2-Methoxyestradiol-3,17-O,O-bis-sulfamate (STX140) Inhibits Proliferation and Invasion via Senescence Pathway Induction in Human BRAFi-Resistant Melanoma Cells.

The endogenous estradiol derivative 2-Methoxyestradiol (2-ME) has shown good and wide anticancer activity but suffers from poor oral bioavailability and extensive metabolic conjugation. However, its sulfamoylated derivative, 2-methoxyestradiol-3,17-O,O-bis-sulfamate (STX140), has superior potential as a therapeutic agent, acts by disrupting microtubule polymerization, leading to cell cycle arrest and apoptosis in cancer cells and possesses much better pharmaceutical properties. This study investigated the antiproliferative and anti-invasive activities of STX140 in both SKMEL-28 naïve melanoma (SKMEL28-P) cells and resistant melanoma cells (SKMEL-28R). STX140 inhibited cell proliferation in the nanomolar range while having a less pronounced effect on human melanocytes. Additionally, STX140 induced cell cycle arrest in the G2/M phase and sub-G1, reduced migration, and clonogenic potential in monolayer models, and inhibited invasion in a 3D human skin model with melanoma cells. Furthermore, STX140 induced senescence features in melanoma and activated the senescence machinery by upregulating the expression of senescence genes and proteins related to senescence signaling. These findings suggest that STX140 may hold potential as a therapeutic agent for melanoma treatment.

The Interplay of GPER1 with 17ß-Aminoestrogens in the Regulation of the Proliferation of Cervical and Breast Cancer Cells: A Pharmacological Approach.

The G-protein-coupled receptor for estrogen (GPER1) is a transmembrane receptor involved in the progression and development of various neoplasms whose ligand is estradiol (E2). 17ß-aminoestrogens (17ß-AEs) compounds, analogs to E2, are possible candidates for use in hormone replacement therapy (HRT), but our knowledge of their pharmacological profile is limited. Thus, we explored the molecular recognition of GPER1 with different synthetic 17ß-AEs: prolame, butolame, and pentolame. We compared the structure and ligand recognition sites previously reported for a specific agonist (G1), antagonists (G15 and G36), and the natural ligand (E2). Then, the biological effects of 17ß-AEs were analyzed through cell viability and cell-cycle assays in two types of female cancer. In addition, the effect of 17ß-AEs on the phosphorylation of the oncoprotein c-fos was evaluated, because this molecule is modulated by GPER1. Molecular docking analysis showed that 17ß-AEs interacted with GPER1, suggesting that prolame joins GPER1 in a hydrophobic cavity, similarly to G1, G15, and E2. Prolame induced cell proliferation in breast (MCF-7) and cervical cancer (SIHA) cells; meanwhile, butolame and pentolame did not affect cell proliferation. Neither 17ß-AEs nor E2 changed the activation of c-fos in MCF-7 cells. Meanwhile, in SIHA cells, E2 and 17ß-AEs reduced c-fos phosphorylation. Thus, our data suggest that butolame and pentolame, but not prolame, could be used for HRT without presenting a potential risk of inducing breast- or cervical-cancer-cell proliferation. The novelty of this work lies in its study of compound analogs to E2 that may represent important therapeutic strategies for women in menopause, with non-significant effects on the cell viability of cancer cells. The research focused on the interactions of GPER1, a molecule recently associated with promoting and maintaining various neoplasms.

Effects of post-ovariectomy time frame and age on the antidepressant-like actions of estradiol and prolame in female rats.

Estrogen replacement therapy (ERT) is an effective treatment for symptoms associated with climacteric and depression some women experience during perimenopause and menopause. The antidepressant-like effects of ERT may depend on the type of estrogen, age, and time when restitution is initiated after hormonal decline. Prolame is a synthetic steroid with estrogenic and antidepressant-like effects that may produce fewer adverse effects. We hypothesize that such actions of prolame on females depend on age and the duration of hormone deprivation period. We assessed the antidepressant-like effects of 17ß-estradiol (E2) and prolame in young and middle-aged rats across different post-ovariectomy (Ovx) time frames. Independent groups of young adults and middle-aged female rats were tested in the forced swimming test (FST) at 3, 8, 16, and 24 weeks post-Ovx. Prolame and E2 were administered in a sub-chronic schedule consisting of three injections before the FST. Likewise, the utero-trophic effects of these hormones were analyzed. We found that E2 and prolame reduced immobility in young rats 3 and 8 weeks after Ovx; in contrast, only prolame produced this effect in middle-aged rats three weeks post-Ovx. E2 and prolame increased the animals' utero-somatic index at all post-Ovx times, but the action of E2 and prolame produced a greater response in young adult rats. Our findings showed that the antidepressant-like effects of E2 and prolame depend on the post-Ovx time frame, age, and estrogen type. Interestingly, our results indicate that, in contrast to E2, prolame maintained its antidepressant effect in middle-aged rats.

