β-catenin confers resistance to PI3K and AKT inhibitors and subverts FOXO3a to promote metastasis in colon cancer

The Wnt–β-catenin and PI3K-AKT-FOXO3a pathways have a central role in cancer. AKT phosporylates FOXO3a, relocating it from the cell nucleus to the cytoplasm, an effect that is reversed by PI3K and AKT inhibitors. Simultaneous hyperactivation of the Wnt–β-catenin pathway and inhibition of PI3K-AKT signaling promote nuclear accumulation of β-catenin and FOXO3a, respectively, promoting cell scattering and metastasis by regulating a defined set of target genes. Indeed, the anti-tumoral AKT inhibitor API-2 promotes nuclear FOXO3a accumulation and metastasis of cells with high nuclear β-catenin content. Nuclear β-catenin confers resistance to the FOXO3a-mediated apoptosis induced by PI3K and AKT inhibitors in patient-derived primary cultures and in corresponding xenograft tumors in mice. This resistance is reversed by XAV-939, an inhibitor of Wnt–β-catenin signaling. In the presence of high nuclear β-catenin content, activation of FOXO3a by PI3K or AKT inhibitors makes it behave as a metastasis inductor rather than a proapoptotic tumor suppressor. We show that it is possible to evaluate the β-catenin status of patients' carcinomas and the response of patient-derived cells to target-directed drugs that accumulate FOXO3a in the nucleus before deciding on a course of treatment. We propose that this evaluation could be essential to the provision of a safer and more effective personalized treatment.     

High-fat diets rich in soy or fish oil distinctly alter hypothalamic insulin signaling in rats

Hypothalamic insulin inhibits food intake, preventing obesity. High-fat feeding with polyunsaturated fats may be obesogenic, but their effect on insulin action has not been elucidated. The present study evaluated insulin hypophagia and hypothalamic signaling after central injection in rats fed either control diet (15% energy from fat) or high-fat diets (50% energy from fat) enriched with either soy or fish oil. Soy rats had increased fat pad weight and serum leptin with normal body weight, serum lipid profile and peripheral insulin sensitivity. Fish rats had decreased body and fat pad weight, low leptin and corticosterone levels, and improved serum lipid profile. A 20-mU dose of intracerebroventricular (ICV) insulin inhibited food intake in control and fish groups, but failed to do so in the soy group. Hypothalamic protein levels of IR, IRS-1, IRS-2, Akt, mTOR, p70S6K and AMPK were similar among groups. ICV insulin stimulated IR tyrosine phosphorylation in control (68%), soy (36%) and fish (34%) groups. Tyrosine phosphorylation of the pp185 band was significantly stimulated in control (78%) and soy (53%) rats, but not in fish rats. IRS-1 phosphorylation was stimulated only in control rats (94%). Akt serine phosphorylation was significantly stimulated only in control (90%) and fish (78%) rats. The results showed that, rather than the energy density, the fat type was a relevant aspect of high-fat feeding, since blockade of hypothalamic insulin signal transmission and insulin hypophagia was promoted only by the high-fat soy diet, while they were preserved in the rats fed with the high-fat fish diet.     

Differential expression of cruzipain- and gp63-like molecules in the phytoflagellate trypanosomatid Phytomonas serpens induced by exogenous proteins

