Activated AMP-protein kinase (pAMPK) is overexpressed in human somatotroph pituitary adenomas.

INTRODUCTION: AMPK (AMP-activated protein kinase) is an enzyme that acts as a metabolic sensor and regulates multiple pathways via phosphorylating proteins in metabolic and proliferative pathways. The aim of this work was to study the activated cellular AMPK (phosphorylated-AMPK at Thr172, pAMPK) levels in pituitary tumor samples from patients with sporadic and familial acromegaly, as well as in samples from normal human pituitary gland. METHODS: We studied pituitary adenoma tissue from patients with sporadic somatotroph adenomas, familial acromegaly with heterozygote germline variants in the aryl hydrocarbon receptor interacting protein (AIP) gene (p.Q164*, p.R304* and p.F269_H275dup) and autopsy from normal pituitary glands without structural alterations. RESULTS: Cellular levels of pAMPK were significantly higher in patients with sporadic acromegaly compared to normal pituitary glands (p < 0.0001). Tissues samples from patients with germline AIP mutations also showed higher cellular levels of pAMPK compared to normal pituitary glands. We did not observe a significant difference in cellular levels of pAMPK according to the cytokeratin (CAM5.2) pattern (sparsely or densely granulated) for tumor samples of sporadic acromegaly. CONCLUSION: Our data show, for the first time in human cells, an increase of cellular levels of pAMPK in sporadic somatotropinomas, regardless of cytokeratin pattern, as well as in GH-secreting adenomas from patients with germline AIP mutations.

Perfil de sensibilidade antimicrobiana e formação de biofilme por amostras de bacilos gram-negativos derivados de serpentes

Introduction: Gram-negative bacilli, both enterobacteria and nonfermenters, are the main responsible for infections in snakes, and have been presenting an increasing number of multiresistant antimicrobial isolates, through the production of beta- lactamases and other mechanisms, in addition to the ability to form a biofilm. Objectives: This work aimed to realize the microbiological diagnosis of bacteria strains isolated from snakes, to verify the sensitivity profile to antimicrobials and the ability to form a biofilm; as well as to determine the minimum inhibitory concentration of selected strains against gentamicin and the antimicrobial activity of silver nanoparticles. Methodology: Identification tests were carried out on 37 bacterial strains, followed by disc-diffusion antibiogram tests and disc-approximation tests with antibiotics on representative strains. Biofilm formation was verified by staining with crystal violet, in addition to the minimum inhibitory concentration of gentamicin and antimicrobial activity of silver nanoparticles. Results and discussion: The isolates showed high sensitivity to Aztreonam, Ceftazedime, Cefepime, Ertapenem, Gentamicin and Levofloxacin (72,9% a 97,22%), with lower susceptibility to: Imipinem, Cefotaxin and Tetraciclin (56,75% a 65,16%). Five strains (13,5%) showed multidrug resistance and 91.67% of the 12 tested clinical strains were producers of extended spectrum beta lactamase. The nanoparticles showed bacteriostatic as well as bactericidal potential, in addition to inhibiting biofilm formation in approximately 77.77% of the tested strains. These results demonstrate the variation in the pattern of resistance, ranging from a high level of sensitivity to multidrug-resistant strains. Biofilm formation capacity is an important factor for bacterial persistence in the host and in the environment, and resistance to antibiotics. Conclusion: Monitoring antibiotic resistance provides data for more appropriate antibiotic therapy and resistance control in infections caused by Gram-negative bacilli. Silver nanoparticles were effective in inducing bacterial death and inhibiting biofilm formation in most of the tested strains, proving to be an important treatment alternative.