Lysophosphatidic acid (LPA) as a modulator of plasma membrane Ca2+-ATPase from basolateral membranes of kidney proximal tubules.

Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.

G protein-coupled receptor signal transduction and Ca2+ signaling pathways of the allatotropin/orexin system in Hydra.

Allatotropin is a pleiotropic peptide originally characterized in insects. The existence of AT neuropeptide signaling was proposed in other invertebrates. In fact, we previously proposed the presence of an AT-like system regulating feeding behavior in Hydra sp. Even in insects, the information about the AT signaling pathway is incomplete. The aim of this study is to analyze the signaling cascade activated by AT in Hydra plagiodesmica using a pharmacological approach. The results show the involvement of Ca2+ and IP3 signaling in the transduction pathway of the peptide. Furthermore, we confirm the existence of a GPCR system involved in this pathway, that would be coupled to a Gq subfamily of Gα protein, which activates a PLC, inducing an increase in IP3 and cytosolic Ca2+. To the best of our knowledge, this work represents the first in vivo approach to study the overall signaling pathway and intracellular events involved in the myoregulatory effect of AT in Hydra sp.

Neryl butyrate induces contractile effects on isolated preparations of rat aorta.

Neryl butyrate is a constituent of volatile oils obtained from aromatic plants. Aliphatic organic compound analogues chemically close to neryl butyrate possess vasodilator properties in rat aorta. To evaluate whether neryl butyrate has relaxing properties, this study tested its effects on isolated rat aorta. Unlike the analogues, neryl butyrate did not show relaxant profile in aortic rings precontracted with phenylephrine, but induced a contraction when it stimulated aortic rings under resting tonus. The contractile effect augmented in endothelium-denuded aortic rings. Treatment of endothelium-intact preparations with the nitric oxide synthase inhibitor L-NAME or the guanylyl cyclase inhibitor ODQ also augmented the contractile effect of neryl butyrate. Such phenomenon was absent in the presence of the cyclooxygenase inhibitor indomethacin. Contractile responses decreased in the presence of verapamil, a L-type Ca2+ channel blocker, or when Ca2+ was removed from the extracellular solution. Antagonists of α-adrenergic receptors (prazosin and yohimbine), but not the thromboxane-prostanoid receptor seratrodast, reversed the contraction induced by neryl butyrate. The α1A selective antagonist RS-17053 antagonized the neryl butyrate-induced contraction. The contraction caused by neryl butyrate was decreased by inhibiting the phospholipase C or the rho-associated kinase with U-73122 or Y-27632, respectively. Injected intravenously to awake rats, neryl butyrate induced arterial hypotension and bradycardia. Decreased frequency was also present in isolated right atrium preparations. In conclusion, the contractile effects of neryl butyrate were inhibited by α-adrenergic antagonists, indicating the involvement of α-adrenoceptors in the mechanism of action. In vivo, neryl butyrate caused hypotension, suggesting that other systemic influence than vasoconstriction may occur.

Inhibitory mechanism of 17ß-aminoestrogens in the formation of Aß aggregates.

