Distinct signaling pathways mediate stimulation of cell cycle progression and prevention of apoptotic cell death by estrogen in rat pituitary tumor PR1 cells

Estrogens control cell growth and viability in target cells via an interplay of genomic and extragenomic pathways not yet elucidated. Here, we show evidence that cell proliferation and survival are differentially regulated by estrogen in rat pituitary tumor PR1 cells. Pico- to femtomolar concentrations of 17beta-estradiol (E2) are sufficient to foster PR1 cell proliferation, whereas nanomolar concentrations of the same are needed to prevent cell death that occurs at a high rate in these cells in the absence of hormone. Activation of endogenous (PRL) or transfected estrogen-responsive genes occurs at the same, higher concentrations of E2 required to promote cell survival, whereas stimulation of cyclin D3 expression and DNA synthesis occur at lower E2 concentrations. Similarly, the pure antiestrogen ICI 182,780 inhibits estrogen response element-dependent trans-activation and cell death more effectively than cyclin-cdk activity, G1-S transition, or DNA synthesis rate. In antiestrogen-treated and/or estrogen-deprived cells, death is due predominantly to apoptosis. Estrogen-induced cell survival, but not E2-dependent cell cycle progression, can be prevented by an inhibitor of c-Src kinase or by blockade of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathway. These data indicate the coexistence of two distinguishable estrogen signaling pathways in PR1 cells, characterized by different functions and sensitivity to hormones and antihormones.     

Chylomicron remnant induction of lipid accumulation in J774 macrophages is associated with up-regulation of triacylglycerol synthesis which is not dependent on oxidation of the particles

The influence of chylomicron remnants on lipid accumulation and synthesis and the activity and/or expression of mRNA for some of the key enzymes involved was investigated in the murine macrophage cell line J774. The effects of varying the polyunsaturated fatty acid (PUFA) composition and oxidation state of the remnants were also examined. Chylomicron remnants derived from corn oil (rich in n-6 PUFA) or fish oil (rich in n-3 PUFA) were prepared in vivo and oxidised by incubation with CuSO(4). The native and oxidised remnants caused a marked rise in intracellular triacylglycerol levels, but the rise induced by corn oil remnants (four- to sixfold) was greater than that observed with fish oil remnants (<2-fold). Triacylglycerol synthesis, as measured by the incorporation of [3H]oleate and [3H]glycerol into cellular triacylglycerol, was increased by all four remnant types tested, and corn oil remnants had a significantly greater effect than fish oil remnants. Oxidation of the remnants did not affect the results obtained. Although the incorporation of [3H]oleate into cholesteryl ester by the cells was not significantly changed by any of the four types of remnants tested, the activity and expression of mRNA for acyl Co-enzyme A: cholesterol acyltransferase (ACAT) was increased by corn oil, but not by fish or oxidised corn, remnants. Neutral cholesteryl ester hydrolase (nCEH) activity, however, was also raised by corn oil remnants. These studies indicate that chylomicron remnants induce the accumulation of triacylglycerol in J774 macrophages, and that increased synthesis of triacylglycerol plays a major role in this process. Furthermore, they demonstrate that these effects are enhanced when the remnants are enriched in n-6 PUFA as compared with n-3 PUFA, but not after oxidation of the particles, suggesting that the fatty acid composition of chylomicron remnants may be more important than their oxidation state in their ability to induce foam cell formation.     

Noladin ether, a putative novel endocannabinoid: inactivation mechanisms and a sensitive method for its quantification in rat tissues

The occurrence of the novel proposed endocannabinoid, noladin ether (2-arachidonyl glyceryl ether, 2-AGE) in various rat organs and brain regions, and its inactivation by intact C6 glioma cells, were studied. 2-AGE was measured by isotope dilution liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry, with a detection limit of 100 fmol. A compound with the same mass and chromatographic/chemical properties as 2-AGE was found in whole brain, with the highest amounts in the thalamus and hippocampus. Synthetic [(3)H]2-AGE was inactivated by intact rat C6 glioma cells by a time- and temperature-dependent process consisting of cellular uptake and partial incorporation into phospholipids. Further data suggested that 2-AGE is taken up by cells via the anandamide/2-arachidonoyl glycerol (2-AG) membrane transporter(s), and biosynthesized in a different way as compared to 2-AG.     

