Decorin Core Protein (Decoron) Shape Complements Collagen Fibril Surface Structure and Mediates Its Binding

Decorin is the archetypal small leucine rich repeat proteoglycan of the vertebrate extracellular matrix (ECM). With its glycosaminoglycuronan chain, it is responsible for stabilizing inter-fibrillar organization. Type I collagen is the predominant member of the fibrillar collagen family, fulfilling both organizational and structural roles in animal ECMs. In this study, interactions between decoron (the decorin core protein) and binding sites in the d and e₁ bands of the type I collagen fibril were investigated through molecular modeling of their respective X-ray diffraction structures. Previously, it was proposed that a model-based, highly curved concave decoron interacts with a single collagen molecule, which would form extensive van der Waals contacts and give rise to strong non-specific binding. However, the large well-ordered aggregate that is the collagen fibril places significant restraints on modes of ligand binding and necessitates multi-collagen molecular contacts. We present here a relatively high-resolution model of the decoron-fibril collagen complex. We find that the respective crystal structures complement each other well, although it is the monomeric form of decoron that shows the most appropriate shape complementarity with the fibril surface and favorable calculated energies of interaction. One molecule of decoron interacts with four to six collagen molecules, and the binding specificity relies on a large number of hydrogen bonds and electrostatic interactions, primarily with the collagen motifs KXGDRGE and AKGDRGE (d and e₁ bands). This work helps us to understand collagen-decorin interactions and the molecular architecture of the fibrillar ECM in health and disease.     

Detergent extraction of the human alpha-beta interferon receptor: a soluble form capable of binding interferon.

After examining a variety of detergents we find that receptors for human alpha interferon can be solubilized, in active form, from plasma membranes of lymphoid cells using the detergent CHAPS. The complexes formed, in solution, with interferon are stable enough to be separated chromatographically.     

A validated antibody panel for the characterization of tau post-translational modifications

Background

Tau is a microtubule-binding protein, which is subject to various post-translational modifications (PTMs) including phosphorylation, methylation, acetylation, glycosylation, nitration, sumoylation and truncation. Aberrant PTMs such as hyperphosphorylation result in tau aggregation and the formation of neurofibrillary tangles, which are a hallmark of Alzheimer’s disease (AD). In order to study the importance of PTMs on tau function, antibodies raised against specific modification sites are widely used. However, quality control of these antibodies is lacking and their specificity for particular modifications is often unclear.

Methods

In this study, we first designed an online tool called ‘TauPTM’, which enables the visualization of PTMs and their interactions on human tau. Using TauPTM, we next searched for commercially available antibodies against tau PTMs and characterized their specificity by peptide array, immunoblotting, electrochemiluminescence ELISA and immunofluorescence technologies.

Results

We demonstrate that commercially available antibodies can show a significant lack of specificity, and PTM-specific antibodies in particular often recognize non-modified versions of the protein. In addition, detection may be hindered by other PTMs in close vicinity, complicating the interpretation of results. Finally, we compiled a panel of specific antibodies and show that they are useful to detect PTM-modified endogenous tau in hiPSC-derived neurons and mouse brains.

Conclusion

This study has created a platform to reliably and robustly detect changes in localization and abundance of post-translationally modified tau in health and disease. A web-based version of TauPTM is fully available at http://www.tauptm.org.

     

Cyclin D1 gene amplification in proliferating haemangioma

Cyclin D1 gene amplification has been reported to promote abnormal endothelial cell proliferation and angiogenesis; these findings constantly present in proliferating haemangiomas. The present study was conducted to evaluate cyclin D1 gene amplification by fluorescence in situ hybridization analysis in tissue biopsies of 22 proliferating haemangiomas from 20 infants. Two significant correlations of cyclin D1 gene amplification with the early onset and the duplication of proliferating haemangiomas have been observed. Moreover, a significant correlation (P≤0.05) has been found between the treatment parameters of proliferating haemangiomas with the amplified versus the normal cyclin D1 gene. Proliferating haemangiomas with the amplified cyclin D1 gene required more frequent flashlamp pulsed dye laser treatment sessions at the maximum dosimetry and more frequent intralesional steroid injections at the maximum dose/injection but treatment outcomes were limited. The more frequent post-treatment complications among proliferating haemangiomas with cyclin D1 gene amplification might be attributable not only to the associated more aggressive natural course, but also to the higher treatment parameters needed for effective treatment. Within the limitations of the present study, cyclin D1 gene amplification was seen for the first time in proliferating haemangiomas. We have found that the amplification of the cyclin D1 gene can predict the more aggressive natural course of proliferating haemangiomas and the limited outcome and higher incidence of complications after non–excision treatment modalities. The present findings reflect the possible usefulness of antisense cyclin D1 to improve the therapeutic outcome of proliferating haemangiomas.     

First generation 5-vinyl-3-pyridinecarbonitrile PKCθ inhibitors

A series of 5-vinyl-3-pyridinecarbonitriles were synthesized and evaluated as PKCθ inhibitors. The systematic optimization of 4-[(4-methyl-1H-indol-5-yl)amino]-5-[(E)-2-phenylvinyl]-3-pyridinecarbonitrile 3 resulted in the identification of compound 23e as a potent PKCθ inhibitor with good selectivity over PKCδ.     