Alzheimer's disease (AD) is a complex neurodegenerative disorder associated with the aggregation of the amyloid-beta peptide (Aß) into large oligomers and fibrils that damage healthy brain cells. The predominant peptide fragments in the plaques are mainly formed by the Aß1-40 and Aß1-42 peptides, albeit the eleven-residue Aß25-35 segment is largely used in biological studies because it retains the neurotoxic properties of the longer Aß peptides. Recent studies indicate that treatment with therapeutic steroid hormones reduces the progress of the disease in AD models. Particularly, treatment with 17ß-aminoestrogens (AEs) has shown a significant alleviation of the AD development by inhibiting oxidative stress and neuronal death. Yet, the mechanism by which the AE molecules exhibit their beneficial effects remains speculative. To shed light into the molecular mechanism of inhibition of the AD development by AEs, we investigated the possibility of direct interaction with the Aß25-35 peptide. First, we calculate various interacting electronic properties of three AE derivatives as follows: prolame, butolame, and pentolame by performing DFT calculations. To account for the polymorphic nature of the Aß aggregates, we considered four different Aß25-35 systems extracted from AD relevant fibril structures. From the calculation of different electron density properties, specific interacting loci were identified that guided the construction and optimization of various complexes. Interestingly, the results suggest a similar inhibitory mechanism based on the direct interaction between the AEs and the M35 residue that seems to be general and independent of the polymorphic properties of the Aß aggregates. Our analysis of the complex formation provides a structural framework for understanding the AE therapeutic properties in the molecular inhibitory mechanism of Aß aggregation.

Progesterone and ultrasonographic changes during aglepristone or cloprosternol treatment in queens at 21 to 22 or 35 to 38 days of pregnancy.

Progesterone (P4) is a requirement for pregnancy development. Previous reports observed a maximal value of serum P4 concentration on 21 days after the first mating after which it slowly declines throughout the rest of pregnancy. Ultrasound examination should be performed to ensure that pregnancy interruption is complete. Limited information is available on the ultrasonic appearance of conceptuses during pregnancy termination in cats The objective was to study serum P4 concentration and ultrasonographic changes during aglepristone (ALI) or cloprostenol (CLO) treatment and to evaluate the fertility after treatment. Two experiments (EXP) were carried out to accomplish this aim. Sixty queens, 12- to 36-month-old, were used. On Days 21 to 22 of pregnancy (EXP I) or 35 to 38 of pregnancy (EXP II), queens were divided into three groups (G). Queens in G1 received ALI (10 mg/kg, sc; EXP I, n = 10; EXP II, n = 10) for 2 consecutive days. Queens in G2 received CLO (5 µg/kg, sc; EXP I, n = 10; EXP II = 10) for 3 consecutive days. Queens in G3 received 1 mL of saline solution (PLA, sc; EXP I, n = 10; EXP II = 10). Blood samples were taken before treatment (Day 0) and every day during 10 days after the treatment to measure serum P4 concentrations. Likewise, after treatment, queens were monitored daily by ultrasonography for 10 days and weekly until the end of gestation to obtain gestational sacs measurements (GS), fetal measurements, and fetal biophysical profile. Data were analyzed by ANOVA. Serum P4 concentrations were significantly different on Day 6 (EXP I) and on Day 1 (EXP II) in ALI and CLO groups compared with PLA group (P < 0.05 and P < 0.01; respectively). The ultrasonographic monitoring during treatment allowed assessing changes in the GS and fetal measurements, embryo-fetal viability, and risk of pregnancy loss. In conclusion, the results from this study reported changes in serum P4 concentration and in ultrasonography measurements during pregnancy interruption with ALI or CLO treatment. Also it was observed that ALI and CLO are safe drugs and can preserve posttreatment queen fertility. Therefore, the results obtained in our work will be applied in feline reproduction practice.

Participation of PLA2 and PLC in DhL-induced activation of Rhinella arenarum oocytes.

Rhinella arenarum oocytes can be artificially activated, a process known as parthenogenesis, by a sesquiterpenic lactone of the guaianolide group, dehydroleucodine (DhL). Transient increases in the concentration of cytosolic Ca2+ are essential to trigger egg activation events. In this sense, the 1-4-5 inositol triphosphate receptors (IP3R) seem to be involved in the Ca2+ transient release induced by DhL in this species. We analyzed the involvement of phosphoinositide metabolism, especially the participation of phospholipase A2 (PLA2) and phospholipase C (PLC) in DhL-induced activation. Different doses of quinacrine, aristolochic acid (ATA) (PLA2 inhibitors) or neomycin, an antibiotic that binds to PIP2, thus preventing its hydrolysis, were used in mature Rhinella arenarum oocytes. In order to assay the participation of PI-PLC and PC- PLC we used U73122, a competitive inhibitor of PI-PLC dependent events and D609, an inhibitor of PC-PLC. We found that PLA2 inhibits quinacrine more effectively than ATA. This difference could be explained by the fact that quinacrine is not a specific inhibitor for PLA2 while ATA is specific for this enzyme. With respect to the participation of PLC, a higher decrease in oocyte activation was detected when cells were exposed to neomycin. Inhibition of PC-PLC with D609 and IP-PLC with U73122 indicated that the last PLC has a significant participation in the effect of DhL-induced activation. Results would indicate that DhL induces activation of in vitro matured oocytes of Rhinella arenarum by activation of IP-PLC, which in turn may induce IP3 formation which produces Ca2+ release.

