Variation in heat-shock proteins among species of desert fishes (Poeciliidae, Poeciliopsis)

Analysis of the heat-shock proteins (hsps) of six closely related species of Poeciliopsis demonstrated the existence of biochemical diversity in the hsp100, hsp70, hsp60, and hsp30 protein families among species. Each species expressed five to seven hsp70-related isoforms. Constitutive 70-kD isoforms were identical among species, but four different patterns of heat-inducible isoforms were seen in these six species. Members of the hsp70 family of molecular chaperones are included among the most highly conserved proteins known, and the possibility of variation in hsp70 among closely related species has rarely been addressed. The hsp30 family is known to be less conserved than the hsp70 family, and, as expected, the Poeciliopsis hsp30 patterns showed more variation. Most of the hsp30 isoforms characteristic of a particular species were unique to that species. Hsp100 and hsp60 were identical in five of the species, but alternate isoforms were found in P. monacha. The small size and limited geographical distribution of the P. monacha population have probably contributed to the uniqueness of the monacha pattern. Two of the species were shown to acquire thermotolerance, the ability to withstand normally lethal temperatures when subjected to a gradual temperature increase. Rapid-heating protocols commonly used to establish critical thermal maxima of organisms do not include this inducible component of thermoresistance and therefore do not adequately assess an organism's capacity to withstand thermal stress.      

Improving the clinical assessment of consciousness with advances in electrophysiological and neuroimaging techniques

In clinical neurology, a comprehensive understanding of consciousness has been regarded as an abstract concept - best left to philosophers. However, times are changing and the need to clinically assess consciousness is increasingly becoming a real-world, practical challenge. Current methods for evaluating altered levels of consciousness are highly reliant on either behavioural measures or anatomical imaging. While these methods have some utility, estimates of misdiagnosis are worrisome (as high as 43%) - clearly this is a major clinical problem. The solution must involve objective, physiologically based measures that do not rely on behaviour. This paper reviews recent advances in physiologically based measures that enable better evaluation of consciousness states (coma, vegetative state, minimally conscious state, and locked in syndrome). Based on the evidence to-date, electroencephalographic and neuroimaging based assessments of consciousness provide valuable information for evaluation of residual function, formation of differential diagnoses, and estimation of prognosis.     

A proteomic approach to study parathyroid glands

Parathyroid tumours are heterogeneous and in some cases the diagnosis may be difficult using histological features. In this study we used a two-dimensional electrophoresis (2D)/mass spectrometry (MS)-based approach to examine the global changes of parathyroid adenoma tissues protein profile compared to the parathyroid normal tissues. Validation of protein expression was performed by immunoblotting using specific antibodies. Ingenuity software was used to identify the biological processes to which these proteins belong and to construct a potential network. A total of 30 proteins were found to be differentially expressed, of which 22 resulted in being over-expressed. Proteins identified by 2D/MS/MS proteomics were classified into functional categories and a major change (≥ 2-fold) in terms of expression was found in proteins involved in response to biotic stimuli, cell organization and signal transduction. After Ingenuity analysis, 14-3-3 ζ/δ appears to be a key protein in the network of parathyroid adenoma, where it is linked to other proteins such as annexin A2, B box and SPRY domain-containing protein (BSPRY), p53 and epidermal growth factor receptor (EGFR). Our results suggest that the proteomic approach was able to differentiate the protein profiles of normal parathyroid and parathyroid adenoma and identify a panel of proteins which are differentially expressed. The functional role of these proteins in the network of intracellular pathways is discussed.     

Synthesis, structural characterization and biological studies of novel mixed ligand Ag(I) complexes with triphenylphosphine and aspirin or salicylic acid

Two new mixed ligand silver(I) complexes of formulae {[Ag(tpp)₃(asp)](dmf)} (1) (aspH = o-acetylsalicylic acid and tpp = triphenylphosphine) and [Ag(tpp)₂(o-Hbza)] (2) (o-HbzaH = o-hydroxy-benzoic acid) were synthesized and characterized by elemental analyses, spectroscopic techniques and X-ray crystallography at ambient conditions. Three phosphorus and one carboxylic oxygen atoms from a de-protonated aspirin ligand in complex 1 and two phosphorus and two carboxylic oxygen atoms from a chelating o-Hbza anion in complex 2 form a tetrahedral geometry around Ag(I) ions in both complexes.Complexes 1 and 2 and the silver(I) nitrate, tpp, aspNa and o-HbzaH were tested for their in vitro cytotoxic activity against leiomyosarcoma cells (LMS), human breast adenocarcinoma cells (MCF-7) and normal human fetal lung fibroblasts (MRC-5) cells with Thiazolyl Blue Tetrazolium Bromide (MTT) assay. For both cell lines 1 and 2 were found to be more active than cisplatin. Additionally, 1 and 2 exhibit lower activity on cell growth proliferation of MRC-5 cells. The type of LMS cell death caused by 1 and 2 were evaluated in vitro by use of flow cytometry assay. The results show that at concentrations of 1.5 and 1.9 μΜ of complex 1, 44.1% and 69.4%, respectively of LMS cells undergo programmed cell death (apoptosis). When LMS cells were treated with 1.6 and 2.3 μM of 2, LMS cells death was by 29.6% and 81.3%, respectively apoptotic. Finally, the influence of the complexes 1 and 2, upon the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically and theoretically studied. The binding of 1 and 2 towards LOX was also investigated by Saturation Transfer Difference (STD) ¹H NMR experiments.     

