Differentiation of the endemic Greek genus <Emphasis Type="Italic">Hymenonema</Emphasis> and its relatives of subtribe Scolyminae (Compositae,Cichorieae) based on a multilocus species tree reconstruction

Hymenonema (Compositae, tribe Cichorieae) together with the genera Catananche, Gundelia, and Scolymus forms the subtribe Scolyminae. It is endemic to Greece and consists of two species, Hymenonema laconicum and Hymenonema graecum, which occur in the south Peloponnisos and central Aegean area, respectively. The present contribution aims at a phylogenetic reconstruction of evolutionary relationships among the 12 species of the subtribe, focusing on the temporal and spatial framework for its evolution. The phylogenetic relationships among the members of Scolyminae were inferred from molecular data based on the multi-copy region of the nrDNA internal transcribed spacers ITS1 and ITS2, two intergenic spacers of the cpDNA (trnL-trnF, rpl32-trnL), and one single-copy nuclear region (D10). The gene trees were reconstructed using Bayesian phylogenetic methods. All gene trees support the monophyly of Hymenonema and the sister-group relationship with the genus Scolymus. The further sister-group relationship of this group (Hymenonema–Scolymus) with Catananche is also supported by nrDNA and cpDNA analyses. Finally, a species tree (inferred in a Bayesian coalescent framework) was reconstructed and dates the divergence time between the two Hymenonema species to the Pleistocene (around 1.3 Ma ago). Maximum likelihood-based biogeographical reconstructions suggest a Miocene (pre-Messinian) differentiation of the subtribe on the northern Tethyan platform, followed by Miocene/Pliocene dispersal events to the western Mediterranean and North-African platforms and final, small-scale vicariance events within the genera in the Pleistocene.     

Mechanisms of divergent effects of activated peroxisome proliferator-activated receptor-γ on mitochondrial citrate carrier expression in 3T3-L1 fibroblasts and mature adipocytes

The citrate carrier (CIC), a nuclear-encoded protein located in the mitochondrial inner membrane, plays an important metabolic role in the transport of acetyl-CoA from the mitochondrion to the cytosol in the form of citrate for fatty acid and cholesterol synthesis. Citrate has been reported to be essential for fibroblast differentiation into fat cells. Because peroxisome proliferator-activated receptor-gamma (PPARγ) is known to be one of the master regulators of adipogenesis, we aimed to study the regulation of CIC by the PPARγ ligand rosiglitazone (BRL) in 3T3-L1 fibroblasts and in adipocytes. We demonstrated that BRL up-regulated CIC mRNA and protein levels in fibroblasts, while it did not elicit any effects in mature adipocytes. The enhancement of CIC levels upon BRL treatment was reversed using the PPARγ antagonist GW9662, addressing how this effect was mediated by PPARγ. Functional experiments using a reporter gene containing rat CIC promoter showed that BRL enhanced CIC promoter activity. Mutagenesis studies, electrophoretic-mobility-shift assay and chromatin-immunoprecipitation analysis revealed that upon BRL treatment, PPARγ and Sp1 are recruited on the Sp1-containing region within the CIC promoter, leading to an increase in CIC expression. In addition, mithramycin, a specific inhibitor for Sp1-DNA binding activity, abolished the PPARγ-mediated up-regulation of CIC in fibroblasts. The stimulatory effects of BRL disappeared in mature adipocytes in which PPARγ/Sp1 complex recruited SMRT corepressor to the Sp1 site of the CIC promoter. Taken together, our results contribute to clarify the molecular mechanisms by which PPARγ regulates CIC expression during the differentiation stages of fibroblasts into mature adipocytes.     

Immunosuppression-Independent Role of Regulatory T Cells against Hypertension-Driven Renal Dysfunctions

Hypertension-associated cardiorenal diseases represent one of the heaviest burdens for current health systems. In addition to hemodynamic damage, recent results have revealed that hematopoietic cells contribute to the development of these diseases by generating proinflammatory and profibrotic environments in the heart and kidney. However, the cell subtypes involved remain poorly characterized. Here we report that CD39⁺ regulatory T (T_REG) cells utilize an immunosuppression-independent mechanism to counteract renal and possibly cardiac damage during angiotensin II (AngII)-dependent hypertension. This mechanism relies on the direct apoptosis of tissue-resident neutrophils by the ecto-ATP diphosphohydrolase activity of CD39. In agreement with this, experimental and genetic alterations in T_REG/T_H cell ratios have a direct impact on tissue-resident neutrophil numbers, cardiomyocyte hypertrophy, cardiorenal fibrosis, and, to a lesser extent, arterial pressure elevation during AngII-driven hypertension. These results indicate that T_REG cells constitute a first protective barrier against hypertension-driven tissue fibrosis and, in addition, suggest new therapeutic avenues to prevent hypertension-linked cardiorenal diseases.     