Differential effect of the 17ß-aminoestrogens prolame, butolame and pentolame in anxiety and depression models in rats.

Estrogens of clinical use produce consistent antidepressant- and anxiolytic-like effects in animal models of menopause. Regulation of the hypothalamic-pituitary-adrenal (HPA) or stress axis, has been proposed as a pathway through which estrogens improve affective-like behaviors. Anticoagulant 17ß-aminoestrogens (17ß-AEs) butolame and pentolame mimic some effects of estradiol (E2), i.e., on female rodent sexual behavior, with opposite actions on coagulation. However, their psychoactive actions have not been explored. On the basis of similitude with E2's effects, we hypothesized that these 17ß-AEs would induce anxiolytic- and antidepressant-like effects, which would be reflected in a reduction of activity in the HPA axis. In ovariectomized female rats, chronic treatment with prolame (60 µg/kg), butolame (65 µg/kg) and pentolame (70 µg/kg) reduced anxiety-like behavior in the elevated plus maze (evidenced by an increase in time in open arms, E2 (40 µg/kg) +176%; prolame +201%; butolame, +237%; and pentolame +295%, in comparison to the control vehicle group 100%). Pentolame also decreased significantly anxiety-like behavior in the burying behavior test. Prolame and E2 produced a significantly antidepressant-like action, which was not induced by butolame and pentolame. Behavioral effects of 17ß-AEs (and E2) on anxiety and depression did not follow the same pattern than corticosterone or E2 levels; they also were associated to changes in locomotor activity, evaluated by the open field test. These results constitute the first evidence of specific and selective actions of butolame and pentolame as anxiolytics for females with a hypoestrogenic condition. Results also confirm the potential of prolame as an antidepressant steroid with equivalent actions to E2. Psychoactive properties of 17ß-AEs in combinations with reduced adverse effects on coagulation, suggest that 17ß-AEs may be a good alternative replacement therapy for women with symptoms associated with menopause.

DDRT-PCR approaches applied for preeminent results in the isolation of DETs from fish brain tissues.

Differential Display (DD) is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored. Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs (Differentially Expressed Transcripts), using Leporinus macrocephalus, a large commercially exploited South American species, as a fish design. Different DDRT-PCR approaches were applied to brain samples and the products of the reactions were analyzed on 6% polyacrylamide gels stained with 0.17% Silver Nitrate (AgNO3). The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR (Variable Number of Tandem Repeats) core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides. The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. macrocephalus. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach.

Cloning, identification and functional characterization of bovine free fatty acid receptor-1 (FFAR1/GPR40) in neutrophils.

Long chain fatty acids (LCFAs), which are ligands for the G-protein coupled receptor FFAR1 (GPR40), are increased in cow plasma after parturition, a period in which they are highly susceptible to infectious diseases. This study identified and analyzed the functional role of the FFAR1 receptor in bovine neutrophils, the first line of host defense against infectious agents. We cloned the putative FFAR1 receptor from bovine neutrophils and analyzed the sequence to construct a homology model. Our results revealed that the sequence of bovine FFAR1 shares 84% identity with human FFAR1 and 31% with human FFAR3/GPR41. Therefore, we constructed a homology model of bovine FFAR1 using human as the template. Expression of the bovine FFAR1 receptor in Chinese hamster ovary (CHO)-K1 cells increased the levels of intracellular calcium induced by the LCFAs, oleic acid (OA) and linoleic acid (LA); no increase in calcium mobilization was observed in the presence of the short chain fatty acid propionic acid. Additionally, the synthetic agonist GW9508 increased intracellular calcium in CHO-K1/bFFAR1 cells. OA and LA increased intracellular calcium in bovine neutrophils. Furthermore, GW1100 (antagonist of FFAR1) and U73122 (phospholipase C (PLC) inhibitor) reduced FFAR1 ligand-induced intracellular calcium in CHO-K1/bFFAR1 cells and neutrophils. Additionally, inhibition of FFAR1, PLC and PKC reduced the FFAR1 ligand-induced release of matrix metalloproteinase (MMP)-9 granules and reactive oxygen species (ROS) production. Thus, we identified the bovine FFAR1 receptor and demonstrate a functional role for this receptor in neutrophils activated with oleic or linoleic acid.

