Differential Pharmacologic Properties of the Two C75 Enantiomers: (+)‐C75 Is a Strong Anorectic Drug; (−)‐C75 Has Antitumor Activity

C75 is a synthetic compound described as having antitumoral properties. It produces hypophagia and weight loss in rodents, limiting its use in cancer therapy but identifying it as a potential anti‐obesity drug. C75 is a fatty acid synthase (FAS) inhibitor and, through its coenzyme A (CoA) derivative, it acts as a carnitine palmitoyltransferase (CPT) 1 inhibitor. Racemic mixtures of C75 have been used in all the previous studies; however, the potential different biological activities of C75 enantiomers have not been examined yet. To address this question we synthesized the two C75 enantiomers separately. Our results showed that (?)‐C75 inhibits FAS activity in vitro and has a cytotoxic effect on tumor cell lines, without affecting food consumption. (+)‐C75 inhibits CPT1 and its administration produces anorexia, suggesting that central inhibition of CPT1 is essential for the anorectic effect of C75. The differential activity of C75 enantiomers may lead to the development of potential new specific drugs for cancer and obesity. Chirality 25:281–287, 2013. © 2013 Wiley Periodicals, Inc.     

Prevalence of <Emphasis Type="Italic">Candida dubliniensis</Emphasis> Fungemia in Argentina: Identification by a Novel Multiplex PCR and Comparison of Different Phenotypic Methods

Candida dubliniensis is an emerging pathogen that can cause invasive disease in patients who have a variety of clinical conditions. C. dubliniensis is often misidentified as Candida albicans by clinical laboratories. In Argentina, incidence data are still scarce, and only one systemic infection has been reported. This study aims to determine the prevalence of C. dubliniensis in blood samples in Argentina, to evaluate a novel PCR multiplex as well as several phenotypic methods for the identification of this yeast, and to know the susceptibility profile of isolates against seven antifungal drugs. We have found that prevalence in Argentina appears to be lower than that reported in other countries, occurring only in 0.96% of the Candidemia cases recovered in 47 hospitals during a 1-year period. All C. dubliniensis clinical isolates included in this study were genetically identical when comparing ITS genes sequences. This is in agreement with the previous studies suggesting little genetic variation within this species. The novel multiplex PCR proved to be 100% sensitive and specific for the identification of C. dubliniensis. Therefore, we propose its use as a rapid and inexpensive method for laboratories having access to molecular techniques. Although no single phenotypic test has proved to be infallible, both colony morphology on tobacco agar, as well as abundant chlamydospore formation on both tobacco agar and on sunflower seed agar, may be used as a presumptive differentiation method in routine mycology laboratories. It has been suggested that C. dubliniensis may have higher propensity to develop azole antifungal drug resistance than C. albicans. In this study, one of the five clinical isolates of C. dubliniensis was resistant to fluconazole.     

Type II arginine methyltransferase PRMT5 regulates gene expression of inhibitors of differentiation/DNA binding Id2 and Id4 during glial cell differentiation

PRMT5 is a type II protein arginine methyltranferase that catalyzes monomethylation and symmetric dimethylation of arginine residues. PRMT5 is functionally involved in a variety of biological processes including embryo development and circadian clock regulation. However, the role of PRMT5 in oligodendrocyte differentiation and central nervous system myelination is unknown. Here we show that PRMT5 expression gradually increases throughout postnatal brain development, coinciding with the period of active myelination. PRMT5 expression was observed in neurons, astrocytes, and oligodendrocytes. siRNA-mediated depletion of PRMT5 in mouse primary oligodendrocyte progenitor cells abrogated oligodendrocyte differentiation. In addition, the PRMT5-depleted oligodendrocyte progenitor and C6 glioma cells expressed high levels of the inhibitors of differentiation/DNA binding, Id2 and Id4, known repressors of glial cell differentiation. We observed that CpG-rich islands within the Id2 and Id4 genes were bound by PRMT5 and were hypomethylated in PRMT5-deficient cells, suggesting that PRMT5 plays a role in gene silencing during glial cell differentiation. Our findings define a role of PRMT5 in glial cell differentiation and link PRMT5 to epigenetic changes during oligodendrocyte differentiation.     

Cytochemical immuno-localization of allene oxide cyclase, a jasmonic acid biosynthetic enzyme, in developing potato stolons

The involvement of jasmonates in the tuber development has been proved by the presence of many of these compounds in potato stolons, modification of their levels during the transition of the stolon into tuber, and induction of cell expansion upon exogenous jasmonates treatment. However, to date there is only little evidence of the presence of the jasmonic acid-biosynthetic enzymes in stolons or young tubers. As allene oxide cyclase represents the major control point for jasmonic acid biosynthesis, we studied the occurrence of allene oxide cyclase by immunological approaches in the early stages of tuber formation. In developing stolons, allene oxide cyclase as well as lipoxygenase were clearly detectable, but their levels did not change during development. Jasmonic acid treatment for 24h, however, increased lipoxygenase and allene oxide cyclase protein levels in both developmental stages analyzed. In longitudinal sections of stolons of stages 1 and 2, allene oxide cyclase and lipoxygenase occurred in the apex and along the stolon axis. Allene oxide cyclase was clearly detectable in epidermal, cortical and pith parenchymatic cells, showing the highest levels in vascular tissues surrounding cells. Lipoxygenase was mainly located in the parenchymatic cortex cells. The occurrence of allene oxide cyclase in stolons together with the previous identification of jasmonates from developing stolons reveals that these organs are capable to synthesize and metabolize jasmonates.     