The HIV-1 integrase α4-helix involved in LTR-DNA recognition is also a highly antigenic peptide element

Monoclonal antibodies (MAbas) constitute remarkable tools to analyze the relationship between the structure and the function of a protein. By immunizing a mouse with a 29mer peptide (K159) formed by residues 147 to 175 of the HIV-1 integrase (IN), we obtained a monoclonal antibody (MAba4) recognizing an epitope lying in the N-terminal portion of K159 (residues 147-166 of IN). The boundaries of the epitope were determined in ELISA assays using peptide truncation and amino acid substitutions. The epitope in K159 or as a free peptide (pep-a4) was mostly a random coil in solution, while in the CCD (catalytic core domain) crystal, the homologous segment displayed an amphipathic helix structure (α4-helix) at the protein surface. Despite this conformational difference, a strong antigenic crossreactivity was observed between pep-a4 and the protein segment, as well as K156, a stabilized analogue of pep-a4 constrained into helix by seven helicogenic mutations, most of them involving hydrophobic residues. We concluded that the epitope is freely accessible to the antibody inside the protein and that its recognition by the antibody is not influenced by the conformation of its backbone and the chemistry of amino acids submitted to helicogenic mutations. In contrast, the AA →Glu mutations of the hydrophilic residues Gln148, Lys156 and Lys159, known for their interactions with LTRs (long terminal repeats) and inhibitors (5CITEP, for instance), significantly impaired the binding of K156 to the antibody. Moreover, we found that in competition ELISAs, the processed and unprocessed LTR oligonucleotides interfered with the binding of MAba4 to IN and K156, confirming that the IN α4-helix uses common residues to interact with the DNA target and the MAba4 antibody. This also explains why, in our standard in vitro concerted integration assays, MAba4 strongly impaired the IN enzymatic activity.     

Lead acetate and arsenic trioxide induce instability of microsatellites at three different fragile sites (6q21, 9q32-9q33 and 15p14) within the genome of the rat

Human exposure to metals is of increasing concern due to the well-documented toxic and carcinogenic effects of metals and metal compounds, and the rising environmental levels due to industrial processes and pollution. It has been reported that metals can be genotoxic by several modes of action including generation of reactive oxygen species and inhibition of DNA repair. The aim of this study was to evaluate microsatellite instability (MSI) in three microsatellite loci (D6mit3, D9mit2 and D15Mgh1) located within three common fragile sites in the genome of the laboratory rat (6q21, 9q32-9q33 and 15p14) exposed to acute and chronic doses of a metal salt (lead acetate trihydrate) and a metalloid oxide (arsenic trioxide). In the acute and sub-chronic studies with the two compounds, MSI was observed in the three loci as deletions or additions of microsatellite repeats. The percentages of MSI were 36.4% and 42.1% for lead acetate and arsenic trioxide, respectively. Results of the present work indicate that the microsatellites located within fragile sites provide a convenient assay system to detect changes in DNA sequences resulting from exposure to genotoxic agents.     

Carnosol Induces ROS-Mediated Beclin1-Independent Autophagy and Apoptosis in Triple Negative Breast Cancer

Background

In this study we investigated the in vitro and in vivo anticancer effect of carnosol, a naturally occurring polyphenol, in triple negative breast cancer.

Results

We found that carnosol significantly inhibited the viability and colony growth induced G2 arrest in the triple negative MDA-MB-231. Blockade of the cell cycle was associated with increased p21/WAF1 expression and downregulation of p27. Interestingly, carnosol was found to induce beclin1-independent autophagy and apoptosis in MDA-MB-231 cells. The coexistence of both events, autophagy and apoptosis, was confirmed by electron micrography. Induction of autophagy was found to be an early event, detected within 3 h post-treatment, which subsequently led to apoptosis. Carnosol treatment also caused a dose-dependent increase in the levels of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2). Moreover, we show that carnosol induced DNA damage, reduced the mitochondrial potential and triggered the activation of the intrinsic and extrinsic apoptotic pathway. Furthermore, we found that carnosol induced a dose-dependent generation of reactive oxygen species (ROS) and inhibition of ROS by tiron, a ROS scavenger, blocked the induction of autophagy and apoptosis and attenuated DNA damage. To our knowledge, this is the first report to identify the induction of autophagy by carnosol.

Conclusion

In conclusion our findings provide strong evidence that carnosol may be an alternative therapeutic candidate against the aggressive form of breast cancer and hence deserves more exploration.     

Ten years primary succession on a newly created landfill at a lagoon of the Mediterranean Sea (Lake Burullus RAMSAR site)

This study was carried out on the transported bed soil dredged from the outlet of Lake Burullus to the Mediterranean Sea and deposited nearby, forming by this way new land that underwent a primary plant succession. The multi-methodological approach comprised floristic inventories, vegetation sampling and soil composition analyses of the study site in order to detect the crucial parameters controlling the plant resettlement on recently deposited soil as related to time, local micro-topography and substrate characteristics. Floristic composition was assessed for the first 10 years of primary succession (2001–2010) on 18 stands of the area, distributed on basement, slope stands and plateau of the landfill, respectively. Vegetation surveys were the basis of multivariate analyses of the vegetation and soil data using TWINSPAN, DCA and CCA. Relationships between the edaphic gradients, floristic composition and species diversity were assessed.