DDRT-PCR approaches applied for preeminent results in the isolation of DETs from fish brain tissues / Aplicação de abordagens de DDRT-PCR para aprimoramento de resultados no isolamento de DETs a partir de tecido cerebral de peixes

Differential Display (DD) is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored. Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs (Differentially Expressed Transcripts), using Leporinus macrocephalus, a large commercially exploited South American species, as a fish design. Different DDRT-PCR approaches were applied to brain samples and the products of the reactions were analyzed on 6% polyacrylamide gels stained with 0.17% Silver Nitrate (AgNO3). The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR (Variable Number of Tandem Repeats) core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides. The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. macrocephalus. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach.(AU)

Display Diferencial (DD) é uma técnica amplamente utilizada em estudos de expressão diferencial. A maioria desses estudos envolvendo espécies de peixes está restrita a espécies da América do Norte e Europa ou a espécies comerciais, como os salmoniformes. Estudos relacionados a peixes da América do Sul são ainda pouco explorados. Desse modo, o presente trabalho teve como objetivo descrever modificações na técnica de DD, a fim de melhorar os resultados relacionados ao isolamento de DETs (Transcritos Diferencialmente Expressos), utilizando Leporinus macrocephalus, peixe explorado comercialmente na América do Sul, como espécie para tal delineamento. Diferentes abordagens de DDRT-PCR foram desenvolvidas a partir de amostras de tecido cerebral e os produtos das reações foram analisados em gel de poliacrilamida 6% corados com 0,17% de nitrato de Prata (AgNO3). A utilização de reações de PCR sob condições de elevada estringência e oligonucleotídeos mais longos, com base em sequências cerne de VNTR (Número Variável de Repetições em Tandem), mostrou melhores resultados quando comparada a condições de baixa estringência e ao uso de oligonucleotídeos decâmeros. A estratégia empregada permitiu o isolamento de transcritos diferencialmente expressos em machos e fêmeas adultos de L. macrocephalus. Este estudo evidencia que modificações no método de DDRT-PCR garantem o melhor isolamento de DETs a partir de diferentes tecidos de peixes e asseguram a obtenção de dados mais sólidos relacionados a essa abordagem.(AU)

DDRT-PCR approaches applied for preeminent results in the isolation of DETs from fish brain tissues / Aplicação de abordagens de DDRT-PCR para aprimoramento de resultados no isolamento de DETs a partir de tecido cerebral de peixes

Differential Display (DD) is a technique widely used in studies of differential expression. Most of these analyses, especially those involving fish species, are restricted to species from North America and Europe or to commercial species, as salmonids. Studies related to South American fish species are underexplored. Thus, the present work aimed to describe DD technique modifications in order to improve outcomes related to the isolation of DETs (Differentially Expressed Transcripts), using Leporinus macrocephalus, a large commercially exploited South American species, as a fish design. Different DDRT-PCR approaches were applied to brain samples and the products of the reactions were analyzed on 6% polyacrylamide gels stained with 0.17% Silver Nitrate (AgNO3). The use of PCR reactions under high stringency conditions and longer oligonucleotides based on VNTR (Variable Number of Tandem Repeats) core sequences led to better results when compared to low stringency PCR conditions and the use of decamer oligonucleotides. The improved approach led to the isolation of differentially expressed transcripts on adult males and females of L. macrocephalus. This study indicates that some modifications on the DDRT-PCR method can ensure isolation of DETs from different fish tissues and the development of robust data related to this approach.

