Further Withaphysalin Derivatives from Acnistus arborescens

Two new epimeric chlorinated withaphysalins, rel-(4 beta,5 beta,6 alpha,18S,22R)- and rel-(4 beta,5 beta,6 alpha,18R,22R)-6-chloro-18,20-epoxy-18-ethoxy-4,5-dihydroxy-1- oxowitha-2,24-diene-26,22-lactone (1 and 2 resp.), together with the new rel-(4 beta,5 beta,6a,18R,22R)-6-chloro-18,20-epoxy-4,5-dihydroxy-18-methoxy-1-oxowitha-2,24-diene-26,22-lactone (3) and rel-(3 beta,4 beta,5 beta,6 beta,18R,22R)-5,6:18,20-diepoxy-3,18-diethoxy-4-hydroxy-1-oxowith-24-ene-26,22-lactone (4) were isolated from the leaves of Acnistus arborescens and named withaphysalins TW, respectively. The final structures and the complete 1H- and 13C-NMR assignments of the three chlorowithaphysalins 13 were performed by means of HR-ESI-MS and 1D- and 2D-NMR experiments, including COSY, HSQC, and HMBC, beside comparison with spectral data of analogous compounds from the literature. The structure of 4 was also confirmed by means of a single-crystal X-ray diffraction analysis.

Expression of laminin-5 and integrins in actinic cheilitis and superficially invasive squamous cell carcinomas of the lip

The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5 gamma 2 chain as well as alpha 3, beta 1 subunits of alpha 3 beta 1 heterodimer and beta 4 subunit of integrin alpha 6 beta 4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of beta 1, beta 4 and alpha 3 integrins, and SISCCs lacked beta 1, beta 4 and alpha 3 integrins in the invasive front. AC cases were negative for the Ln-5 gamma 2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5 gamma 2 chain expression in the invasive front of the tumor. Integrin beta 1, beta 4 and alpha 3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5 gamma 2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype. (C) 2012 Elsevier GmbH. All rights reserved.

Diversidade genética em germoplasma tropical de cebola estimada via marcadores RAPD

A cebola é uma cultura de expressiva importância socioeconômica para o Brasil. Marcantes contribuições para o desenvolvimento da cultura têm sido feitas utilizando-se germoplasma de cebola adaptado às regiões tropicais e subtropicais. Nesse contexto, o presente trabalho teve como objetivo estudar a diversidade genética existente em uma coleção de germoplasma potencialmente útil ao desenvolvimento de cultivares para essas regiões. Para isso, a variabilidade genética de um grupo de 21 acessos foi analisada via marcadores RAPD. Esses acessos ('Red Creole', 'Roxa IPA-3', 'Valenciana 14', 'Beta Cristal', 'Diamante', 'Composto IPA-6', 'Aurora', 'Bojuda Rio Grande', 'Alfa Tropical', 'Pêra IPA-4', 'Primavera', 'Belém IPA-9', 'Crioula Alto Vale', 'Conquista', 'Pira-Ouro', 'Vale-Ouro IPA-11', 'Franciscana IPA-10', 'Serrana', 'CNPH 6400', 'Petroline' e 'Baia Periforme') têm sido empregados como germoplasma e/ou foram desenvolvidos pelos programas de melhoramento genético de cebola conduzidos no Brasil. Dos 520 iniciadores ('primers') utilizados na triagem inicial, somente 38 confirmaram polimorfismos entre os 21 acessos. Esses 38 'primers' produziram 624 amplicons, dos quais 522 (83,7%) foram monomórficos e 102 (16,3%) polimórficos. Com base nos padrões revelados, seis grupos foram formados de acordo com a similaridade média global entre os acessos (= 0,72). Somente um desses seis grupos englobou mais de um acesso. O grupo principal (formado por 16 acessos) incluiu, predominantemente, as cultivares que apresentam no seu pedigree a contribuição de 'Baia Periforme' ('Diamante', 'Composto IPA-6', 'Aurora', 'Bojuda Rio Grande', 'Conquista', 'Pira-Ouro', 'Serrana', 'Vale-Ouro IPA-11', 'Baia Periforme', 'Primavera', 'Franciscana IPA-10', 'Belém IPA-9', 'Crioula Alto Vale', 'Petroline', 'Pêra IPA-4' e 'Alfa Tropical'). As cultivares 'Red Creole', 'Roxa IPA-3', 'Beta Cristal', 'CNPH 6400' e 'Valenciana 14' formaram agrupamentos isolados e distintos do grupo 'Baia Periforme', revelando, dessa forma, divergência genética entre essas cinco populações e o grupo principal. Verificou-se que os materiais estudados possuem base genética relativamente estreita, apresentando, em sua grande maioria origem na população 'Baia Periforme'. Existem, no entanto, alguns materiais divergentes, cuja diversidade pode ser explorada em programas de melhoramento.

