Lipofuscin-mediated photic stress inhibits phagocytic activity of ARPE-19 cells; effect of donors’ age and antioxidants

The risk of chronic oxidative stress in the retinal pigment epithelium (RPE) increases with age due to accumulation of the photoreactive age pigment lipofuscin (LFG). Here, we asked whether sublethal and weakly lethal photic stress, induced by irradiation of ARPE-19 cells containing phagocytised LFG, affected the cell specific phagocytic activity, which is critically important for proper functioning and survival of the retina, and if natural antioxidants could modify the observed outcomes. ARPE-19 cells preloaded with LFG isolated from human donors of different age or containing LFG enriched with zeaxanthin and α-tocopherol (LFG-A), were irradiated with blue light. Phagocytosis of fluorescein-5-isothiocyanate (FITC)-labelled photoreceptor outer segments was determined by flow cytometry. Photoreactivity of LFG and LFG-A was analysed by measuring photoconsumption of oxygen and photogeneration of singlet oxygen mediated by the granules. LFG-mediated photic stress in ARPE-19 cells induced significant inhibition of their specific phagocytosis. The inhibitory effect increased with age of LFG donors and was reduced by enrichment of the granules with antioxidants. Oxygen consumption and generation of singlet oxygen induced by the photoexcited LFG increased with donor’s age and was partially quenched by antioxidants. Although the phototoxic potential of lipofuscin increased with age, natural antioxidants reduced photoreactivity of LFG and their efficiency to induce oxidative stress. This study has demonstrated, for the first time, that mild oxidative stress, mediated by the age pigment lipofuscin, impairs specific phagocytic activity of RPE, and that natural antioxidants can protect this important cellular function by reducing lipofuscin photoreactivity.     

Gene structure of the pregnancy-associated glycoprotein-like (PAG-L) in the Eurasian beaver (<Emphasis Type="Italic">Castor fiber</Emphasis> L.)

The pregnancy-associated glycoprotein-like family (PAG-L) is a large group of chorionic products, expressed in the pre-placental trophoblast and later in the post-implantational chorionic epithelium, and are involved in proper placenta development and embryo-maternal interaction in eutherians. This study describes identification of the PAG-L family in the genome of the Eurasian beaver (Castor fiber L.), named CfPAG-L. We identified 7657 bp of the CfPAG-L gDNA sequence (Acc. No. KX377932), encompassing nine exons (1–9) and eight introns (A–H). The length of the CfPAG-L exons (59–200 bp) was equivalently similar to the only known counterparts of bPAG1, bPAG2, and pPAG2. The length of the CfPAG-L introns ranged 288–1937 bp and was completely different from previously known PAG introns. The exonic CfPAG-L regions revealed 50.3–72.9% homology with equivalent segments of bPAG1 and pPAG2 structure. The intronic CfPAG-L regions alignments revealed a lack of homology. Within the entire CfPAG-L gene, 31 potential single nucleotide variants (SNV: 7 transversions and 24 transitions) were predicted. The identified exonic polymorphic loci did not affect the amino acid sequence of the CfPAG-L polypeptide precursor. This is the first report describing the CfPAG-L gene sequence, structural organization, and SNVs in the Eurasian beaver, one of the largest rodents.     

Application of physical and genetic map of Rhizobium leguminosarum bv. trifolii TA1 to comparison of three closely related rhizobial genomes

A combined physical and genetic map of Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) genome was constructed and used in comparison of chromosomal organization with the closely related R. leguminosarum bv. viciae 3841 (Rlv) and Rhizobium etli CNF42 (Rhe). This approach allowed evaluation of chromosome and genome plasticity and provided important insights into R. leguminosarum lineage diversity. MssI, SmiI, PacI, and I-CeuI restriction endonucleases were chosen for the analysis, generating fragments with suitable size distributions for RtTA1 genome mapping. The fragments were assembled into a physical map using a combination of complementary methods, including multiple and partial digests of genomic DNA, hybridization with homologous gene probes, and cross-Southern hybridization. About 100 genetic markers were located on the RtTA1 restriction map. Comparison of genetic maps of RtTA1, Rlv, and Rhe revealed extensive chromosomal colinearity despite differences in the physical maps. The comparison provides bases for comprehensive analysis of the evolution of R. leguminosarum genome, indicating that, at least on the chromosomal level, no major rearrangements had occurred after the evolutionary divergence of R. leguminosarum biovars. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Integrating the discovery pipeline for novel compounds targeting ion channels

Many ion channels are attractive therapeutic targets for the treatment of neurological or cardiovascular diseases; there is a continuous need for selective channel antagonists and/or agonists. Recently, several technologies have been developed that make exploration of the enormous diversity of venom-derived peptidic toxins more feasible. Integration of exogenomics with synthetic methods such as diselenide or fluorous bridges, backbone spacers, and N-to-C cyclization provides an emerging technology that promises to accelerate discovery and development of natural products based compounds. These drug-discovery efforts are complemented by novel approaches to modulate the activities of ion channels and receptors. Taken together, these technologies will advance our knowledge and understanding of ion channels and will accelerate their expansion as targets for first-in-class therapeutics.     