Display Diferencial (DD) é uma técnica amplamente utilizada em estudos de expressão diferencial. A maioria desses estudos envolvendo espécies de peixes está restrita a espécies da América do Norte e Europa ou a espécies comerciais, como os salmoniformes. Estudos relacionados a peixes da América do Sul são ainda pouco explorados. Desse modo, o presente trabalho teve como objetivo descrever modificações na técnica de DD, a fim de melhorar os resultados relacionados ao isolamento de DETs (Transcritos Diferencialmente Expressos), utilizando Leporinus macrocephalus, peixe explorado comercialmente na América do Sul, como espécie para tal delineamento. Diferentes abordagens de DDRT-PCR foram desenvolvidas a partir de amostras de tecido cerebral e os produtos das reações foram analisados em gel de poliacrilamida 6% corados com 0,17% de nitrato de Prata (AgNO3). A utilização de reações de PCR sob condições de elevada estringência e oligonucleotídeos mais longos, com base em sequências cerne de VNTR (Número Variável de Repetições em Tandem), mostrou melhores resultados quando comparada a condições de baixa estringência e ao uso de oligonucleotídeos decâmeros. A estratégia empregada permitiu o isolamento de transcritos diferencialmente expressos em machos e fêmeas adultos de L. macrocephalus. Este estudo evidencia que modificações no método de DDRT-PCR garantem o melhor isolamento de DETs a partir de diferentes tecidos de peixes e asseguram a obtenção de dados mais sólidos relacionados a essa abordagem.

Reduction of no-reflow and reperfusion injury with the synthetic 17ß-aminoestrogen compound Prolame is associated with PI3K/Akt/eNOS signaling cascade.

A high proportion of primary percutaneous coronary interventions performed in the setting of acute myocardial infarction, concur with inadequate myocardial perfusion at the microvascular level. This phenomenon, known as "no-reflow" contributes to reperfusion injury, poor prognosis and to unfavorable clinical outcome. In this study, we evaluated the hypothesis that the synthetic 17ß-aminoestrogen Prolame, may confer cardioprotection and prevent against no-reflow. In an open-chest model of 30-min ischemia and 90-min reperfusion, male Wistar rats were randomly assigned to different groups: Control, Prolame, Prolame followed by the nitric oxide synthase inhibitor (L-NAME), and 17ß-estradiol. Areas of risk, infarct size and no-reflow were determined by planimetry with triphenyltetrazolium chloride and thioflavin-S stains. Structural damage of the vasculature was measured as capillary compression in clarified tissue after intra-atrial injection of Microfil. Hemodynamic function was obtained at the end of stabilization, ischemia and reperfusion; nitric oxide (NO·) content was determined indirectly using the Griess reaction. Activation of the eNOS signaling cascade was determined by western blot. Prolame reduced the infarcted area, decreased the zones of no-reflow and capillary compression by activating the PI3K/Akt/eNOS signaling pathway in correlation with NO· increase. Prolame also activated endothelial cells augmenting NO· production, which was inhibited by ICI182780 (a selective estrogen receptor down-regulator), supporting the notion that the cardioprotective effect of Prolame involves the preservation of endothelium through the activation of estrogen receptor downstream signaling. Our results provide evidence that Prolame has potential therapeutic application in patients with AMI, as it prevents from both vascular and cardiac tissue damage.

Ryanodine and inositol triphosphate receptors modulate facilitation and tetanic depression at the frog neuromuscular junction.

INTRODUCTION: Short-term plasticity of synaptic function is an important physiological control of transmitter release. Short-term plasticity can be regulated by intracellular calcium released by ryanodine and inositol triphosphate (IP3) receptors, but the role of these receptors at the neuromuscular junction is understood incompletely. METHODS: We measured short-term plasticity of evoked endplate potential (EPP) amplitudes from frog neuromuscular junctions treated with ryanodine, 2-aminoethoxydiphenylborane (2-APB), or 1-[6-[[(17ß)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U- 73122). RESULTS: Ryanodine decreases paired-pulse facilitation for intervals <20 ms and markedly decreases tetanic depression. Treatment with 2-APB reduces EPP amplitude, increases paired-pulse facilitation for intervals of <20 ms, and significantly reduces tetanic depression. U-73122 decreases EPP amplitude and decreases paired-pulse depression for intervals <20 ms. CONCLUSIONS: Ryanodine, IP3 receptors, and phospholipase C modulate short-term plasticity of transmitter release at the neuromuscular junction. These results suggest possible targets for improving the safety factor of neuromuscular transmission during repetitive activity of the neuromuscular junction.