Phytomonas serpens synthesizes metallo- and cysteine-proteases that are related to gp63 and cruzipain, respectively, two virulence factors produced by pathogenic trypanosomatids. Here, we described the cellular distribution of gp63- and cruzipain-like molecules in P. serpens through immunocytochemistry and confocal fluorescence microscopy. Both proteases were detected in distinct cellular compartments, presenting co-localization in membrane domains and intracellular regions. Subsequently, we showed that exogenous proteins modulated the production of both protease classes, but in different ways. Regarding the metalloprotease, only fetal bovine serum (FBS) influenced the gp63 expression, reducing its surface exposition (≈30%). Conversely, the cruzipain-like molecule was differentially modulated according to the proteins: human and bovine albumins reduced its expression around 50% and 35%, respectively; mucin and FBS did not alter its production, while IgG and hemoglobin drastically enhanced its surface exposition around 7- and 11-fold, respectively. Additionally, hemoglobin induced an augmentation in the cell-associated cruzipain-like activity in a dose-dependent manner. A twofold increase of the secreted cruzipain-like protein was detected after parasite incubation with 1% hemoglobin compared to the parasites incubated in PBS-glucose. The results showed the ability of P. serpens in modulating the expression and the activity of proteolytic enzymes after exposition to exogenous proteins, with emphasis in its cruzipain-like molecules.     

Supressive effect of maslinic acid from pomace olive oil on oxidative stress and cytokine production in stimulated murine macrophages

The pentacyclic triterpene maslinic acid (MA) is a natural compound present in the non glyceride fraction of pomace olive oil, also called orujo olive oil. This compound has previously demonstrated antioxidant properties against lipid peroxidation in vitro, but its effects on reactive oxygen and nitrogen-derived species and pro-inflammatory cytokines generated by a cell system have not yet been investigated. In this study, we have tested the effect of MA upon oxidative stress and cytokine production using peritoneal murine macrophages. MA significantly inhibited the enhanced production of nitric oxide (NO) induced by lypopolysaccharide (LPS) when it was measured by the nitrite production with an inhibitory concentration 50% value (IC(50)) of 25.4 microM. This inhibiting effect seems to be consequence of an action at the level of the LPS-induction of the inducible nitric oxide synthethase (iNOS) gene enzyme expression rather than to a direct inhibitory action on enzyme activity. The secretion of the inflammatory cytokines interleukine-6 and TNF-a from LPS-stimulated murine macrophages was also significantly reduced (p < 0.05 and 0.01) by 50 and 100 microM of MA. In addition, reactive oxygen species were measured after stimulation with phorbol-12-myristate-13-acetate (PMA). Thus, pre-treatment with MA reduced the generation of hydrogen peroxide from stimulated macrophages in a dose-dependent manner (IC(50): 43.6 microM) as assayed by the oxidation of the peroxidase enzyme. However, no inhibitory effect on superoxide release, measured by the reduction of ferricytochrome c, was observed after the pretreatment with MA in the culture medium.These results suggest a potential biopharmaceutical use of this hydroxy-pentacyclic triterpene derivative, present in orujo olive oil, on preventing oxidative stress and pro-inflammatory cytokine generation.     

Relationship between Debaryomyces pseudopolymorphus enzymatic extracts and release of terpenes in wine

As a result of the interest that exists in the liberation of aromas in young wines, we obtained some different enzymatic extracts (purified extract, P; lyophilized purified extract, LP; immobilized purified extract, IP; and immobilized lyophilized purified extract, ILP) with beta-glucosidase activity from Debaryomyces pseudopolymorphus, which excreted the enzyme in the growth medium. The extracts were added to natural glycosides isolated from different grape varieties. The results were compared with the effect of seven commercial enzyme preparations (CEP), obtained from molds used in wine making. It was shown that some yeast extracts had effects similar to those of the CEP, and the next step was to use them on wine samples elaborated in the laboratory. The effect was studied at 9 and 16 days of contact, quantifying both the precursors that were retained and the liberated terpenes. The results were compared with a control wine (without any extract) and with wine treated with a commercial enzyme preparation specially indicated for the liberation of aromas. It was observed that the enzymatic extracts from Db. pseudopolymorphus hydrolyzed the precursors in wine and that they could compete with the commercial preparations since the liberation was produced in even less time.     

