Membrane-insertion fragments of Bcl-xL, Bax, and Bid

Apoptosis regulators of the Bcl-2 family associate with intracellular membranes from mitochondria and the endoplasmic reticulum, where they perform their function. The activity of these proteins is related to the release of apoptogenic factors, sequestered in the mitochondria, to the cytoplasm, probably through the formation of ion and/or protein transport channels. Most of these proteins contain a C-terminal putative transmembrane (TM) fragment and a pair of hydrophobic alpha helices (alpha5-alpha6) similar to the membrane insertion fragments of the ion-channel domain of diphtheria toxin and colicins. Here, we report on the membrane-insertion properties of different segments from antiapoptotic Bcl-x(L) and proapoptotic Bax and Bid, that correspond to defined alpha helices in the structure of their soluble forms. According to prediction methods, there are only two putative TM fragments in Bcl-x(L) and Bax (the C-terminal alpha helix and alpha-helix 5) and one in activated tBid (alpha-helix 6). The rest of their sequence, including the second helix of the pore-forming domain, displays only weak hydrophobic peaks, which are below the prediction threshold. Subsequent analysis by glycosylation mapping of single alpha-helix segments in a model chimeric system confirms the above predictions and allows finding an extra TM fragment made of helix alpha1 of Bax. Surprisingly, the amphipathic helices alpha6 of Bcl-x(L) and Bax and alpha7 of Bid do insert in membranes only as part of the alpha5-alpha6 (Bcl-x(L) and Bax) or alpha6-alpha7 (Bid) hairpins but not when assayed individually. This behavior suggests a synergistic insertion and folding of the two helices of the hairpin that could be due to charge complementarity and additional stability provided by turn-inducing residues present at the interhelical region. Although these data come from chimeric systems, they show direct potentiality for acquiring a membrane inserted state. Thus, the above fragments should be considered for the definition of plausible models of the active, membrane-bound species of Bcl-2 proteins.     

A geminivirus-induced gene silencing system for gene function validation in cassava

We have constructed an African cassava mosaic virus (ACMV) based gene-silencing vector as a reverse genetics tool for gene function analysis in cassava. The vector carrying a fragment from the Nicotiana tabacumsulfur gene (su), encoding one unit of the chloroplast enzyme magnesium chelatase, was used to induce the silencing of the cassava orthologous gene resulting in yellow–white spots characteristic of the inhibition of su expression. This result suggests that well developed sequence databases from model plants like Arabidopsis thaliana, Nicotiana benthamiana, N. tabacum, Lycopersicon esculentum and others could be used as a major source of information and sequences for functional genomics in cassava. Furthermore, a fragment of the cassava CYP79D2endogenous gene, sharing 89% homology with CYP79D1endogenous gene was inserted into the ACMV vector. The resultant vector was inducing the down regulation of the expression of these two genes which catalyze the first-dedicated step in the synthesis of linamarin, the major cyanogenic glycoside in cassava. At 21 days post-inoculation (dpi), a 76% reduction of linamarin content was observed in silenced leaves. Using transgenic plants expressing antisense RNA of CYP79D1and CYP79D2, Siritunga and Sayre (2003) obtained several lines with a reduction level varying from 60% to 94%. This result provides the first example of direct comparison of the efficiency of a virus-induced gene silencing (VIGS) system and the transgenic approach for suppression of a biosynthetic pathway. The ACMV VIGS system will certainly be a complement and in some cases an alternative to the transgenic approach, for gene discovery and gene function analysis in cassava.     

Merychippus (Mammalia, Perissodactyla, Equidae) from the Middle Miocene of state of Oaxaca, southeastern Mexico

Mexican material referable to Merychippus from two localities in eastern Oaxaca was described first nearly 50 years ago. Subsequent work there and in Central Oaxaca, spanning some 30 years, has allowed to establish the detail stratigraphy in both regions, and assembled a collection of merychippine material from the Matatlán (Central Oaxaca) and El Camarón (eastern Oaxaca) Formations, both K-Ar dated ~15 Ma (late early Barstovian). Detailed taxonomic analysis of this collection indicate the presence of two subhypsodont horse species referable to “Merychippus” cf. “M.” primus and “M.” cf. “M.” sejunctus in both regions. These records document the coexistence in tropical southern North America of basal and hipparionine affinity merychippine grade species, and provide a glimpse in to the diversity of subhypsodont equids in this region.     

Synthesis and in vitro anti-proliferative activity of some novel isatins conjugated with quinazoline/phthalazine hydrazines against triple-negative breast cancer MDA-MB-231 cells as apoptosis-inducing agents

Treatment of patients with triple-negative breast cancer (TNBC) is challenging due to the absence of well- defined molecular targets and the heterogeneity of such disease. In our endeavor to develop potent isatin-based anti-proliferative agents, we utilized the hybrid-pharmacophore approach to synthesize three series of novel isatin-based hybrids 5a–h, 10a–h and 13a–c, with the prime goal of developing potent anti-proliferative agents toward TNBC MDA-MB-231 cell line. In particular, compounds 5e and 10g were the most active hybrids against MDA-MB-231 cells (IC₅₀?=?12.35?±?0.12 and 12.00?±?0.13?μM), with 2.37- and 2.44-fold increased activity than 5-fluorouracil (5-FU) (IC₅₀?=?29.38?±?1.24?μM). Compounds 5e and 10g induced the intrinsic apoptotic mitochondrial pathway in MDA-MB-231; evidenced by the reduced expression of the anti-apoptotic protein Bcl-2, the enhanced expression of the pro-apoptotic protein Bax and the up-regulated active caspase-9 and caspase-3 levels. Furthermore, 10g showed significant increase in the percent of annexin V-FITC positive apoptotic cells from 3.88 to 31.21% (8.4 folds compared to control).     

