Maria F. Duarte

Lipophilic Extracts of Cynara cardunculus L. var. altilis (DC): A Source of Valuable Bioactive Terpenic Compounds

Lipophilic extracts of Cynara cardunculus L. var. altilis (DC) from the south of Portugal (Baixo Alentejo) were studied by gas chromatography-mass spectrometry. One sesquiterpene lactone, four pentacyclic triterpenes, and four sterols were reported for the first time as cultivated cardoon components, namely, deacylcynaropicrin, β- and α-amyrin, lupenyl and ψ-taraxasteryl acetates, stigmasterol, 24-methylenecholesterol, campesterol, and Δ(5)-avenasterol. In addition, other new compounds were identified: ten fatty acids, eight long-chain aliphatic alcohols, and six aromatic compounds. Four triterpenyl fatty acid esters were also detected. Sesquiterpene lactones and pentacyclic triterpenes were the major lipophilic families, representing respectively 2-46% and 10-89% of the detected compounds. Cynaropicrin was the most abundant sesquiterpene lactone, while taraxasteryl acetate was the main pentacyclic triterpene. Fatty acids and sterols, mainly hexadecanoic acid and β-sitosterol, were present at lower amounts (1-20% and 1-11% of the detected compounds). Long-chain aliphatic alcohols and aromatic compounds were detected at reduced abundances (1-6% of the detected compounds).

The intracellular domain of Notch ligand Delta1 induces cell growth arrest

Notch signaling involves proteolytic cleavage of the transmembrane Notch receptor after binding to its transmembrane ligands, Delta or Jagged; and the resultant soluble intracellular domain of Notch stimulates a cascade of transcriptional events. The Delta1 ligand also undergoes proteolytic cleavage upon Notch binding, resulting in the production of a free intracellular domain. We demonstrate that the expression of the intracellular domain of Delta1 results in a non‐proliferating senescent‐like cell phenotype which is dependent on the expression of the cell cycle inhibitor, p21, and is abolished by co‐expression of constitutively active Notch1. These data suggest a new intracellular role for Delta1.

Oleanolic, Ursolic, and Betulinic Acids as Food Supplements or Pharmaceutical Agents for Type 2 Diabetes: Promise or Illusion?

Oleanolic (OA), ursolic (UA), and betulinic (BA) acids are three triterpenic acids (TAs) with potential effects for treatment of type 2 diabetes (T2DM). Mechanistic studies showed that these TAs act as hypoglycemic and antiobesity agents mainly through (i) reducing the absorption of glucose; (ii) decreasing endogenous glucose production; (iii) increasing insulin sensitivity; (iv) improving lipid homeostasis; and (v) promoting body weight regulation. Besides these promising beneficial effects, it is believed that OA, UA, and BA protect against diabetes-related comorbidities due to their antiatherogenic, anti-inflammatory, and antioxidant properties. We also highlight the protective effect of OA, UA, and BA against oxidative damage, which may be very relevant for the treatment and/or prevention of T2DM. In the present review, we provide an integrative description of the antidiabetic properties of OA, UA, and BA, evaluating the potential use of these TAs as food supplements or pharmaceutical agents to prevent and/or treat T2DM.

Soluble Jagged1 Attenuates Lateral Inhibition, Allowing for the Clonal Expansion of Neural Crest Stem Cells

The activation of Notch signaling in neural crest stem cells (NCSCs) results in the rapid loss of neurogenic potential and differentiation into glia. We now show that the attenuation of endogenous Notch signaling within expanding NCSC clones by the Notch ligand soluble Jagged1 (sJ1), maintains NCSCs in a clonal self‐renewing state in vitro without affecting their sensitivity to instructive differentiation signals observed previously during NCSC self‐renewal. sJ1 functions as a competitive inhibitor of Notch signaling to modulate endogenous cell‐cell communication to levels sufficient to inhibit neural differentiation but insufficient to instruct gliogenic differentiation. Attenuated Notch signaling promotes the induction and nonclassic release of fibroblast growth factor 1 (FGF1). The functions of sJ1 and FGF1 signaling are complementary, as abrogation of FGF signaling diminishes the ability of sJ1 to promote NCSC expansion, yet the secondary NCSCs maintain the dosage sensitivity of the founder. These results validate and build upon previous studies on the role of Notch signaling in stem cell self‐renewal and suggest that the differentiation bias or self‐renewal potential of NCSCs is intrinsically linked to the level of endogenous Notch signaling. This should provide a unique opportunity for the expansion of NCSCs ex vivo without altering their differentiation bias for clinical cell replacement or transplant strategies in tissue repair.