Introdução: Bacilos gram-negativos, tanto enterobactérias como não fermentadores são os principais responsáveis por infecções em serpentes, e vêm apresentando um número crescente de isolados multirresistentes a antimicrobianos, através da produção de beta-lactamases e de outros mecanismos, além da capacidade de formação de biofilme. Objetivos: Este trabalho teve como objetivo realizar o diagnóstico microbiológico de amostras de bactérias isoladas de serpentes, verificar o perfil de sensibilidade a antimicrobianos e a capacidade de formação de biofilme; assim como determinar a concentração inibitória mínima de cepas selecionadas frente à gentamicina e a atividade antimicrobiana de nanopartículas de prata. Metodologia: Foram realizados testes de identificação de 37 amostras bacterianas, posteriormente os testes de antibiograma por disco-difusão e de disco-aproximação com antibióticos de amostras representativas. Foi verificada a formação de biofilme através da coloração com cristal violeta, além da concentração inibitória mínima de gentamicina e atividade antimicrobiana de nanopartículas de prata. Resultados e Discussão: Os isolados apresentaram alta sensibilidade a Aztreonam, Ceftazedime, Cefepime, Ertapenem, Gentamicina e Levofloxacin (72,9% a 97,22%), apresentando menor suscetibilidade a: Imipinem, Cefotaxin e Tetraciclina (56,75% a 65,16%). Cinco cepas (13,5%) apresentaram multirresistência e 91,67% de 12 amostras clínicas testadas se apresentaram como produtoras de beta lactamases de espectro estendido. As nanopartículas apresentaram potencial bacteriostático, tanto quanto bactericida, além de inibir a formação de biofilme em aproximadamente 77,77% das amostras retestadas. Esses resultados demonstram a variação no padrão de resistência, apresentando desde alto nível de sensibilidade até cepas multirresistentes. A capacidade de formação de biofilme é um fator importante para a persistência bacteriana no hospedeiro e no ambiente, e a resistência a antibióticos. Conclusão: O monitoramento da resistência aos antibióticos fornece dados para antibioticoterapia mais adequada e controle da resistência em infecções causadas por bacilos Gram-negativos. As nanopartículas de prata foram eficazes ao induzir morte bacteriana, e inibir a formação de biofilme na maioria das amostras testadas, mostrando-se uma importante alternativa de tratamento.

Preclinical Pharmacokinetics and Acute Toxicity in Rats of 5-{[(2E)-3-Bromo-3-carboxyprop-2-enoyl]amino}-2-hydroxybenzoic Acid: A Novel 5-Aminosalicylic Acid Derivative with Potent Anti-Inflammatory Activity.

Compound 5-{[(2E)-3-bromo-3-carboxyprop-2-enoyl]amino}-2-hydroxybenzoic acid (C1), a new 5-aminosalicylic acid (5-ASA) derivative, has proven to be an antioxidant in vitro and an anti-inflammatory agent in mice. The in vivo inhibition of myeloperoxidase was comparable to that of indomethacin. The aim of this study was to take another step in the preclinical evaluation of C1 by examining acute toxicity with the up-and-down OECD method and pharmacokinetic profiles by administration of the compound to Wistar rats through intravenous (i.v.), oral (p.o.), and intraperitoneal (i.p.) routes. According to the Globally Harmonized System, C1 belongs to categories 4 and 5 for the i.p. and p.o. routes, respectively. An RP-HPLC method for C1 quantification in plasma was successfully validated. Regarding the pharmacokinetic profile, the elimination half-life was approximately 0.9 h with a clearance of 24 mL/min after i.v. administration of C1 (50 mg/kg). After p.o. administration (50 mg/kg), the maximum plasma concentration was reached at 33 min, the oral bioavailability was about 77%, and the compound was amply distributed to all tissues evaluated. Therefore, C1 administered p.o. in rats is suitable for reaching the colon where it can exert its effect, suggesting an important advantage over 5-ASA and indomethacin in treating ulcerative colitis and Crohn's disease.

Racial/Ethnic Disparities in Meeting 5-2-1-0 Recommendations among Children and Adolescents in the United States.

OBJECTIVE: To evaluate racial/ethnic disparities among children and adolescents in meeting the 4 daily 5-2-1-0 nutrition and activity targets in a nationally representative sample. The 5-2-1-0 message summarizes 4 target daily behaviors for obesity prevention: consuming ≥5 servings of fruit and vegetables, engaging in ≤2 hours of screen time, engaging in ≥1 hour of physical activity, and consuming 0 sugar-sweetened beverages daily. STUDY DESIGN: The National Health and Nutrition Examination Survey (2011-2012) data were used. The study sample included Hispanic (n = 608), non-Hispanic black (n = 609), Asian (n = 253), and non-Hispanic white (n = 484) youth 6-19 years old. The 5-2-1-0 targets were assessed using 24-hour dietary recalls, the Global Physical Activity Questionnaire, and sedentary behavior items. Outcomes included meeting all targets, no targets, and individual targets. Multivariable logistic regression models accounting for the complex sampling design were used to evaluate the association of race/ethnicity with each outcome among children and adolescents separately. RESULTS: None of the adolescents and <1% of children met all 4 of the 5-2-1-0 targets, and 19% and 33%, of children and adolescents, respectively, met zero targets. No racial/ethnic differences in meeting zero targets were observed among children. Hispanic (aOR, 1.76 [95% CI, 1.04-2.98]), non-Hispanic black (aOR, 1.82 [95% CI, 1.04-3.17]), and Asian (aOR, 1.48 [95% CI, 1.08-2.04]) adolescents had greater odds of meeting zero targets compared with non-Hispanic whites. Racial/ethnic differences in meeting individual targets were observed among children and adolescents. CONCLUSIONS: Despite national initiatives, youth in the US are far from meeting 5-2-1-0 targets. Racial/ethnic disparities exist, particularly among adolescents.