Reliability of FEV1/FEV6 to Diagnose Airflow Obstruction Compared with FEV1/FVC: The PLATINO Longitudinal Study

QUESTION

A 6-second spirometry test is easier than full exhalations. We compared the reliability of the ratio of the Forced expiratory volume in 1 second/Forced expiratory volume in 6 seconds (FEV₁/FEV₆) to the ratio of the FEV₁/Forced vital capacity (FEV₁/FVC) for the detection of airway obstruction.

METHODS

The PLATINO population-based survey in individuals aged 40 years and over designed to estimate the prevalence of post-Bronchodilator airway obstruction repeated for the same study participants after 5–9 years in three Latin-American cities.

RESULTS

Using the FEV₁/FVC<Lower limit of normal (LLN) index, COPD prevalence apparently changed from 9.8 to 13.2% in Montevideo, from 9.7 to 6.0% in São Paulo and from 8.5 to 6.6% in Santiago, despite only slight declines in smoking prevalence (from 30.8% to 24.3%). These changes were associated with differences in Forced expiratory time (FET) between the two surveys. In contrast, by using the FEV₁/FEV₆ to define airway obstruction, the changes in prevalence were smaller: 9.7 to 10.6% in Montevideo, 8.6 to 9.0% in São Paulo, and 7.5 to 7.9% in Santiago. Changes in the prevalence of COPD with criteria based on FEV₁/FVC correlated strongly with changes in the FET of the tests (R² 0.92) unlike the prevalence based on a low FEV₁/FEV₆ (R² = 0.40).

CONCLUSION

The FEV₁/FEV₆ is a more reliable index than FEV₁/FVC because FVC varies with the duration of the forced exhalation. Reporting FET and FEV₁/FEV₆<LLN helps to understand differences in prevalence of COPD obtained from FEV₁/FVC-derived indices.     

Hypoxic cardiorespiratory reflexes in the facultative air-breathing fish jeju (Hoplerythrinus unitaeniatus): role of branchial O2 chemoreceptors

In one series of experiments, heart frequency (f _H), blood pressure (P _a), gill ventilation frequency (f _R ), ventilation amplitude (V _AMP) and total gill ventilation (V _TOT) were measured in intact jeju (Hoplerythrinus unitaeniatus) and jeju with progressive denervation of the branchial branches of cranial nerves IX (glossopharyngeal) and X (vagus) without access to air. When these fish were submitted to graded hypoxia (water PO₂ ~140, normoxia to 17 mmHg, severe hypoxia), they increased f _R , V _AMP, V _TOT and P _a and decreased f _H. In a second series of experiments, air-breathing frequency (f _RA), measured in fish with access to the surface, increased with graded hypoxia. In both series, bilateral denervation of all gill arches eliminated the responses to graded hypoxia. Based on the effects of internal (caudal vein, 150 μg NaCN in 0.2 mL saline) and external (buccal) injections of NaCN (500 μg NaCN in 1.0 mL water) on f _R , V _AMP, V _TOT, P _a and f _H we conclude that the O₂ receptors involved in eliciting changes in gill ventilation and associated cardiovascular responses are present on all gill arches and monitor the O₂ levels of both inspired water and blood perfusing the gills. We also conclude that air breathing arises solely from stimulation of branchial chemoreceptors and support the hypothesis that internal hypoxaemia is the primary drive to air breathing.     

Roles of alternative splicing in the functional properties of inner ear-specific KCNQ4 channels