Fresh-wet storage accelerates aortic homograft calcification

The studies were focused on the influence of the fresh-wet storage procedure on aortic valve homograft (AVH) mineralization. Thirty-four aortic valves excised at autopsy were investigated. The cusps were divided into halves, the first one was used as a control while the second half was stored in Morgan, Morton, and Parker’s Medium 199 supplemented with an antibiotic mixture at 4°C for 28 days. The elemental compositions of the samples were determined by the energy dispersive X-ray fluorescence method. The Ca/P ratio and Ca concentration was used as markers of the mineralization development. It was found that the AVH mineralization was accelerated by the applied fresh-wet storage procedure. The aggravation of the AVH mineralization was correlated with the Ca content before storage especially in case of old donors. For donors older than 40 years an increase of Ca concentrations by approximately 40% was observed. To limit AVH mineralization due to fresh-wet storage, age of donors should not exceed ∼40 years. Another method to reduce AVH mineralization relies on a modification of the medium commonly applied in the fresh-wet storage procedure, and research are ongoing.     

Forced expression of MMP9 rescues the loss of angiogenesis and abrogates metastasis of pancreatic tumors triggered by the absence of host SPARC

Pancreatic adenocarcinoma is characterized by desmoplasia, local invasion, and metastasis. These features are regulated in part by MMP9 and SPARC. To explore the interaction of SPARC and MMP9 in cancer, we first established orthotopic pancreatic tumors in SPARC-null and wild-type mice with the murine pancreatic adenocarcinoma cell line, PAN02. MMP9 expression was higher in tumors from wild-type compared to SPARC-null mice. Coincident with lower MMP9 expression, tumors grown in SPARC-null mice were significantly larger, had decreased ECM deposition and reduced microvessel density compared to wild-type controls. In addition, metastasis was enhanced in the absence of host SPARC. Therefore, we next analyzed the orthotopic tumor growth of PAN02 cells transduced with MMP9 or a control empty vector. Forced expression of MMP9 by the PAN02 cells resulted in larger tumors in both wild-type and SPARC-null animals compared to empty vector controls and further diminished ECM deposition. Importantly, forced expression of MMP9 within the tumor reversed the decrease in angiogenesis and abrogated the metastatic potential displayed by control tumors grown in SPARC-null mice. Finally, contrary to the in vivo results, MMP9 increased cell migration in vitro, which was blocked by the addition of SPARC. These results suggest that SPARC and MMP9 interact to regulate many stages of tumor progression including ECM deposition, angiogenesis and metastasis.     

Hierarchically structured scleractinian coral biocrystals

Microscopic (AFM and FESEM) observations show that scleractinian coral biomineral fibers in extant Desmophyllum and Favia, and fossil Jurassic Isastrea are composed of nanocrystalline grains of about 30-100 nm in size. In contrast to these findings, SR diffraction data on the same coral materials exhibit narrow Bragg peaks suggesting much larger crystallite size. These seemingly contradicting results of microscopic and diffraction studies are reconciled within a new, minute-scale model of scleractinian biomineral fibers. In this model, nanocrystalline aragonite units are interconnected by mineral bridges and form aggregates usually larger than 200 nm. Most likely, the size of the aggregates is resulting from physiological biomineralization cycles that control cellular secretion of ions and biopolymeric species. Intercalation of biopolymers into crystal lattice may influence consistently several structural parameters of the scleractinian coral bio-aragonite in all studied samples: (i) the lattice parameters and internal strains of the bio-aragonite are larger than in mineral aragonite, (ii) lattice parameter elongations and internal strains reveal directional anisotropy with respect to crystallographic axes.     

Starmaker exhibits properties of an intrinsically disordered protein

Fish otoliths composed of calcium carbonate and an organic matrix play a primary role in gravity sensing and the perception of sound. Starmaker (Stm) was the first protein found to be capable of influencing the process of biomineralization of otoliths. Stm dictates the shape, size, and selection of calcium carbonate polymorphs in a concentration-dependent manner. To facilitate exploration of the molecular basis of Stm function, we have developed and optimized a protocol for efficient expression and purification of the homogeneous nontagged Stm. The homogeneous nontagged Stm corresponds to its functional form, which is devoid of a signal peptide. A comprehensive biochemical and biophysical analysis of recombinant Stm, along with in silico examinations, indicate for the first time that Stm exhibits the properties of intrinsically disordered proteins. The functional significance of Stm having intrinsically disordered protein properties and its possible role in controlling the formation of otoliths is discussed.