Cytotoxic steroidal saponins from the rhizomes of Tacca integrifolia

Three new steroid saponins (3beta,25R)-spirost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)-6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (1), (3beta,22R,25R)-26-(beta-D-glucopyranosyloxy)-22-hydroxyfurost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (3), and (3beta,22R,25R)-26-(beta-D-glucopyranosyloxy)-22-hydroxyfurost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[beta-D-glucopyranosyl-(1-->4)-6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (5), as well as the new pregnane glycoside (3beta,16beta)-3-{[6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranosyl]oxy}-20-oxopregn-5-en-16-yl (4R)-5-(beta-D-glucopyranosyloxy)-4-methylpentanoate (6), were isolated from the rhizomes of Tacca integrifolia together with two known (25R) configurated steroid saponins (3beta,25R)-spirost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (2) and (3beta,22R,25R)-26-(beta-D-glucopyranosyloxy)-22-methoxyfurost-5-en-3-yl 6-deoxy-alpha-L-mannopyranosyl-(1-->2)-[6-deoxy-alpha-L-mannopyranosyl-(1-->3)]-beta-D-glucopyranoside (4). The cytotoxic activity of the isolated compounds was evaluated in HeLa cells and showed the highest cytotoxicity value for compound 2 with an IC(50) of 1.2+/-0.4 muM. Intriguingly, while compounds 1-5 exhibited similar cytotoxic properties between 1.2+/-0.4 (2) and 4.0+/-0.6 muM (5), only compound 2 showed a significant microtubule-stabilizing activity in vitro.

Patterns of molecular response to and relapse after combination of sorafenib, idarubicin, and cytarabine in patients with FLT3 mutant acute myeloid leukemia

BACKGROUND: FMS-like tyrosine kinase 3 (FLT3) is a class III receptor tyrosine kinase involved in hematopoietic progenitor cell development. Mutations of FLT3 have been reported in about a third of patients with acute myeloid leukemia (AML), and inhibitors of FLT3 are of clinical interest. Sorafenib is an orally active multikinase inhibitor with potent activity against FLT3 and the Raf/ERK/MEK kinase pathway. METHODS: We studied the patterns of molecular response and relapse in 18 patients with mutated FLT3 treated with the combination of sorafenib, idarubicin, and cytarabine. RESULTS: The median follow-up was 9 months. Sixteen patients achieved complete remission (CR), and the other 2 patients achieved CR but lacked platelet recovery for an overall response rate of 100%. Ten patients had their FLT3-mutated clone eradicated, with 6 patients who showed some residual FLT3-mutated cells, and 2 patients who showed persistent FLT3-mutated cells. The elimination of FLT3-mutated population at the time of morphologic CR, however, was not predictive of relapse. After a median follow-up of 9 months (range, 1-16 months), 10 (55%) patients had relapsed, with a median CR duration of 8.8 months (range, 1-9.5 months). By DNA sequencing, there was no evidence of an acquired FLT3 point mutation at the time of relapse in 7 patients tested, which suggested the presence of other mechanisms of sorafenib resistance. CONCLUSION: Sorafenib, combined with chemotherapy, is effective in attaining CR, but relapses still occur.

Regulation of endothelial cell cytoskeletal reorganization by a secreted frizzled-related protein-1 and frizzled 4- and frizzled 7-dependent pathway: role in neovessel formation

Consistent with findings of Wnt pathway members involved in vascular cells, a role for Wnt/Frizzled signaling has recently emerged in vascular cell development. Among the few Wnt family members implicated in vessel formation in adult, Wnt7b and Frizzled 4 have been shown as involved in vessel formation in the lung and in the retina, respectively. Our previous work has shown a role for secreted Frizzled-related protein-1 (sFRP-1), a proposed Wnt signaling inhibitor, in neovascularization after an ischemic event and demonstrated its role as a potent angiogenic factor. However the mechanisms involved have not been investigated. Here, we show that sFRP-1 treatment increases endothelial cell spreading on extracellular matrix as revealed by actin stress fiber reorganization in an integrin-dependent manner. We demonstrate that sFRP-1 can interact with Wnt receptors Frizzled 4 and 7 on endothelial cells to transduce downstream to cellular machineries requiring Rac-1 activity in cooperation with GSK-3beta. sFRP-1 overexpression in endothelium specifically reversed the inactivation of GSK-3 beta and increased neovascularization in ischemia-induced angiogenesis in mouse hindlimb. This study illustrates a regulated pathway by sFRP-1 involving GSK-3beta and Rac-1 in endothelial cell cytoskeletal reorganization and in neovessel formation.