Haploinsufficiency of Mdm2 and Mdm4 in tumorigenesis and development

The tumor suppressor p53 is inactivated by multiple mechanisms that include mutations of the p53 gene itself and increased levels of the p53 inhibitors MDM2 and MDM4. Mice lacking Mdm2 or Mdm4 exhibit embryo-lethal phenotypes that are completely rescued by concomitant deletion of p53. Here we show that Mdm2 and Mdm4 haploinsufficiency leads to increased p53 activity, exhibited as increased sensitivity to DNA damage and decreased transformation potential. Moreover, in in vivo tumor development, Emu-myc Mdm4+/- mice show a delayed onset of B-cell lymphomas compared to Emu-myc mice. Additionally, Mdm2+/- Mdm4+/- double-heterozygous mice are not viable and exhibit defects in hematopoiesis and cerebellar development. The defects in Mdm2+/- Mdm4+/- mice are corrected by deletion of a single p53 allele. These findings highlight the exquisite sensitivity of p53 to Mdm2 and Mdm4 levels and suggest that some cell types may be more sensitive to therapeutic drugs that inhibit the Mdm-p53 interaction.     

Prediction of hydraulic conductivity loss from relative water loss: new insights into water storage of tree stems and branches

More frequently occurring, drought waves call for a deeper understanding of tree hydraulics and fast and easily applicable methods to measure drought stress. The aim of this study was to establish empirical relationships between the percent loss of hydraulic conductivity (PLC) and the relative water loss (RWL) in woody stem axes with different P₅₀, i.e. the water potential (Ψ) that causes 50% conductivity loss. Branches and saplings of temperate conifer (Picea abies, Larix decidua) and angiosperm species (Acer campestre, Fagus sylvatica, Populus x canescens, Populus tremula, Sorbus torminalis) and trunk wood of mature P. abies trees were analyzed. P₅₀ was calculated from hydraulic measurements following bench top dehydration or air injection. RWL and PLC were fitted by linear, quadratic or cubic equations. Species‐ or age‐specific RWLs at P₅₀ varied between 10 and 25% and P₈₈, the Ψ that causes 88% conductivity loss, between 18 and 44%. P₅₀ was predicted from the relationship between Ψ and the RWL. The predictive quality for P₅₀ across species was almost 1:1 (r² = 0.99). The approach presented allows thus reliable and fast prediction of PLC from RWL. Branches and saplings with high hydraulic vulnerability tended to have lower RWLs at P₅₀ and at P₈₈. The results are discussed with regard to the different water storage capacities in sapwood and survival strategies under drought stress. Potential applications are screening trees for drought sensitivity and a fast interpretation of diurnal, seasonal or drought induced changes in xylem water content upon their impact on conductivity loss.     

Identifying Ligand Binding Conformations of the β2-Adrenergic Receptor by Using Its Agonists as Computational Probes

Recently available G-protein coupled receptor (GPCR) structures and biophysical studies suggest that the difference between the effects of various agonists and antagonists cannot be explained by single structures alone, but rather that the conformational ensembles of the proteins need to be considered. Here we use an elastic network model-guided molecular dynamics simulation protocol to generate an ensemble of conformers of a prototypical GPCR, β₂-adrenergic receptor (β₂AR). The resulting conformers are clustered into groups based on the conformations of the ligand binding site, and distinct conformers from each group are assessed for their binding to known agonists of β₂AR. We show that the select ligands bind preferentially to different predicted conformers of β₂AR, and identify a role of β₂AR extracellular region as an allosteric binding site for larger drugs such as salmeterol. Thus, drugs and ligands can be used as “computational probes” to systematically identify protein conformers with likely biological significance.     

Dopamine-induced toxicity is synergistically potentiated by simultaneous HSP-90 and Akt inhibition in oligodendrocyte progenitors

Oligodendrocyte progenitors are highly susceptible to various insults. Their limited antioxidant defenses and high levels of apoptotic factors, such as Bax and pro-caspase-3 contribute to their sensitivity. We previously showed that dopamine (DA) is toxic to oligodendrocyte progenitors by inducing superoxide generation, lowering glutathione levels and promoting apoptosis through caspase-3 activation. In contrast, factors that contribute to cell survival and defense against dopamine (DA) toxicity are less studied. Here, we explored the role of two molecules which play important roles in cell survival, namely the heat shock protein 90 (HSP-90) and the protein kinase Akt, using the selective inhibitors, 17-AAG and Akt inhibitor III, respectively. The HSP-90 inhibitor caused a decrease in P-Akt level, induced caspase-3 activation, increased nuclear condensation and caused a loss in cell viability. Furthermore, 17-AAG potentiated DA-induced apoptosis by enhancing caspase-3 activation. In addition, the Akt inhibitor alone exacerbated DA toxicity and in combination with 17-AAG caused synergistic potentiation of DA toxicity by enhancing caspase-3 activation. Together, these results indicate that HSP-90 is essential for oligodendrocyte progenitor survival. Both HSP-90 and Akt play important roles in concert in the defense against DA-induced apoptosis.     

Expression and functional analysis of an Entamoeba histolytica truncated adhesin in tomato plants

Several antigens from Entamoeba histolytica the agent causing human amoebiasis have been identified as potential vaccine candidates. One of them is the EhADH112 adhesin, which plays an important role during trophozoite adherence to and phagocytosis of target cells. We report here the expression of the EhADH112 carboxy-terminus of the gene (Adh240) in tomato plants. A 28 kDa band is recognized by specific antibodies in total proteins from transformed tomatoes. The plant-based adhesin conserves the ability to inhibit trophozoite adherence and phagocytosis of target cells by an average of 32.8 and 44.75% respectively.