The function of the KCNQ4 channel in the auditory setting is crucial to hearing, underpinned by the finding that mutations of the channel result in an autosomal dominant form of nonsyndromic progressive high frequency hearing loss. The precise function of KCNQ4 in the inner ear has not been established. However, recently we demonstrated that there is differential expression among four splice variants of KCNQ4 (KCNQ4_v1-v4) along the tonotopic axis of the cochlea. Alternative splicing specifies the outcome of functional channels by modifying the amino acid sequences within the C terminus at a site designated as the membrane proximal region. We show that variations within the C terminus of splice variants produce profound differences in the voltage-dependent phenotype and functional expression of the channel. KCNQ4_v4 lacks exons 9-11, resulting in deletion of 54 amino acid residues adjacent to the S6 domain compared with KCNQ4_v1. Consequently, the voltage-dependent activation of KCNQ4_v4 is shifted leftward by approximately 20 mV, and the number of functional channels is increased severalfold compared with KCNQ4_v1. The properties of KCNQ4_v2 and KCNQ4_v3 fall between KCNQ4_v1 and KCNQ4_v4. Because of variations in the calmodulin binding domains of the splice variants, the channels are differentially modulated by calmodulin. Co-expression of these splice variants yielded current magnitudes suggesting that the channels are composed of heterotetramers. Indeed, a dominant negative mutant of KCNQ4_v1 cripples the currents of the entire KCNQ4 channel family. Furthermore, the dominant negative KCNQ4 mutant stifles the activity of KCNQ2-5, raising the possibility of a global disruption of KCNQ channel activity and the ensuing auditory phenotype.     

Ligands and signaling of the G-protein-coupled receptor GPR14, expressed in human kidney cells.

Activation of the urotensin II (U-II) receptor, GPR14, leads to an increase in Ca(2+), activation of phospholipase A(2) (PLA(2)) and an increase in arachidonic acid. The signaling pathway for guanylin peptides in the kidney involves an unknown G-protein coupled receptor which activates PLA(2) and increases arachidonic acid as well. To test if guanylin peptides could be, as U-II, agonists for the GPR14 receptor in the kidney, we used HEK293 and CHO cells transfected with hGPR14 (HEK293+hGPR14, CHO+hGPR14, respectively). Effects of guanylin peptides and U-II were studied by slow-whole-cell patch-clamp analysis and microfluorimetric measurements of intracellular Ca(2+). Guanylin peptides and U-II depolarized HEK293+hGPR14 significantly more than wild type cells. These effects were inhibited in the presence of Ba(2+) or PLA(2) inhibition (AACOCF(3)), suggesting that guanylin peptides and U-II increase arachidonic acid and inhibit ROMK channels in these cells. However, only U-II was capable to increase the cellular Ca(2+), suggesting different mechanism of GPR14 activation by guanylin peptides and U-II. This signaling pathway of U-II involves PKC, because U-II effects in HEK293+hGPR14 cells were inhibited by calphostin C. Guanylin peptides activate PLA(2) and inhibit ROMK channels in HEK293 cells transfected with the human GPR14 receptor. Since GPR14 is present in mouse and human CCD it is a candidate for the guanylate cyclase independent receptor for guanylin peptides.     

Taenia solium metacestode immunodominant peptides recognized by IgG antibodies in cerebrospinal fluid and serum paired samples from patients with active and inactive neurocysticercosis

The aim of this study was to test if serological distinction between patients with active and inactive neurocysticercosis (NCC), could be accomplished by the recognition of immunodominant peptides in total saline antigenic extract of Taenia solium metacestodes by IgG antibody in cerebrospinal fluid (CSF) and serum paired samples. CSF and serum samples of 10 each, active NCC patients, inactive NCC, and individuals with other neurological disorders, were used to recognize the antigenic peptides by western blot (WB). In the active NCC the 28-32 and 39-42 kDa peptides were more frequently detected in CSF than in sera (p < 0.05). The 47-52, 64-68, and 70 kDa antigens showed high frequencies in both samples from patients with active NCC. All the CSF samples of inactive NCC and other neurological disorder (control) patients tested negative, while serum samples from these last two groups recognized mainly the 80, 86, 95, and 98 kDa bands. This finding eliminates the use of the high molecular weigh bands (>or= 80 kDa) for diagnosis of NCC. The final conclusions were that the difference between active and inactive NCC may be done with the detection of peptides only in the CSF samples and that the 47-52, 64-68, and 70 kDa bands may be included as specific markers for active NCC when detected in CSF samples by WB using total saline extract of T. solium metacestode.