Unfolded Protein Response Is Required for the Definitive Endodermal Specification of Mouse Embryonic Stem Cells via Smad2 and β-Catenin Signaling

Tremendous efforts have been made to elucidate the molecular mechanisms that control the specification of definitive endoderm cell fate in gene knockout mouse models and ES cell (ESC) differentiation models. However, the impact of the unfolded protein response (UPR), because of the stress of the endoplasmic reticulum on endodermal specification, is not well addressed. We employed UPR-inducing agents, thapsigargin and tunicamycin, in vitro to induce endodermal differentiation of mouse ESCs. Apart from the endodermal specification of ESCs, Western blotting demonstrated the enhanced phosphorylation of Smad2 and nuclear translocation of β-catenin in ESC-derived cells. The inclusion of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid to the induction cultures prevented the differentiation of ESCs into definitive endodermal cells even when Activin A was supplemented. Also, the addition of the TGF-β inhibitor SB431542 and the Wnt/β-catenin antagonist IWP-2 negated the endodermal differentiation of ESCs mediated by thapsigargin and tunicamycin. These data suggest that the activation of the UPR appears to orchestrate the induction of the definitive endodermal cell fate of ESCs via both the Smad2 and β-catenin signaling pathways. The prospective regulatory machinery may be helpful for directing ESCs to differentiate into definitive endodermal cells for cellular therapy in the future.     

Mixed ligand-silver(I) complexes with anti-inflammatory agents which can bind to lipoxygenase and calf-thymus DNA, modulating their function and inducing apoptosis

A new mixed ligand-silver(I) complex of formula [Ag(tpp)(2)(p-Hbza)] (1) (p-HbzaH = 4-hydroxybenzoic acid and tpp = triphenylphosphine) has been synthesized and characterized by elemental analysis, mp, vibrational spectroscopy (mid- and far-FT-IR), (1)H-NMR, UV-vis, ESI-MS spectroscopic techniques and X-ray crystallography. Complex 1 and the already known mixed ligand-silver(I) complexes of formulae [Ag(tpp)(2)(salH)] (2) (salH(2) = salicylic acid or 2-hydroxy-benzoic acid) and {[Ag(tpp)(3)(asp)](dmf)} (3) (aspH = o-acetylsalicylic acid) were used for the clarification of the cytostatic activity mechanism. Thus, 1-3 were tested for their in vitro cytotoxic activity against leiomyosarcoma (LMS) and human breast adenocarcinoma (MCF-7) cells with trypan blue and Thiazolyl Blue Tetrazolium Bromide (MTT) assays. For both cell lines, complexes 1-3 were found to be more active than cisplatin. Due to the morphology of the LMS cells after incubation with 1-3, the type of cell death was evaluated by flow cytometry assay and DNA fragmentation. The results show that LMS cells undergo programmed cell death (apoptosis). DNA binding tests indicate the ability of complexes 1-3 to modify the activity of the cells. The binding constants of 1-3 towards calf-thymus DNA (CT-DNA) ((27.7 ± 7.9) × 10(4) (1), (13.3 ± 6.5) × 10(4) (2) and (11 ± 2.8) × 10(4) (3) M(-1)) indicate strong interaction. Moreover, the influence of complexes 1-3 on the catalytic peroxidation of linoleic acid to hydroperoxylinoleic acid by the enzyme lipoxygenase (LOX) was kinetically studied. Finally, docking studies on DNA binding interactions were performed.     

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin–Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.     

Renal Carcinogenesis After Uninephrectomy

Nephrectomized rats have widely been used to study chronic renal failure. Interestingly, renal cell carcinoma occurred in the remnant kidney after uninephrectomy (UNX). In this study, we probed insulin-like growth factor (IGF)-1 signaling pathway in UNX-induced renal cancer. Adult male Sprague-Dawley rats were randomized into two groups: UNX rats (n = 22) and sham-operated rats (n = 12). Rats were killed at 3, 7, and 10 months. After 7 months after nephrectomy, the UNX rats developed renal cell carcinoma with increased expression of proliferating cell nuclear antigen, and 68.2% (15/22) of the animals exhibited invasive carcinoma. Western blot demonstrated significant down-regulation of IGF binding protein 3 contrasting with the up-regulation of protein kinase Cζ and Akt/protein kinase B in the renal cancer tissues. These findings indicate a unique rat model of UNX-induced renal cancer associated with enhanced IGF-1 signaling pathway.