Novel Cross-Talk between Three Cardiovascular Regulators: Thrombin Cleavage Fragment of Jagged1 Induces Fibroblast Growth Factor 1 Expression and Release

Angiogenesis is controlled by several regulatory mechanisms, including the Notch and fibroblast growth factor (FGF) signaling pathways. FGF1, a prototype member of FGF family, lacks a signal peptide and is released through an endoplasmic reticulum-Golgi-independent mechanism. A soluble extracellular domain of the Notch ligand Jagged1 (sJ1) inhibits Notch signaling and induces FGF1 release. Thrombin, a key protease of the blood coagulation cascade and a potent inducer of angiogenesis, stimulates rapid FGF1 release through a mechanism dependent on the major thrombin receptor protease-activated receptor (PAR) 1. This study demonstrates that thrombin cleaves Jagged1 in its extracellular domain. The sJ1 form produced as a result of thrombin cleavage inhibits Notch-mediated CBF1/Suppressor of Hairless [(Su(H)]/Lag-1-dependent transcription and induces FGF1 expression and release. The overexpression of Jagged1 in PAR1 null cells results in a rapid thrombin-induced export of FGF1. These data demonstrate the existence of novel cross-talk between thrombin, FGF, and Notch signaling pathways, which play important roles in vascular formation and remodeling.

Transgenic Expression of Nonclassically Secreted FGF Suppresses Kidney Repair

FGF1 is a signal peptide-less nonclassically released growth factor that is involved in angiogenesis, tissue repair, inflammation, and carcinogenesis. The effects of nonclassical FGF export in vivo are not sufficiently studied. We produced transgenic mice expressing FGF1 in endothelial cells (EC), which allowed the detection of FGF1 export to the vasculature, and studied the efficiency of postischemic kidney repair in these animals. Although FGF1 transgenic mice had a normal phenotype with unperturbed kidney structure, they showed a severely inhibited kidney repair after unilateral ischemia/reperfusion. This was manifested by a strong decrease of postischemic kidney size and weight, whereas the undamaged contralateral kidney exhibited an enhanced compensatory size increase. In addition, the postischemic kidneys of transgenic mice were characterized by hyperplasia of interstitial cells, paucity of epithelial tubular structures, increase of the areas occupied by connective tissue, and neutrophil and macrophage infiltration. The continuous treatment of transgenic mice with the cell membrane stabilizer, taurine, inhibited nonclassical FGF1 export and significantly rescued postischemic kidney repair. It was also found that similar to EC, the transgenic expression of FGF1 in monocytes and macrophages suppresses kidney repair. We suggest that nonclassical export may be used as a target for the treatment of pathologies involving signal peptide-less FGFs.

Angiogenesis, haemostasis and cancer: new paradigms and old concerns

Neovascularization is a crucial phenomenon for the continuous growing of neoplastic cells and cancer progression. The growth of new blood vessels from pre-existing vessels (angiogenesis) occurs in several physiological and pathological conditions, including cancer, where it is critical for tumor-cells nutrition. Recently, new remarkable insights regarding angiogenesis and blood coagulation (key events in vascular biology) have been described. The serine protease thrombin, which plays a central role in blood coagulation cascade through its ability to cleave fibrinogen conducting to fibrin clot formation, is also known to be involved in embryogenesis, inflammation, wound healing, through its active role on vascular remodeling. Although the increased knowledge of factors regulating angiogenesis and coagulation led to the understanding that angiogenesis, homeostasis and carcinogenesis are three close team players, little is still known about how these pathways support each other in the process of angiogenesis in vivo. This review summarizes current understanding of blood coagulation cascade role in conducting angiogenesis and tumor progression, as well as provides an overview of the emerging anti-angiogenic and anti-coagulation therapies inducing tumor regression.A neovascularizacao e um processo fundamental para a sobrevivencia e a progressao das celulas neoplasicas malignas. O crescimento de novos vasos sanguineos a partir de vasos ja existentes, fenomeno designado como angiogenese, esta envolvido em varios processos fisiologicos e patologicos, incluindo o crescimento tumoral, onde a angiogenese desempenha papel critico na nutricao das celulas tumorais. Tal como a angiogenese, o sistema de coagulacao sanguinea exerce importante funcao na biologia vascular. A trombina, uma serina protease, tem papel fundamental na cascata de coagulacao, pela quebra enzimatica do fibrinogenio e pela consequente producao de fibrina. Essa protease encontra-se tambem implicada em desenvolvimento embrionario, inflamacao e cicatrizacao, processos nos quais a remodelacao vascular esta altamente ativa. Apesar de o crescente conhecimento de fatores reguladores da angiogenese e da coagulacao demonstrar que a carcinogenese, a coagulacao e a angiogenese sao tres close team players, ainda muito pouco se sabe sobre o modo como esses players comunicam-se e interagem no processo de angiogenese in vivo. Esta revisao sumariza os conhecimentos atuais quanto ao papel da cascata de coagulacao na conducao do processo angiogenico e do crescimento tumoral, bem como oferece uma visao geral sobre recentes terapias antiangiogenicas e anticoagulantes envolvidas na regressao tumoral.