Nutrient-responsive mTOR signalling grows on Sterile ground

The control of cell growth, that is cell size, is largely controlled by mTOR (the mammalian target of rapamycin), a large serine/threonine protein kinase that regulates ribosome biogenesis and protein translation. mTOR activity is regulated both by the availability of growth factors, such as insulin/IGF-1 (insulin-like growth factor 1), and by nutrients, notably the supply of certain key amino acids. The last few years have seen a remarkable increase in our understanding of the canonical, growth factor-regulated pathway for mTOR activation, which is mediated by the class I PI3Ks (phosphoinositide 3-kinases), PKB (protein kinase B), TSC1/2 (the tuberous sclerosis complex) and the small GTPase, Rheb. However, the nutrient-responsive input into mTOR is important in its own right and is also required for maximal activation of mTOR signalling by growth factors. Despite this, the details of the nutrient-responsive signalling pathway(s) controlling mTOR have remained elusive, although recent studies have suggested a role for the class III PI3K hVps34. In this issue of the Biochemical Journal, Findlay et al. demonstrate that the protein kinase MAP4K3 [mitogen-activated protein kinase kinase kinase kinase-3, a Ste20 family protein kinase also known as GLK (germinal centre-like kinase)] is a new component of the nutrient-responsive pathway. MAP4K3 activity is stimulated by administration of amino acids, but not growth factors, and this is insensitive to rapamycin, most likely placing MAP4K3 upstream of mTOR. Indeed, MAP4K3 is required for phosphorylation of known mTOR targets such as S6K1 (S6 kinase 1), and overexpression of MAP4K3 promotes the rapamycin-sensitive phosphorylation of these same targets. Finally, knockdown of MAP4K3 levels causes a decrease in cell size. The results suggest that MAP4K3 is a new component in the nutrient-responsive pathway for mTOR activation and reveal a completely new function for MAP4K3 in promoting cell growth. Given that mTOR activity is frequently deregulated in cancer, there is much interest in new strategies for inhibition of this pathway. In this context, MAP4K3 looks like an attractive drug target since inhibitors of this enzyme should switch off mTOR, thereby inhibiting cell growth and proliferation, and promoting apoptosis.

Calcineurin inhibition or mutation enhances cell wall inhibitors against Aspergillus fumigatus

Calcineurin mutation or inhibition enhanced the antifungal morphological effect of cell wall inhibitors caspofungin or nikkomycin Z against Aspergillus fumigatus. Quantification of 1,3-beta-d-glucan revealed decreased amounts in the calcineurin A (DeltacnaA) mutant. Calcineurin can be an excellent adjunct therapeutic target in combination with other cell wall inhibitors against A. fumigatus.

Activation of the unfolded protein response is associated with favorable prognosis in acute myeloid leukemia

PURPOSE: The unfolded protein response is triggered by the accumulation of misfolded proteins within the endoplasmic reticulum. Previous studies suggest that the unfolded protein response is activated in some cancer cell lines and involved in tumor development. The role of the unfolded protein response during leukemogenesis is unknown thus far. EXPERIMENTAL DESIGN: Here, we assessed the induction of key effectors of the unfolded protein response in leukemic cells at diagnosis of 105 acute myeloid leukemia (AML) patients comprising all subtypes. We determined the formation of the spliced variant of the X-box-binding protein 1 (XBP1) mRNA, as well as expression levels of calreticulin, GRP78, and CHOP mRNA. RESULTS: The formation of the spliced variant of XBP1s was detectable in 16.2% (17 of 105) of AML patients. Consistent with activated unfolded protein response, this group also had significantly increased expression of calreticulin, GRP78, and CHOP. AML patients with activated unfolded protein response had lower WBC counts, lactate dehydrogenase levels, and more frequently, secondary AML. The incidence of fms-related tyrosine kinase 3 (FLT3) mutations was significantly lower in patients with activated unfolded protein response. In addition, an association was observed between activated unfolded protein response and deletion of chromosome 7. Finally, the clinical course of AML patients with activated unfolded protein response was more favorable with lower relapse rate (P = 0.0182) and better overall (P = 0.041) and disease-free survival (P = 0.022). CONCLUSIONS: These results suggest that the unfolded protein response is activated in a considerable subset of AML patients. AML patients with activated unfolded protein response present specific clinical characteristics and a more favorable course of the disease.