Thioredoxin-Interacting Protein (TXNIP) in Cerebrovascular and Neurodegenerative Diseases: Regulation and Implication

Neurological diseases, including acute attacks (e.g., ischemic stroke) and chronic neurodegenerative diseases (e.g., Alzheimer’s disease), have always been one of the leading cause of morbidity and mortality worldwide. These debilitating diseases represent an enormous disease burden, not only in terms of health suffering but also in economic costs. Although the clinical presentations differ for these diseases, a growing body of evidence suggests that oxidative stress and inflammatory responses in brain tissue significantly contribute to their pathology. However, therapies attempting to prevent oxidative damage or inhibiting inflammation have shown little success. Identification and targeting endogenous “upstream” mediators that normalize such processes will lead to improve therapeutic strategy of these diseases. Thioredoxin-interacting protein (TXNIP) is an endogenous inhibitor of the thioredoxin (TRX) system, a major cellular thiol-reducing and antioxidant system. TXNIP regulating redox/glucose-induced stress and inflammation, now is known to get upregulated in stroke and other brain diseases, and represents a promising therapeutic target. In particular, there is growing evidence that glucose strongly induces TXNIP in multiple cell types, suggesting possible physiological roles of TXNIP in glucose metabolism. Recently, a significant body of literature has supported an essential role of TXNIP in the activation of the NOD-like receptor protein (NLRP3)-inflammasome, a well-established multi-molecular protein complex and a pivotal mediator of sterile inflammation. Accordingly, TXNIP has been postulated to reside centrally in detecting cellular damage and mediating inflammatory responses to tissue injury. The majority of recent studies have shown that pharmacological inhibition or genetic deletion of TXNIP is neuroprotective and able to reduce detrimental aspects of pathology following cerebrovascular and neurodegenerative diseases. Conspicuously, the mainstream of the emerging evidences is highlighting TXNIP link to damaging signals in endothelial cells. Thereby, here, we keep the trend to present the accumulative data on CNS diseases dealing with vascular integrity. This review aims to summarize evidence supporting the significant contribution of regulatory mechanisms of TXNIP with the development of brain diseases, explore pharmacological strategies of targeting TXNIP, and outline obstacles to be considered for efficient clinical translation.     

Cryopreservation of an artificial human oral mucosa stroma. A viability and rheological study

The aim of this study was to evaluate the viability and biomechanical properties of artificial human oral mucosa stroma (HOMS) subjected to cryopreservation with different cryoprotectant solutions.     

The role of the VQIVYK peptide in tau protein phosphorylation

Although it remains unclear whether they are related to one another, tau aggregation and phosphorylation are the main pathological hallmarks of the neuronal disorders known as tauopathies. The capacity to aggregate is impaired in a variant of the tau 3R isoform that lacks residues 306–311 (nomenclature for the largest CNS tau isoform) and hence, we have taken advantage of this feature to study how phosphorylation and aggregation may be related as well as the role of this six amino acid peptide (VQIVYK). Through these analyses, we found that the phosphorylation of the tau variant was higher than that of the complete tau protein and that not only the deletion of these residues, but also the interaction of these residues, in tau 3R, with thioflavin-S augmented tau phosphorylation by glycogen synthase kinase 3. In addition, the binding of the peptide containing the residues 306–311 to the whole tau protein provoked an increase in tau phosphorylation. This observation could be physiologically relevant as may suggest that tau–tau interactions, through those residues, facilitate tau phosphorylation. In summary, our data indicate that deletion of residues VQIVYK, in tau protein produces an increase in tau phosphorylation, without tau aggregation, because the VQIVYK peptide, that favors aggregation, is missing. On the other hand, when the whole tau protein interacts with thioflavin-S or the peptide VQIVYK, an increase in both aggregation and phosphorylation occurs.     

Differential impact of the most extreme drought event over the last half century on growth and sap flow in two coexisting Mediterranean trees

Extreme climatic events such as intense droughts are becoming more frequent in Mediterranean regions, but our understanding of their impact on tree performance is still fragmentary. We analyzed growth and sap flow responses for a 3-year period including the most stressful drought over the last half century in the evergreen Pinus nigra and the deciduous Quercus faginea, two dominant tree species in the continental plateau of the Iberian Peninsula. Our aim was to quantify the differential impacts of this event on the performance of both species and their modulation by local microclimate. Growth was registered with digital dendrometers, and water use was assessed by continuously recording sap flow in 8–9 coexisting adult individuals of each species in two sites. Q. faginea spring growth rate decreased by 60 % during the dry year at the dry site, while the decrease in P. nigra was around 36 %. P. nigra exhibited larger sap flow reductions during the dry season and also larger decreases during the extreme year, but in contrast to Q. faginea, it was able to recover growth and sap flow values after the extreme drought. Minor microclimatic differences between sites had significant effects on growth and water use, with slightly more mesic conditions significantly attenuating the impact of drought on both species. Findings suggest that the study species were near to their tolerance thresholds, so that even moderate increases in the intensity and frequency of unusual droughts have important consequences for individual tree performance, and eventually species coexistence and ecosystem processes.