Relationship between the climbing up and climbing down stairs domain scores on the FES-DMD, the score on the Vignos Scale, age and timed performance of functional activities in boys with Duchenne muscular dystrophy / Relação entre escore FES-DMD-subir e descer escada com escore Escala Vignos, idade e tempo de realização das atividades em meninos com Distrofia Muscular de Duchenne

BACKGROUND: Knowing the potential for and limitations of information generated using different evaluation instruments favors the development of more accurate functional diagnoses and therapeutic decision-making. OBJECTIVE: To investigate the relationship between the number of compensatory movements when climbing up and going down stairs, age, functional classification and time taken to perform a tested activity (TA) of going up and down stairs in boys with Duchenne muscular dystrophy (DMD). METHOD: A bank of movies featuring 30 boys with DMD performing functional activities was evaluated. Compensatory movements were assessed using the climbing up and going down stairs domain of the Functional Evaluation Scale for Duchenne Muscular Dystrophy (FES-DMD); age in years; functional classification using the Vignos Scale (VS), and TA using a timer. Statistical analyses were performed using the Spearman correlation test. RESULTS: There is a moderate relationship between the climbing up stairs domain of the FES-DMD and age (r=0.53, p=0.004) and strong relationships with VS (r=0.72, p=0.001) and TA for this task (r=0.83, p<0.001). There were weak relationships between the going down stairs domain of the FES-DMD-going down stairs with age (r=0.40, p=0.032), VS (r=0.65, p=0.002) and TA for this task (r=0.40, p=0.034). CONCLUSION: These findings indicate that the evaluation of compensatory movements used when climbing up stairs can provide more relevant information about the evolution of the disease, although the activity of going down stairs should be investigated, with the aim of enriching guidance and strengthening accident prevention. Data from the FES-DMD, age, VS and TA can be used in a complementary way to formulate functional diagnoses. Longitudinal studies and with broader age groups may supplement this information. .

CONTEXTUALIZAÇÃO: Conhecer as potencialidades e limitações das informações geradas por diferentes instrumentos de avaliação favorece o desenvolvimento mais preciso do diagnóstico funcional e da tomada de decisão terapêutica. OBJETIVO : Investigar a relação entre o número de movimentos compensatórios ao subir e descer escadas, idade, classificação funcional e tempo de realização de atividade (TA) em meninos com Distrofia Muscular de Duchenne (DMD). MÉTODO : Foi utilizado banco de filmes de 30 meninos com DMD realizando atividades funcionais. Os movimentos compensatórios foram avaliados pela Escala de Avaliação Funcional para Distrofia Muscular de Duchenne (FES-DMD), domínio subir e descer escada; a idade, mensurada em anos; a classificação funcional foi pesquisada pela Escala de Vignos (EV), e o TA foi cronometrado. Foi utilizado o teste de correlação de Spearman. RESULTADOS : Existe moderada relação entre a FES-DMD-subir escada e a idade (r=0,53, p=0,004) e forte relação com a EV (r=0,72, p=0,001) e TA dessa tarefa (r=0,83, p<0,001). Houve fraca relação entre a FES-DMD-descer escada e a idade (r=0,40, p=0,032), EV (r=0,65, p=0,002) e o TA dessa tarefa (r=0,40, p=0,034). CONCLUSÃO : Esses achados indicam que a avaliação da tarefa de subir escada pode trazer informações mais relevantes sobre a evolução da doença, embora a atividade de descer escada deva ser pesquisada visando à orientação e prevenção de acidentes. A utilização conjunta de dados provenientes da FES-DMD, da idade e do TA pode se complementar para formulação do diagnóstico funcional. Estudos longitudinais e com outras faixas etárias mais amplas podem complementar tal informação. .

Ultrastructural changes of erythrocytes in whole blood after exposure to prospective in silico-designed anticancer agents: a qualitative case study.

