Inhibition of VCAM-1 expression in endothelial cells by CORM-3: the role of the ubiquitin-proteasome system, p38, and mitochondrial respiration

Carbon monoxide (CO) abrogates TNF-α-mediated inflammatory responses in endothelial cells, yet the underlying mechanism thereof is still elusive. We have previously shown that the anti-inflammatory effect of CO-releasing molecule-3 (CORM-3) is not completely mediated via deactivation of the NF-κB pathway. In this study, we sought to explore other potential mechanisms by which CORM-3 downregulates VCAM-1 expression on TNF-α-stimulated HUVECs. By genome-wide gene expression profiling and pathway analysis we studied the relevance of particular pathways for the anti-inflammatory effect of CORM-3. In CORM-3-stimulated HUVECs significant changes in expression were found for genes implicated in the proteasome and porphyrin pathways. Although proteasome activities were increased by CORM-3, proteasome inhibitors did not abolish the effect of CORM-3. Likewise, heme oxygenase-1 inhibitors did not abrogate the ability of CORM-3 to downregulate VCAM-1 expression. Interestingly, CORM-3 inhibited MAPK p38, and the p38 inhibitor SB203580 downregulated VCAM-1 expression. However, downregulation of VCAM-1 by CORM-3 occurred only at concentrations that partly inhibit ATP production and sodium azide and oligomycin paralleled the effect of CORM-3 in this regard. Our results indicate that CORM-3-induced downregulation of VCAM-1 is mediated via p38 inhibition and mitochondrial respiration, whereas the ubiquitin-proteasome system seems not to be involved.     

STIM1 tyrosine-phosphorylation is required for STIM1-Orai1 association in human platelets

Stromal interaction molecule 1 (STIM1) is a key element of the store-operated Ca(2+) entry mechanism (SOCE). Recently, regulation of STIM1 by glycosylation and phosphorylation on serine/threonine or proline residues has been described; however other modes of phosphorylation that are important for activating SOCE in platelets, such as tyrosine phosphorylation, have been poorly investigated. Here we investigate the latency of STIM1 phosphorylation on tyrosine residues during the first steps of SOCE activation. Human platelets were stimulated and fixed at desired times using rapid kinetic assays instruments, and immunoprecipitation and western blotting techniques were then used to investigate the pattern of STIM1 tyrosine phosphorylation during the first steps of SOCE activation. We have found that maximal STIM1 tyrosine phosphorylation occurred 2.5s after stimulation of human platelets with thapsigargin (Tg). STIM1 localized in the plasma membrane were also phosphorylated in platelets stimulated with Tg. By using chemical inhibitors that target different members of the Src family of tyrosine kinases (SKFs), two independent signaling pathways involved in STIM1 tyrosine phosphorylation during the first steps of SOCE activation were identified. We finally conclude that STIM1 tyrosine phosphorylation is a key event for the association of STIM1 with plasma membrane Ca(2+) channels such as Orai1, hence it is required for conducting SOCE activation.     

Development of small-molecule probes that selectively kill cells induced to express mutant RAS

Synthetic lethal screening is a chemical biology approach to identify small molecules that selectively kill oncogene-expressing cell lines with the goal of identifying pathways that provide specific targets against cancer cells. We performed a high-throughput screen of 303,282 compounds from the National Institutes of Health-Molecular Libraries Small Molecule Repository (NIH-MLSMR) against immortalized BJ fibroblasts expressing HRAS(G12V) followed by a counterscreen of lethal compounds in a series of isogenic cells lacking the HRAS(G12V) oncogene. This effort led to the identification of two novel molecular probes (PubChem CID 3689413, ML162 and CID 49766530, ML210) with nanomolar potencies and 4-23-fold selectivities, which can potentially be used for identifying oncogene-specific pathways and targets in cancer cells.     

Lipase-catalyzed preparation of chromomycin A₃ analogues and biological evaluation for anticancer activity

Several acyl derivatives of the aureolic acid chromomycin A(3) were obtained via lipase-catalyzed acylation. Lipase B from Candida antarctica (CAL-B) was found to be the only active biocatalyst, directing the acylation regioselectively towards the terminal secondary hydroxyl group of the aglycone side chain. All new chromomycin A(3) derivatives showed antitumor activity at the micromolar or lower level concentration. Particularly, chromomycin A(3) 4'-vinyladipate showed 3-5 times higher activity against the four tumor cell lines assayed as compared to chromomycin A(3).     

Discovery of a novel class of zwitterionic,potent, selective and orally active S1P1 direct agonists

Amido-1,3,4-thiadiazoles have been identified as a novel structural class of potent and selective sphingosine-1-phosphate receptor subtype 1 agonists. Starting from a micromolar HTS hit with the help of an in-house homology model, robust structural–activity relationships were developed to yield compounds with good selectivity and excellent in vivo efficacy in rat models.     

HAK transporters from Physcomitrella patens and Yarrowia lipolytica mediate sodium uptake

The widespread presence of Na(+)-specific uptake systems across plants and fungi is a controversial topic. In this study, we identify two HAK genes, one in the moss Physcomitrella patens and the other in the yeast Yarrowia lipolytica, that encode Na(+)-specific transporters. Because HAK genes are numerous in plants and are duplicated in many fungi, our findings suggest that some HAK genes encode Na(+) transporters and that Na(+) might play physiological roles in plants and fungi more extensively than is currently thought.     