A deleterious gene-by-environment interaction imposed by calcium channel blockers in Marfan syndrome.

Calcium channel blockers (CCBs) are prescribed to patients with Marfan syndrome for prophylaxis against aortic aneurysm progression, despite limited evidence for their efficacy and safety in the disorder. Unexpectedly, Marfan mice treated with CCBs show accelerated aneurysm expansion, rupture, and premature lethality. This effect is both extracellular signal-regulated kinase (ERK1/2) dependent and angiotensin-II type 1 receptor (AT1R) dependent. We have identified protein kinase C beta (PKCβ) as a critical mediator of this pathway and demonstrate that the PKCβ inhibitor enzastaurin, and the clinically available anti-hypertensive agent hydralazine, both normalize aortic growth in Marfan mice, in association with reduced PKCβ and ERK1/2 activation. Furthermore, patients with Marfan syndrome and other forms of inherited thoracic aortic aneurysm taking CCBs display increased risk of aortic dissection and need for aortic surgery, compared to patients on other antihypertensive agents.

NET formation can occur independently of RIPK3 and MLKL signaling

The importance of neutrophil extracellular traps (NETs) in innate immunity is well established but the molecular mechanisms responsible for their formation are still a matter of scientific dispute. Here, we aim to characterize a possible role of the receptor-interacting protein kinase 3 (RIPK3) and the mixed lineage kinase domain-like (MLKL) signaling pathway, which are known to cause necroptosis, in NET formation. Using genetic and pharmacological approaches, we investigated whether this programmed form of necrosis is a prerequisite for NET formation. NETs have been defined as extracellular DNA scaffolds associated with the neutrophil granule protein elastase that are capable of killing bacteria. Neither Ripk3-deficient mouse neutrophils nor human neutrophils in which MLKL had been pharmacologically inactivated, exhibited abnormalities in NET formation upon physiological activation or exposure to low concentrations of PMA. These data indicate that NET formation occurs independently of both RIPK3 and MLKL signaling.

Activation of dendritic cells from patients with epithelial ovarian cancer

Dendritic cells (DCs) are the most potent antigen-presenting cells for inducing immune responses to tumor cells. Lin−HLA-DR + DC populations in peripheral blood mononuclear cells (PBMCs) and in ascites mononuclear leukocytes (MNLs) of patients with epithelial ovarian cancer (EOC) are phenotypically immature. Lin−HLA-DR + DCs from PBMCs of normal subjects and EOC patients and MNLs from ascites cells of patients were examined for specific cell surface markers or indicators of differentiation or activation. Separating Lin− HLA-DR+ DCs into subsets based on their HLA-DR intensity provided an additional method for identifying the two major lineages of DCs, myeloid and plasmacytoid. The activation potential of these DCs following exposure to the maturation agents CD40 ligand (CD40L) and lipopolysaccharide (LPS) was examined by measurement of IL-12 and IL-10 concentrations in DC culture supernatants in addition to their ability to stimulate allogeneic T cells. DCs from PBMCs of normal subjects and EOC patients and DCs isolated from ascites MNLs of EOC patients were separated into subsets based on CD11c and CD123 cell surface marker expression identifying the major DC types. These subsets were then compared with cells sorted on the basis of HLA-DR intensity. The in vivo behavior of DCs and DC subsets in peripheral blood and ascites following treatment of peritoneal carcinoma patients with the growth factor fins-like tyrosine kinase 3 ligand (Flt3L) was also examined. Increases in proportions and total numbers of DCs from peripheral blood and ascites were associated with increased secretion of IL-12 and IL-10 following in vitro activation of cultured DCs. There were differences between DCs from PBMCs and ascites and between DC subsets in expression of cell surface markers, cytokine profile, and the ability of Lin−HLA-DR + cells to stimulate proliferation of allogeneic T cells from EOC patients. These Lin−HLA-DR+ cells have certain functional properties that suggest that they could have the potential to facilitate an adaptive anti-tumor immune response. ^