Population shift vs induced fit: the case of bovine seminal ribonuclease swapping dimer

Bovine seminal ribonuclease (BS-RNase) is a unique member of the pancreatic-like ribonuclease superfamily. This enzyme exists as two conformational isomers with distinctive biological properties. The structure of the major isomer is characterized by the swapping of the N-terminal segment (MxM BS-RNase). In this article, the crystal structures of the ligand-free MxM BS-RNase and its complex with 2'-deoxycitidylyl(3',5')-2'-deoxyadenosine derived from isomorphous crystals have been refined. Interestingly, the comparison between this novel ligand-free form and the previously published sulfate-bound structure reveals significant differences. In particular, the ligand-free MxM BS-RNase is closer to the structure of MxM BS-RNase productive complexes than to the sulfate-bound form. These results reveal that MxM BS-RNase presents a remarkable flexibility, despite the structural constraints of the interchain disulfide bridges and the swapping of the N-terminal helices. These findings have important implications to the ligand binding mechanism of MxM BS-RNase. Indeed, a population shift rather than a substrate-induced conformational transition may occur in the MxM BS-RNase ligand binding process.     

Influence of conformational flexibility on biological activity in cyclic astin analogues

The astins, a family of natural antitumor cyclopeptides, from the roots of Aster tataricus, consist of a 16-membered ring system containing uncoded amino acid residues. The backbone conformation, with a cis-3,4-dichlorinated proline residue, plays an important role in antineoplastic activity. The acyclic astins, on the other hand, do not show antitumor activity, suggesting that the cyclic nature of astins may be a key role in their biological properties. Although the antineoplastic activity of natural astins has been screened in vitro and in vivo, the mechanism of action has never been investigated. With the aim at elucidating the influence of conformational flexibility on biological activity, we have designed and synthesized several astin analogues containing either Aib and the nonproteinogenic Abu and (S)beta3-hPhe residues, able to modify the peptide backbone structure, or the peptide bond surrogate -SO2-NH-. Tested for their antitumor effect, our astin-related cyclopeptides are able to inhibit the growth of tumor cell lines, while the acyclic astins are inefficacious. The present work reports on the structure-activity study of a selected synthetic cyclotetrapeptide corresponding to the sequence c[Thr-Aib-(S)beta3-hPhePsi(CH2-SO2-NH)-Abu], synthesized by classical methods and characterized conformationally by two-dimensional NMR and molecular dynamics analyses.     

Novel isoforms of Pru av 1 with diverging immunoglobulin E binding properties identified by a synergistic combination of molecular biology and proteomics

Birch pollen-related food allergies are mainly associated to Bet v 1. Little is known about isoforms of Bet v 1 homologous in fruit of the Rosaceae family. We attempted to identify novel isoforms of Pru av 1, the major cherry allergen, at the cDNA and the protein level by a combination of molecular biology and proteomic tools. A cDNA library was screened with patients immunoglobulin E (IgE) and a specific hybridization probe. Edman sequencing, mass spectrometry (MS), and MS/MS were performed after detecting Pru av 1 on 2-D maps by immunoblotting using patients IgE and a monoclonal antibody. Partial amino acid sequences were completed with a polymerase chain reaction (PCR) strategy. The IgE-binding properties of the Pru av 1 spots were analyzed by 2-D blot inhibition. cDNA library analysis revealed a novel Pru av 1 isoform. MS and N-terminal sequencing confirmed the cDNA sequences at the protein level. A series of spots were confirmed as the already known Pru av 1. One spot, exclusively detected with patients sera, was identified as the novel isoform. A partial amino acid sequence detected with MS/MS was completed by PCR-cloning. The 2-D blot inhibition revealed epitope differences between the novel isoform and the previously published Pru av 1. Our data demonstrate that a synergistic combination of molecular biology and proteomics represents a powerful tool for reliable and comprehensive identification of allergen isoforms and variants. The newly identified isoform showed diverging IgE-binding properties and may be relevant for the diagnosis or therapy of cherry allergy.     