Reduction of the sevoflurane minimum alveolar concentration induced by methadone, tramadol, butorphanol and morphine in rats

This study aimed to estimate the reduction in the minimum alveolar concentration (MAC) of sevoflurane induced by low and high doses of methadone (5 and 10 mg/kg), tramadol (25 and 50 mg/kg), butorphanol (5 and 10 mg/kg) or morphine (5 and 10 mg/kg) in the rat. A control group received normal saline. Sixty-three adult male Sprague-Dawley rats were anaesthetized with sevoflurane (n = 7 per group). Sevoflurane MAC was then determined before and after intraperitoneal administration of the opioids or saline. The duration of the sevoflurane MAC reduction and basic cardiovascular and respiratory measurements were also recorded. The baseline MAC was 2.5 (0.3) vol%. Methadone, tramadol and morphine reduced the sevoflurane MAC (low dose: 31 ± 10, 38 ± 15 and 30 ± 13% respectively; high dose: 100 ± 0, 83 ± 17 and 77 ± 25%, respectively) in a dose-dependent manner. The low and high doses of butorphanol reduced the sevoflurane MAC to a similar extent (33 ± 7 and 31 ± 4%, low and high doses, respectively). Two rats developed apnoea following administration of high-dose butorphanol and methadone. These anaesthetic-sparing effects are clinically relevant and may reduce the adverse effects associated with higher doses of inhalational anaesthetics.     

Functional impact bias reveals cancer drivers

Identifying cancer driver genes and pathways among all somatic mutations detected in a cohort of tumors is a key challenge in cancer genomics. Traditionally, this is done by prioritizing genes according to the recurrence of alterations that they bear. However, this approach has some known limitations, such as the difficulty to correctly estimate the background mutation rate, and the fact that it cannot identify lowly recurrently mutated driver genes. Here we present a novel approach, Oncodrive-fm, to detect candidate cancer drivers which does not rely on recurrence. First, we hypothesized that any bias toward the accumulation of variants with high functional impact observed in a gene or group of genes may be an indication of positive selection and can thus be used to detect candidate driver genes or gene modules. Next, we developed a method to measure this bias (FM bias) and applied it to three datasets of tumor somatic variants. As a proof of concept of our hypothesis we show that most of the highly recurrent and well-known cancer genes exhibit a clear FM bias. Moreover, this novel approach avoids some known limitations of recurrence-based approaches, and can successfully identify lowly recurrent candidate cancer drivers.     

3-Hydroxypropionic Acid as an Antibacterial Agent from Endophytic Fungi Diaporthe phaseolorum

Endophytic fungi are considered a rich source of active compounds resulting from their secondary metabolism. Fungi from marine environment grow in a habitat with unique conditions that can contribute to the activation of metabolic pathways of synthesis of different unknown molecules. The production of these compounds may support the adaptation and survival of the fungi in the marine ecosystem. Mangroves are ecosystems situated between land and sea. They are frequently found in tropical and subtropical areas and enclose approximately 18.1 million hectares of the planet. The great biodiversity found in these ecosystems shows the importance of researching them, including studies regarding new compounds derived from the endophytic fungi that inhabit these ecosystems. 3-hydroxypropionic acid (3-HPA) has been isolated from the mangrove endophytic fungus Diaporthe phaseolorum, which was obtained from branches of Laguncularia racemosa. The structure of this compound was elucidated by spectroscopic methods, mainly 1D and 2D NMR. In bioassays, 3-HPA showed antimicrobial activities against both Staphylococcus aureus and Salmonella typhi. The structure of this antibiotic was modified by the chemical reaction of Fischer-Speier esterification to evaluate the biologic activity of its chemical analog. The esterified product, 3-hydroxypropanoic ethyl ester, did not exhibit antibiotic activity, suggesting that the free carboxylic acid group is important to the pharmacological activity. The antibiotic-producing strain was identified with internal transcribed spacer sequence data. To the best of our knowledge, this is the first report of antibacterial activity by 3-HPA against the growth of medically important pathogens.     

C3G transgenic mouse models with specific expression in platelets reveal a new role for C3G in platelet clotting through its GEF activity

We have generated mouse transgenic lineages for C3G (tgC3G) and C3GΔCat (tgC3GΔCat, C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 (platelet factor 4) gene promoter. Transgenic platelet activity has been analyzed through in vivo and in vitro approaches, including bleeding time, aggregation assays and flow cytometry. Both transgenes are expressed (RNA and protein) in purified platelets and megakaryocytes and do not modify the number of platelets in peripheral blood. Transgenic C3G animals showed bleeding times significantly shorter than control animals, while tgC3GΔCat mice presented a remarkable bleeding diathesis as compared to their control siblings. Accordingly, platelets from tgC3G mice showed stronger activation in response to platelet agonists such as thrombin, PMA, ADP or collagen than control platelets, while those from tgC3GΔCat animals had a lower response. In addition, we present data indicating that C3G is a mediator in the PKC pathway leading to Rap1 activation. Remarkably, a significant percentage of tgC3G mice presented a higher level of neutrophils than their control siblings. These results indicate that C3G plays an important role in platelet clotting through a mechanism involving its GEF activity and suggest that it might be also involved in neutrophil development.