cAMP regulates IL-2 receptor signaling in human T cells

Proper immune system function is dependent on positive and negative regulation of T cell signaling pathways. Full T cell activation requires sequential signaling through the T cell receptor (TCR), costimulatory molecules and the IL-2 receptor (IL-2R). The IL-2R associated Janus tyrosine kinase 3 (Jak3), as well as Signal transducer and activator of transcription 5 (Stat5), are required for normal T cell function and survival. Constitutive activation of Jak3 and Stat5 have been linked to cancers of hematopoietic origin, including certain lymphomas and leukemias. ^ The production of cAMP by adenylate cyclase has been shown to negatively regulate human TCR mediated cell proliferation. Since cAMP has been shown to negatively regulate T cell activation, we sought to investigate whether crosstalk exists between cAMP and IL-2R signaling. The first objective of this study was to determine the effect of cAMP on the activation of IL-2R signaling molecules Jak3 and Stat5. We found that the potent adenylate cyclase activator, forskolin, inhibited IL-2 activation of Jak3 and Stat5. Indeed, in vitro kinase assays and electrophoretic mobility shift assays verified a loss of Jak3 enzymatic activity and Stat5 DNA binding ability, respectively. Further analysis of IL-2R signaling showed that forskolin treatment reduced IL-2 induced association of the IL-2Rβ and γc chain. ^ Because cAMP activates protein kinase A (PKA), the second objective was to determine the role for PKA in the cAMP directed regulation of IL-2R signaling intermediates. Interestingly, forskolin induced serine phosphorylation of Jak3, suggesting that cAMP can directly regulate Jak3 via activation of a serine/threonine kinase. Indeed, phosphoamino acid analysis revealed that PKA was able to induce Jak3 serine phosphorylation in the human leukemia cell line MT-2. In addition, in vitro kinase assays established that PKA can directly inhibit Jak3 enzymatic activity. Collectively, these data indicate that cAMP negatively regulates IL-2R signaling via various effector molecules by a previously unrecognized mechanism. This new data suggests that the Jak3/Stat5 pathway may be regulated by various pharmacological agents that stimulate cAMP production and thus can be used to uncouple some types of T cell mediated diseases. ^

Twenty-five coregulated transcripts define a sterigmatocystin gene cluster in Aspergillus nidulans.

Sterigmatocystin (ST) and the aflatoxins (AFs), related fungal secondary metabolites, are among the most toxic, mutagenic, and carcinogenic natural products known. The ST biosynthetic pathway in Aspergillus nidulans is estimated to involve at least 15 enzymatic activities, while certain Aspergillus parasiticus, Aspergillus flavus, and Aspergillus nomius strains contain additional activities that convert ST to AF. We have characterized a 60-kb region in the A. nidulans genome and find it contains many, if not all, of the genes needed for ST biosynthesis. This region includes verA, a structural gene previously shown to be required for ST biosynthesis, and 24 additional closely spaced transcripts ranging in size from 0.6 to 7.2 kb that are coordinately induced only under ST-producing conditions. Each end of this gene cluster is demarcated by transcripts that are expressed under both ST-inducing and non-ST-inducing conditions. Deduced polypeptide sequences of regions within this cluster had a high percentage of identity with enzymes that have activities predicted for ST/AF biosynthesis, including a polyketide synthase, a fatty acid synthase (alpha and beta subunits), five monooxygenases, four dehydrogenases, an esterase, an 0-methyltransferase, a reductase, an oxidase, and a zinc cluster DNA binding protein. A revised system for naming the genes of the ST pathway is presented.

PKC alpha is activated but not required during glucose-induced insulin secretion from rat pancreatic islets

The role of protein kinase C (PKC) in glucose-stimulated insulin secretion (GSIS) is controversial. Using recombinant adenoviruses for overexpression of PKCalpha and PKCdelta, in both wild-type (WT) and kinase-dead (KD) forms, we here demonstrate that activation of these two PKCs is neither necessary nor sufficient for GSIS from batch-incubated, rat pancreatic islets. In contrast, responses to the pharmacologic activator 12-O-tetradecanoylphorbol-13-acetate (TPA) were reciprocally modulated by overexpression of the PKCalphaWT or PKCalphaKD but not the corresponding PKCdelta adenoviruses. The kinetics of the secretory response to glucose (monitored by perifusion) were not altered in either cultured islets overexpressing PKCalphaKD or freshly isolated islets stimulated in the presence of the conventional PKC (cPKC) inhibitor Go6976. However, the latter did inhibit the secretory response to TPA. Using phosphorylation state-specific antisera for consensus PKC phosphorylation sites, we also showed that (compared with TPA) glucose causes only a modest and transient functional activation of PKC (maximal at 2-5 min). However, glucose did promote a prolonged (15 min) phosphorylation of PKC substrates in the presence of the phosphatase inhibitor okadaic acid. Overall, the results demonstrate that glucose does stimulate PKCalphain pancreatic islets but that this makes little overall contribution to GSIS.