Peripheral and spinal 5-HT receptors participate in the pronociceptive and antinociceptive effects of fluoxetine in rats.

The role of 5-HT receptors in fluoxetine-induced nociception and antinociception in rats was assessed. Formalin produced a typical pattern of flinching and licking/lifting behaviors. Local peripheral ipsilateral, but not contralateral, pre-treatment with fluoxetine (0.3-3 nmol/paw) increased in a dose-dependent fashion 0.5% formalin-induced nociception. In contrast, intrathecal pretreatment with fluoxetine (0.3-3 nmol/rat) prevented nociception induced by formalin. The peripheral pronociceptive effect of fluoxetine was prevented by the 5-HT2A (ketanserin, 3-10 pmol/paw), 5-HT2B (3-(2-[4-(4-fluorobenzoyl)-1-piperidinyl]ethyl)-2,4(1H,3H)-quinazolinedione(+) tartrate, RS-127445, 3-10 pmol/paw), 5-HT2C (8-[5-(2,4-dimethoxy-5-(4-trifluoromethylphenylsulphonamido) phenyl-5-oxopentyl]1,3,8-triazaspiro[4.5] decane-2,4-dione hydrochloride, RS-102221, 3-10 pmol/paw), 5-HT3 (ondansetron, 3-10 nmol/paw), 5-HT4 ([1-[2-methylsulphonylamino ethyl]-4-piperidinyl]methyl 1-methyl-1H-indole-3-carboxylate, GR-113808, 3-100 fmol/paw), 5-HT6 (4-iodo-N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]benzene-sulfonamide hydrochloride, SB-258585, 3-10 pmol/paw) and 5-HT7 ((R)-3-(2-(2-(4-methylpiperidin-1-yl) ethyl) pyrrolidine-1-sulfonyl) phenol hydrochloride, SB-269970, 0.3-1 nmol/paw), but not by the 5-HT1A (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylcyclohexanecarboxamide maleate, WAY-100635, 0.3-1 nmol/paw), 5-HT1B/1D (N-[4-methoxy-3-(4-methyl-1-piperazinyl)phenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-1,1'-biphenyl-4-carboxamide hydrochloride hydrate, GR-127935, 0.3-1 nmol/paw), 5-HT1B (1'-methyl-5-[[2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]carbonyl]-2,3,6,7-tetrahydrospiro[furo[2,3-f]indole-3,4'-piperidine hydrochloride, SB-224289, 0.3-1 nmol/paw), 5-HT1D (4-(3-chlorophenyl)-α-(diphenylmethyl)-1-piperazineethanol hydrochloride, BRL-15572, 0.3-1nmol/paw) nor 5-HT5A ((N-[2-(dimethylamino)ethyl]-N-[[4'-[[(2-phenylethyl)amino]methyl][1,1'-biphenyl]-4-yl]methyl]cyclopentanepropanamide dihydrochloride, SB-699551, 1-3 nmol/paw), receptor antagonists. In marked contrast, the spinal antinociceptive effect of fluoxetine was prevented by the 5-HT1A (WAY-100635, 0.3-1 nmol/rat), 5-HT1B/1D (GR-127935, 0.3-1 nmol/rat), 5-HT1B (SB-224289, 0.3-1 nmol/rat), 5-HT1D (BRL-15572, 0.3-1 nmol/rat) and 5-HT5A (SB-699551, 1-3 nmol/rat), but not by the 5-HT2A (ketanserin, 3-10 pmol/rat), 5-HT2B (RS-127445, 3-10 pmol/rat), 5-HT2C (RS-102221, 3-10 pmol/rat), 5-HT3 (ondansetron, 3-10 nmol/rat), 5-HT4 (GR-113808, 3-100 fmol/rat), 5-HT6 (SB-258585, 3-10 pmol/rat) nor 5-HT7 (SB-269970, 0.3-1 nmol/rat), receptor antagonists. These results suggest that fluoxetine produces nociception at the periphery by activating peripheral 5-HT2A/2B/2C/3/4/6/7 receptors. In addition, intrathecal fluoxetine produces antinociception by activation of spinal 5-HT1A/1B/1D/5A receptors.

