Structure and expression of genes involved in transport and storage of iron in red-blooded and hemoglobin-less antarctic notothenioids

Antarctic notothenioids are characterized by a drastic reduction of the hemoglobin content, a condition that reaches its extreme in icefish that, following a gene deletion event, are completely devoid of hemoglobin. To answer the question on what type of adaptive changes occurred in icefish to prevent accumulation of potentially dangerous ferrous iron, we investigated the genes of four proteins known to play a key role in iron metabolism. For this purpose, we cloned and sequenced the cDNAs encoding ceruloplasmin, transferrin, ferritin and divalent metal transporter 1. While the inferred amino acid sequences of transferrin from different Antarctic fish species showed a high level of similarity with the homologous proteins from other species, ceruloplasmin sequence featured amino acid substitutions affecting a copper binding site. Another peculiarity was the presence in subunit H of the icefish ferritin of the two sets of sites involved in iron oxidation and iron mineralization, which in mammals are located on two distinct ferritin subunits. Significant differences in the expression levels of the four genes were found between hemoglobinless and red-blooded notothenioids. An increased expression of ceruloplasmin mRNA in icefish was interpreted as a compensatory mechanism to prevent accumulation of ferrous iron in hemoglobinless fish. In icefish, the amounts of ferritin H-chain mRNA measured in liver, blood and head kidney were lower than in the same organs of the red-blooded fish. In the spleen of both fishes, the expression levels of ferritin H-chain were significantly lower than in the spleen of a "pink-blooded" notothenioid with an intermediate hemoglobin content. Finally, the amount of divalent metal transporter mRNA measured in the head-kidney was lower in the icefish than in the same organ of its red-blooded counterpart. These results indicate that the loss of hemoglobin in icefish is accompanied by remodulation of the iron metabolism.     

Absorption flattening as one cause of distortion of circular dichroism spectra of Delta-RuPhen3 . H2TPPS complex

To extend the model that explains why and how much absorption flattening (AF) influences circular dichroism (CD) signals, we have investigated the interesting case of exciton CD in the Soret region of a noncovalent complex formed by (Delta-RuPhen(3))(2+) and the tetraanionic porphyrin H(2)TPPS. Different concentrations have been studied by using an AF emulator and spectra simulation. The CD spectra of this compound occasionally show distortions in the solution sampling mode with the increase of concentration; the inhomogeneous distribution in the cell volume is due to aggregation and is the source of the AF effect. On the basis of these results, we conclude that AF is an important cause of distortions in CD spectra for Delta-RuPhen(3) . H(2)TPPS complexes and might affect the CD bands of other aggregated systems as well.     

Platelet‐Rich Plasma Increases Growth and Motility of Adipose Tissue‐Derived Mesenchymal Stem Cells and Controls Adipocyte Secretory Function

Adipose tissue‐derived mesenchymal stem cells (Ad‐MSC) and platelet derivatives have been used alone or in combination to achieve regeneration of injured tissues. We have tested the effect of platelet‐rich plasma (PRP) on Ad‐MSC and adipocyte function. PRP increased Ad‐MSC viability, proliferation rate and G1‐S cell cycle progression, by at least 7‐, 2‐, and 2.2‐fold, respectively, and reduced caspase 3 cleavage. Higher PRP concentrations or PRPs derived from individuals with higher platelet counts were more effective in increasing Ad‐MSC growth. PRP also accelerated cell migration by at least 1.5‐fold. However, PRP did not significantly affect mature adipocyte viability, differentiation and expression levels of PPAR‐γ and AP‐2 mRNAs, while it increased leptin production by 3.5‐fold. Interestingly, PRP treatment of mature adipocytes also enhanced the release of Interleukin (IL)‐6, IL‐8, IL‐10, Interferon‐γ, and Vascular Endothelial Growth Factor. Thus, data are consistent with a stimulatory effect of platelet derivatives on Ad‐MSC growth and motility. Moreover, PRP did not reduce mature adipocyte survival and increased the release of pro‐angiogenic factors, which may facilitate tissue regeneration processes. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc. J. Cell. Biochem. 116: 2408–2418, 2015. © 2015 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.     

Dual target strategy: combining distinct non‐dopaminergic treatments reduces neuronal cell loss and synergistically modulates l‐DOPA‐induced rotational behavior in a rodent model of Parkinson's disease