An increase in the ATP levels occurs in cerebellar granule cells en route to apoptosis in which ATP derives from both oxidative phosphorylation and anaerobic glycolysis

Although it is recognized that ATP plays a part in apoptosis, whether and how its level changes en route to apoptosis as well as how ATP is synthesized has not been fully investigated. We have addressed these questions using cultured cerebellar granule cells. In particular, we measured the content of ATP, ADP, AMP, IMP, inosine, adenosine and L-lactate in cells undergoing apoptosis during the commitment phase (0-8 h) in the absence or presence of oligomycin or/and of citrate, which can inhibit totally the mitochondrial oxidative phosphorylation and largely the substrate-level phosphorylation in glycolysis, respectively. In the absence of inhibitors, apoptosis was accompanied by an increase in ATP and a decrease in ADP with 1:1 stoichiometry, with maximum ATP level found at 3 h apoptosis, but with no change in levels of AMP and its breakdown products and with a relatively low level of L-lactate production. Consistently, there was an increase in the cell energy charge and in the ratio ([ATP][AMP])/[ADP](2). When the oxidative phosphorylation was completely blocked by oligomycin, a decrease of the ATP content was found both in control cells and in cells undergoing apoptosis, but nonetheless cells still died by apoptosis, as shown by checking DNA laddering and by death prevention due to actinomycin D. In this case, ATP was provided by anaerobic glycolysis, as suggested by the large increase of L-lactate production. On the other hand, citrate itself caused a small decrease in ATP level together with a huge decrease in L-lactate production, but it had no effect on cell survival. When ATP level was further decreased due to the presence of both oligomycin and citrate, death occurred via necrosis at 8 h, as shown by the lack of DNA laddering and by death prevention found due to the NMDA receptor antagonist MK801. However, at a longer time, when ATP level was further decreased, cells died neither via apoptosis nor via glutamate-dependent necrosis, in a manner similar to something like to energy catastrophe. Our results shows that cellular ATP content increases in cerebellar granule cell apoptosis, that the role of oxidative phosphorylation is facultative, i.e. ATP can also derive from anaerobic glycolysis, and that the type of cell death depends on the ATP availability.     

Polyamine Localization and Biosynthesis in Chemically Fractionated Rat Retina

For elucidation of polyamine localization and biosynthesis in various cell types of rat retina, the putrescine, spermidine, and spermine contents as well as the ornithine decarboxylase and S-adenosylmethionine decarboxylase activities have been measured in retinal cell layers obtained by the selective cytotoxic action of iodoacetate on photoreceptor cells and of monosodium glutamate on higher-order retinal neurons. A notable depletion only in spermine content was associated with loss of the visual cell layer. Total ornithine decarboxylase and S-adenosylmethionine decarboxylase activities per retina were significantly lower in all chemically fractionated tissue, but loss of the photoreceptor layer produced the greatest decrease. The specific activities of these enzymes did not show marked changes in rat retinas deprived of inner neurons. The data support the suggestions that polyamine synthesis, storage, and catabolism have different distributions in the retinal layers and that the spermine levels and the high value of the spermine/spermidine molar ratio might depend essentially on the proportion of rods to cones.     

The cellular modifier MOAG‐4/SERF drives amyloid formation through charge complementation

While aggregation‐prone proteins are known to accelerate aging and cause age‐related diseases, the cellular mechanisms that drive their cytotoxicity remain unresolved. The orthologous proteins MOAG‐4, SERF1A, and SERF2 have recently been identified as cellular modifiers of such proteotoxicity. Using a peptide array screening approach on human amyloidogenic proteins, we found that SERF2 interacted with protein segments enriched in negatively charged and hydrophobic, aromatic amino acids. The absence of such segments, or the neutralization of the positive charge in SERF2, prevented these interactions and abolished the amyloid‐promoting activity of SERF2. In protein aggregation models in the nematode worm Caenorhabditis elegans, protein aggregation and toxicity were suppressed by mutating the endogenous locus of MOAG‐4 to neutralize charge. Our data indicate that MOAG‐4 and SERF2 drive protein aggregation and toxicity by interactions with negatively charged segments in aggregation‐prone proteins. Such charge interactions might accelerate primary nucleation of amyloid by initiating structural changes and by decreasing colloidal stability. Our study points at charge interactions between cellular modifiers and amyloidogenic proteins as potential targets for interventions to reduce age‐related protein toxicity.