The antioxidant effect of the mesoionic compound SYD-1 in mitochondria.

The sydnone SYD-1 (3-[4-chloro-3-nitrophenyl]-1,2,3-oxadiazolium-5-olate] possesses important antitumor activity against Sarcoma 180 and Ehrlich tumors. We previously showed that SYD-1 depresses mitochondrial phosphorylation efficiency, which could be involved in its antitumoral activity. Considering the important role of mitochondria in the generation of reactive oxygen species (ROS) and the involvement of ROS in cell death mechanisms, we evaluated the effects of SYD-1 on oxidative stress parameters in rat liver mitochondria. SYD-1 (0.5 and 0.75µmolmg(-1) protein) inhibited the lipoperoxidation induced by Fe(3+)/ADP-oxoglutarate by approximately 75% and promoted total inhibition at the highest concentration tested (1.0µmolmg(-1) protein). However, SYD-1 did not affect lipoperoxidation started by peroxyl radicals generated by α-α'-azodiisobutyramidine dihydrochloride. The mesoionic compound (0.25-1.0µmolmg(-1) protein) demonstrated an ability to scavenge superoxide radicals, decreasing their levels by 9-19%. The activities of catalase and superoxide dismutase did not change in the presence of SYD-1 (0.25-1.0µmolmg(-1) protein). SYD-1 inhibited mitochondrial swelling dependent on the formation/opening of the permeability transition pore induced by Ca(2+)/phosphate by approximately 30% (1.0µmolmg(-1) protein). When Ca(2+)/H2O2 were used as inducers, SYD-1 inhibited swelling only by approximately 12% at the same concentration. NADPH oxidation was also inhibited by SYD-1 (1.0µmolmg(-1) of protein) by approximately 48%. These results show that SYD-1 is able to prevent oxidative stress in isolated mitochondria and suggest that the antitumoral activity of SYD-1 is not mediated by the increasing generation of ROS.

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2.

BACKGROUND: Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation. METHODS: J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases. RESULTS: ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP. CONCLUSIONS: Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin. GENERAL SIGNIFICANCE: ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.

Incremento de disolución de un derivado del furano mediante la técnica de secado por atomización / Improvement of Furane derivate dissolution rate using spray drying technique

Objetivo: incrementar la solubilidad en agua del 2-bromo-5-(2-bromo-2-nitrovinyl)-furano (G1), un ingrediente farmacéuticamente activo sintetizado por el Centro de Bioactivos Químicos de la Universidad Central de Las Villas, con potente acción bactericida y fungicida, mediante la elaboración de macropartículas de dispersiones sólidas utilizando un proceso de secado por atomización. Métodos: se realizó un ensayo preliminar de secado por atomización de la suspensión de G1, compuesta por: 10 g de G1, 1 g de Aerosil (Aerosil®, Degusa, Bélgica), 1 g de laurilsulfato de sodio y 100 mL de agua. La atomización se efectuó en un equipo de laboratorio (Buchi Mini Dryer spray) a 90 ºC. La dispersión sólida obtenida fue caracterizada físico-químicamente mediante difracción de rayos X, granulometría láser por el método de difracción angular, calorimetría diferencial de barrido, microscopia electrónica de barrido y espectrofotometría de absorción infraroja. Resultados: las partículas obtenidas presentaron un pequeño tamaño, forma esférica y un incremento de la cristalinidad del G-1; no se encontraron interacciones entre los componentes de la dispersión ni presencia de productos de degradación, y la solubilidad del G-1 en agua resultó notablemente incrementada. Conclusiones: el producto obtenido por la técnica de secado por atomización incrementó apreciablemente la solubilidad del G1 sin afectar los grupos funcionales, responsables de la actividad terapéutica que se le reportan al ingrediente activo estudiado. Estos alentadores resultados sugieren la necesidad de continuar estudios para la optimización del proceso y realizar al producto obtenido ensayos de estabilidad con el objetivo de su futura inclusión en formas farmacéuticas de dosificación.