BACKGROUND: Novel, in silico-designed anticancer compounds were synthesized in our laboratory namely, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol) and 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). These compounds were designed to have improved bioavailability when compared to their source compound, 2-methoxyestradiol. This theoretically would be due to their increased binding affinity to carbonic anhydrase II, present in erythrocytes. Since the novel compounds under investigation are proposed to be transported within erythrocytes bound to carbonic anhydrase II, the morphological effect which they may exert on whole blood and erythrocytes is of great significance. A secondary outcome included revision of previously reported procedures for the handling of the whole blood sample. The purpose of this study was twofold. Firstly, the ultrastructural morphology of a healthy female's erythrocytes was examined via scanning electron microscopy (SEM) after exposure to the newly in silico-designed compounds. Morphology of erythrocytes following exposure to ESE-15-ol and ESE-16 for 3 minutes and 24 hours at 22°C were described with the use of SEM. The haemolytic activity of the compounds after 24 hours exposure were also determined with the ex vivo haemolysis assay. Secondly, storage conditions of the whole blood sample were investigated by determining morphological changes after a 24 hour storage period at 22°C and 37°C. RESULTS: No significant morphological changes were observed in the erythrocyte morphology after exposure to the novel anticancer compounds. Storage of the whole blood samples at 37°C for 24 hours resulted in visible morphological stress in the erythrocytes. Erythrocytes incubated at 22°C for 24 hours showed no structural deformity or distress. CONCLUSIONS: From this research the optimal temperature for ex vivo exposure of whole blood samples to ESE-15-ol and ESE-16 for 24 hours was determined to be 22°C. Data from this study revealed the potential of these compounds to be applied to ex vivo study techniques, since no damage occurred to erythrocytes ultrastructure under these conditions. As no structural changes were observed in erythrocytes exposed to ESE-15-ol and ESE-16, further ex vivo experiments will be conducted into the potential effects of these compounds on whole blood. Optimal incubation conditions up to 24 hours for whole blood were established as a secondary outcome.

ATP7B activity is stimulated by PKCɛ in porcine liver.

Copper is necessary for all organisms since it acts as a cofactor in different enzymes, although toxic at high concentrations. ATP7B is one of two copper-transporting ATPases in humans, its vital role being manifested in Wilson disease due to a mutation in the gene that encodes this pump. Our objective has been to determine whether pathways involving protein kinase C (PKC) modulate ATP7B activity. Different isoforms of PKC (α, ɛ, ζ) were found in Golgi-enriched membrane fractions obtained from porcine liver. Cu(I)-ATPase activity was assessed in the presence of different activators and inhibitors of PKC signaling pathways. PMA (10(-8) M), a PKC activator, increased Cu(I)-ATPase activity by 60%, whereas calphostin C and U73122 (PKC and PLC inhibitors, respectively) decreased the activity by 40%. Addition of phosphatase λ decreased activity by 60%, irrespective of pre-incubation with PMA. No changes were detected with 2 µM Ca(2+), whereas PMA plus EGTA increased activity. This enhanced activity elicited by PMA decreased with a specific inhibitor of PKCɛ to levels comparable with those found after phosphatase λ treatment, showing that the ɛ isoform is essential for activation of the enzyme. This regulatory phosphorylation enhanced Vmax without modifying affinities for ATP and copper. It can be concluded that signaling pathways leading to DAG formation and PKCɛ activation stimulate the active transport of copper by ATP7B, thus evidencing a central role for this specific kinase-mediated mechanism in hepatic copper handling.

PLCγ2 and PKC are important to myeloid lineage commitment triggered by M-SCF and G-CSF.

Myeloid differentiation is a complex process whereby mature granulocytes or monocytes/macrophages are derived from a common myeloid progenitor through the coordinated action of hematopoietic cytokines. In this study, we explored the role of the Ca(2+)i signaling transduction pathway in the commitment of hematopoietic stem/progenitor cells to either the monocytic or granulocytic lineage in response to macrophage colony-stimulating factor (M-CSF) and granulocyte colony-stimulating factor (G-CSF). M-CSF and G-CSF induce cell expansion and monocyte or granulocyte differentiation, respectively, without affecting the percentage of hematopoietic progenitor cells. Colony-forming units (CFUs) and flow cytometry demonstrated the involvement of phospholipase Cγ (PLCγ) and protein kinase C (PKC) in monocyte/granulocyte commitment. In addition, using flow cytometry and RNA interference, we identified PLCγ2 as the PLCγ isoform that participates in this cell expansion and differentiation. Differences in signaling elicited by M-CSF and G-CSF were observed. The M-CSF-related effects were associated with the activation of ERK1/2 and nuclear factor of activated T-cells (NFAT); the inhibition of both molecules reduced the number of colonies in a CFU assay. In contrast, using flow cytometry and confocal evaluation, we demonstrated that G-CSF activated Jak-1 and STAT-3. Additionally, the effects induced by G-CSF were also related with the participation of Ca(2+) calmodulin kinase II and the transcription factor PU.1. STAT-3 activation and the increase of PU.1 expression were sensitive to PLC inhibition by U73122. These data show that PLCγ2 and PKC are important upstream signals that regulate myelopoiesis through cytokines, and differences in M-CSF and G-CSF downstream signaling were identified.