Molecular screening of ovine mastitis in different breeds.

Clinical and subclinical mastitis directly affect mammary gland function and have a great economic impact on the sheep and goat dairy industries. The present study explores molecular diagnosis of ovine subclinical mastitis as a faster and more precise screening method compared with microbiology and biochemical techniques to assess the molecular and chemical properties of raw milk samples from healthy animals from 3 breeds of sheep raised in Portugal. Based on 16S ribosomal RNA screening by PCR, milk samples from all sheep were categorized as contaminated (n=123) or noncontaminated (n=104). For contaminated milk, different specific primers were used for pathogen identification (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus uberis). Streptococcus agalactiae was identified as the most frequent agent. We further assessed whether contaminated versus noncontaminated samples were chemically different in terms of fat, protein, lactose, pH, and solids-not-fat. This molecular screening method allowed rapid and efficient identification of contaminated raw sheep milk, including pathogen identification, before significant alterations in milk chemical properties could be detected. This methodology may lead to a specific and efficient animal treatment and consequently less expensive flock management.

Antiproliferative Effects of Cynara cardunculus L. var. altilis (DC) Lipophilic Extracts

Besides being traditionally used to relieve hepatobiliary disorders, Cynara cardunculus L. has evidenced anticancer potential on triple-negative breast cancer (TNBC). This study highlights the antiproliferative effects of lipophilic extracts from C. cardunculus L. var. altilis (DC) leaves and florets, and of their major compounds, namely cynaropicrin and taraxasteryl acetate, against MDA-MB-231 cells. Our results demonstrated that MDA-MB-231 cells were much less resistant to leaves extract (IC50 10.39 µg/mL) than to florets extract (IC50 315.22 µg/mL), during 48 h. Moreover, leaves extract and cynaropicrin (IC50 6.19 µg/mL) suppressed MDA-MB-231 cells colonies formation, via an anchorage-independent growth assay. Leaves extract and cynaropicrin were also assessed regarding their regulation on caspase-3 activity, by using a spectrophotometric assay, and expression levels of G2/mitosis checkpoint and Akt signaling pathway proteins, by Western blotting. Leaves extract increased caspase-3 activity, while cynaropicrin did not affect it. Additionally, they caused p21Waf1/Cip1 upregulation, as well as cyclin B1 and phospho(Tyr15)-CDK1 accumulation, which may be related to G2 cell cycle arrest. They also downregulated phospho(Ser473)-Akt, without changing total Akt1 level. Cynaropicrin probably contributed to leaves extract antiproliferative action. These promising insights suggest that cultivated cardoon leaves lipophilic extract and cynaropicrin may be considered toward a natural-based therapeutic approach on TNBC.

Impact of novel SNPs identified in Cynara cardunculus genes on functionality of proteins regulating phenylpropanoid pathway and their association with biological activities

BackgroundCynara cardunculus L. offers a natural source of phenolic compounds with the predominant molecule being chlorogenic acid. Chlorogenic acid is gaining interest due to its involvement in various biological properties such as, antibacterial, antifungal, antioxidant, hepatoprotective, and anticarcinogenic activities.ResultsIn this work we screened a Cynara cardunculus collection for new allelic variants in key genes involved in the chlorogenic acid biosynthesis pathway. The target genes encode p-coumaroyl ester 3′-hydroxylase (C3′H) and hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase (HQT), both participating in the synthesis of chlorogenic acid. Using high-resolution melting, the C3′H gene proved to be highly conserved with only 4 haplotypes while, for HQT, 17 haplotypes were identified de novo. The putative influence of the identified polymorphisms in C3′H and HQT proteins was further evaluated using bioinformatics tools. We could identify some polymorphisms that may lead to protein conformational changes. Chlorogenic acid content, antioxidant and antithrombin activities were also evaluated in Cc leaf extracts and an association analysis was performed to assess a putative correlation between these traits and the identified polymorphisms.ConclusionIn this work we identified allelic variants with putative impact on C3′H and HQT proteins which are significantly associated with chlorogenic acid content and antioxidant activity. Further study of these alleles should be explored to assess putative relevance as genetic markers correlating with Cynara cardunculus biological properties with further confirmation by functional analysis.