transgenic WAP-Her-2 F1 mice, which are characterized by pronounced immunological tolerance to the human self-antigen. When CTLA-4-ErbB2 producer cells were modified to additionally secrete interleukin (IL)-15, antigen-specific antitumoral activity of the vaccine in WAP-Her-2 F1 mice was restored, documented by an increase in survival, and marked inhibition of the growth of established ErbB2-expressing, but not antigen-negative tumors. Our results demonstrate that continuous in vivo expression of an APC-targeted ErbB2 fusion protein results in antigen-specific immune responses and antitumoral activity in tumor-bearing hosts, which is augmented by the pleiotropic cytokine IL-15. This provides a rationale for further development of this approach for specific cancer immunotherapy.     

Amber force field implementation, molecular modelling study, synthesis and MMP-1/MMP-2 inhibition profile of (R)- and (S)-N-hydroxy-2-(N-isopropoxybiphenyl-4-ylsulfonamido)-3-methylbutanamides

Ab initio calculations (B3LYP/Lanl2DZ level of theory) were performed in this study to determine all the structural and catalytic zinc parameters required in order to study MMPs and their complexes with hydroxamate inhibitors by means of the AMBER force field. The parameters thus obtained were used in order to study the docking of some known MMPi (Batimastat, CGS 27023A and Prinomastat) and our previously described inhibitor a which had shown an inhibitory activity for MMP-1, and -2, with the aim of explaining the different selectivity. On this basis the two enantiomers (R)-b and (S)-b were designed and synthesized, as more potent MMP-2 inhibitors than our previously described inhibitor a. Between these two enantiomers the eutomer (R)-b proved to be 24.7 times and 15.3 times more potent than CGS 27023A and the parent compound a on MMP-2, maintaining a higher index of MMP-2/MMP-1 selectivity compared with CGS 27023A and the more potent inhibitor Prinomastat. The hydroxamate (R)-b can be considered as a progenitor of a new class of biphenylsulfonamido-based inhibitors that differ from compound a in the presence of an alkyl side chain on the C alpha atom, and show different potency and selectivity profiles on the two MMPs considered.