A new pentastomid from the black vulture

This article describes a new pentastomid species from the abdominal air sacs of a black vulture (Aegypius monachus Linnaeus, 1766) from central Spain. The parasite's morphological characteristics (as shown by light and scanning electron microscopy) suggest that it should be classified in the new genus. It is the third pentastomid species described in birds and the first for the Accipitridae. The mouth is almost terminal, there are 2 pairs of hooks behind the mouth, and the genital pore is immediately posterior to these structures, placing the new species within the Cephalobaenida. The anterior and posterior hooks are similar in size and are flanked by parapodial lob. Cuticular tubercles are absent, false annulations can be seen, and the parasite's eggs have 2 layers.     

Ca(V)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism

Ca(V)1.2, the cardiac L-type calcium channel, is important for excitation and contraction of the heart. Its role in other tissues is unclear. Here we present Timothy syndrome, a novel disorder characterized by multiorgan dysfunction including lethal arrhythmias, webbing of fingers and toes, congenital heart disease, immune deficiency, intermittent hypoglycemia, cognitive abnormalities, and autism. In every case, Timothy syndrome results from the identical, de novo Ca(V)1.2 missense mutation G406R. Ca(V)1.2 is expressed in all affected tissues. Functional expression reveals that G406R produces maintained inward Ca(2+) currents by causing nearly complete loss of voltage-dependent channel inactivation. This likely induces intracellular Ca(2+) overload in multiple cell types. In the heart, prolonged Ca(2+) current delays cardiomyocyte repolarization and increases risk of arrhythmia, the ultimate cause of death in this disorder. These discoveries establish the importance of Ca(V)1.2 in human physiology and development and implicate Ca(2+) signaling in autism.     

Comparison of the predicted in vivo behaviour of the Sn(II)-APDDMP complex and the results as studied in a rodent model

In a quest for more effective radiopharmaceuticals for pain palliation of metastatic bone cancer, this paper relates results obtained with ((117m)Sn labelled) Sn(II) complexed to the bone seeking bisphosphonate, N,N-dimethylenephosphonate-1-hydroxy-3-aminopropylidenediphosphonate (APDDMP). APDDMP is synthesised from the known bone cancer pain palliation agent 1-hydroxy-3-aminopropylidenediphosphonate (APD, Pamindronate). This work is performed to utilise the idea that the low bone marrow radio toxicity of (117m)Sn could afford a highly effective radiopharmaceutical in pain palliation but also in the curative treatment of bone metastasis. Complex-formation constants of APDDMP with the important blood plasma metal-ions, Ca(2+), Mg(2+), Zn(2+) as well as the added metal ion, Sn(2+) were measured by glass electrode potentiometry at 25 degrees C and I = 150 mM. Blood plasma models were constructed using the computer code ECCLES and the results compared with those gathered from tests on a rodent model. The ((117m)Sn-labelled) Sn(II)-APDDMP complex was found to have only some liver and bone uptake although a high trabecular to normal bone ratio was recorded. From the blood plasma model this was shown to be primarily due to the high affinity of APDDMP for Ca(II) causing some of the Sn(II)-APDDMP complex to dissociate. High kidney uptake and excretion as well as high bladder uptake was recorded which was shown to be due to the dissociation of the Sn(II)-APDDMP complex in blood plasma. Animal model observations could be explained by the blood plasma modelling.