The glutamate metabotropic receptor 5 (mGluR5) and the adenosine A_2A receptor (A_2AR) represent major non‐dopaminergic therapeutic targets in Parkinson's disease (PD) to improve motor symptoms and slow down/revert disease progression. The 6‐hydroxydopamine rat model of PD was used to determine/compare the neuroprotective and behavioral impacts of single and combined administration of one mGluR5 antagonist, 2‐methyl‐6‐(phenylethynyl)pyridine (MPEP), and two A_2AR antagonists, (E)‐phosphoric acid mono‐[3‐[8‐[2‐(3‐methoxyphenyl)vinyl]‐7‐methyl‐2,6‐dioxo‐1‐prop‐2‐ynyl‐1,2,6,7‐tetrahydropurin‐3‐yl]propyl] (MSX‐3) and 8‐ethoxy‐9‐ethyladenine (ANR 94). Chronic treatment with MPEP or MSX‐3 alone, but not with ANR 94, reduced the toxin‐induced loss of dopaminergic neurons in the substantia nigra pars compacta. Combining MSX‐3 and MPEP further improved the neuroprotective effect of either antagonists. At the behavioral level, ANR 94 and MSX‐3 given alone significantly potentiated l ‐DOPA‐induced turning behavior. Combination of either A_2AR antagonists with MPEP synergistically increased L‐DOPA‐induced turning. This effect was dose‐dependent and required subthreshold drug concentration, which per se had no motor stimulating effect. Our findings suggest that co‐treatment with A_2AR and mGluR5 antagonists provides better therapeutic benefits than those produced by either drug alone. Our study sheds some light on the efficacy and advantages of combined non‐dopaminergic PD treatment using low drug concentration and establishes the basis for in‐depth studies to identify optimal doses at which these drugs reach highest efficacy.

     

Epidermal micromorphology in Dioon: did volcanism constrain Dioon evolution?

The genus Dioon occupies an important phylogenetic position as sister to the other Zamiaceae. However, its epidermal morphology is still poorly known. We employed scanning and transmission electron microscopy, confocal microscopy and light microscopy to examine the epidermal and cuticular morphology of 12 of the 14 currently recognized species of Dioon, examining cultivated plants and herbarium specimens. Epidermal characters separate Dioon quite clearly from the other genera of the cycads. Within the genus, the major subgroups can be distinguished by their epidermal morphology. Some characters, such as the degree of stomatal protection, reflect ecological specializations in species within the subgroups. Several epidermal characters of the crown group Dioon seem to be absent in the Cenozoic fossils usually associated with the genus, which have been used as calibrations in many recent molecular dating analyses. The presence of similar characters in a Mesozoic fossil adapted to volcanic stress offers a new key in the interpretation of Dioon evolution. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 179 , 236–254.     

Protocatechuic acid induces antioxidant/detoxifying enzyme expression through JNK-mediated Nrf2 activation in murine macrophages

Protocatechuic acid (PCA) is a main metabolite of anthocyanins, whose daily intake is much higher than that of other polyphenols. PCA has biological effects, e.g., it induces the antioxidant/detoxifying enzyme gene expression. This study was aimed at defining the molecular mechanism responsible for PCA-induced over-expression of glutathione (GSH) peroxidase (GPx) and GSH reductase (GR) in J774 A.1 macrophages. New evidence is provided that PCA increases GPx and GR expression by inducing C-JUN NH₂-terminal kinase (JNK)-mediated phosphorylation of Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). RNA and proteins were extracted from cells treated with PCA (25 μM) for different time points. Quantitative real-time polymerase chain reaction and immunoblotting analyses showed a rapid increase in mRNA (>60%) and protein (>50%) for both the enzymes. This was preceded by the up-regulation of Nrf2, in terms of mRNA and protein, and by its significant activation as assessed by increased Nrf2 phosphorylation and nuclear translocation (+60%). By using specific kinase inhibitors and detecting the activated form, we showed that JNK was the main upstream kinase responsible for Nrf2 activation. Convincing evidence is provided of a causal link between PCA-induced Nrf2 activation and increased enzyme expression. By silencing Nrf2 and using a JNK inhibitor, enzyme enhancement was counteracted. Finally, with the ChIP assay, we demonstrated that PCA-activated Nrf2 specifically bound ARE sequences in enzyme gene promoters. Our study demonstrates for the first time that PCA improves the macrophage endogenous antioxidant potential by a mechanism in which JNK-mediated Nrf2 activation plays an essential role. This knowledge could contribute to novel diet-based approaches aimed at counteracting oxidative injury by reinforcing endogenous defences.     

Synthesis of novel antimitotic agents based on 2-amino-3-aroyl-5-(hetero)arylethynyl thiophene derivatives

Microtubules are dynamic structures that play a crucial role in cellular division and are recognized as an important target for cancer therapy. In search of new compounds with strong antiproliferative activity and simple molecular structure, a new series of 2-amino-3-(3′,4′,5′-trimethoxybenzoyl)-5-(hetero)aryl ethynyl thiophene derivatives was prepared by the Sonogashira coupling reaction of the corresponding 5-bromothiophenes with several (hetero)aryl acetylenes. When these compounds were analyzed in vitro for their inhibition of cell proliferation, the 2- and 3-thiophenyl acetylene derivatives were the most powerful compounds, both of which exerted cytostatic effects at submicromolar concentrations. In contrast, the presence of a more flexible ethyl chain between the (hetero)aryl and the 5-position of the thiophene ring resulted in significant reduction in activity relative to the 5-(hetero)aryl acetylene substituted derivatives. The effects of a selected series of compounds on cell cycle progression correlated well with their strong antiproliferative activity and inhibition of tubulin polymerization. We found that the antiproliferative effects of the most active compounds were associated with increase of the proportion of cells in the G₂/M and sub-G₁ phases of the cell cycle.