Objective: to increase the solubility of 2-bromium-5(2-bromium-2-nitrovinyl)-furane (G1), one pharmaceutically active ingredient with potent bactericidal and fungicidal action, synthesized through the preparation of solid dispersion macroparticles based on spray-drying process in the Center of Chemical Bioactives of the Central University in Las Villas province. Methods: a preliminary spray-drying test of GI suspension made up of 10 g of G1, 1g of Aerosil (Aerosil®, Degusa, Bélgica), 1g of sodium laurylsulphate and 100 ml of water was made. A piece of lab equipment known as Buchi Mini Dryer spray served for the spraying at 90 ºC. The solid dispersion was characterized from the physical and chemical viewpoints through X-ray diffraction, laser granulometry based on angular diffraction method, differential scanning calorimetry, electronic scanning microscopy and infrared spectrophotometry. Results: the obtained particles were small, spherical and had increased G1 crystallinity. No interactions were found in the dispersion components; there were no degradation products, and G1 solubility was significantly increased. Conclusions: the product obtained from spray-drying technique substantially raised the solubility of G1 without affecting the functional groups, which are responsible for the reported therapeutic action of the studied active ingredient. These encouraging results endorse the need for further studies to optimizing the process and carrying out stability tests for the product to be included in the pharmaceutical forms of dosing in the future.

Measurements of LET distribution and dose equivalent onboard the Space Shuttle IML-2 (STS-65) and S/MM#4 (STS-79).

Space radiation dosimetry measurements have been made onboard the Space Shuttle STS-65 in the Second International Microgravity Laboratory (IML-2: 28.5 degrees x 300 km: 14.68 days) and the STS-79 in the 4th Shuttle MIR mission (S/MM#4: 51.6 degrees x 300-400km: 10.2 days). In these measurements, three kinds of detectors were used; one is a newly developed active detector telescope called "Real-time Radiation Monitoring Device (RRMD-I for IML-2 and RRMD-II with improved triggering system for S/MM#4)" utilizing silicon semi-conductor detectors and the other detectors are conventional passive detectors of thermoluminescence dosimeters (TLDs) and CR-39 plastic track detectors. The main contribution to dose equivalent for particles with LET > 5.0 keV/micrometer (IML-2) and LET > 3.5 keV/micrometer (S/MM#4) is seen to be due to galactic cosmic rays (GCRs) and the contribution of the South Atlantic Anomaly (SAA) is less than 5% (IML-2: 28.5 degrees x 300 km) and 15% (S/MM#4: 51.6 degrees x 400 km) in the above RRMD LET detection conditions. For the whole LET range (> 0.2 kev/micrometer) obtained by TLDs and CR-39 in these two typical orbits (a small inclination x low altitude and a large inclination x high altitude), absorbed dose rates range from 94 to 114 microGy/day, dose equivalent rates from 186 to 207 microSv/day and average quality factors from 1.82 to 2.00 depending on the locations and directions of detectors inside the Spacelab at the highly protected IML-2 orbit (28.5 degrees x 300 km), and also, absorbed dose rates range from 290 to 367 microGy/day, dose equivalent rates from 582 to 651 microSv/day and average quality factors from 1.78 to 2.01 depending on the dosimeter packages around the RRMD-II "Detector Unit" at the S/MM#4 orbit (5l.6 degrees x 400km). In general, it is seen that absorbed doses depend on the orbit altitude (SAA trapped particles contribution dominant) and dose equivalents on the orbit inclination (GCR contribution dominant). The LET distributions obtained by two different types of active and passive detectors, RRMDs and CR-39, are in good agreement for LET of 15 - 200 kev/micrometer and difference of these distributions in the regions of LET < 15 kev/micrometer and LET > 200 kev/micrometer can be explained by considering characteristics of CR-39 etched track formation especially for the low LET tracks and chemical etching conditions.

Radiation dosimetry measurements with real time radiation monitoring device (RRMD)-II in Space Shuttle STS-79.

The real-time measurement of radiation environment was made with an improved real-time radiation monitoring device (RRMD)-II onboard Space Shuttle STS-79 (S/MM#4: 4th Shuttle MIR Mission, at an inclination angle of 51.6 degrees and an altitude of 250-400km) for 199 h during 17-25 September, 1996. The observation of the detector covered the linear energy transfer (LET) range of 3.5-6000 keV/micrometer. The Shuttle orbital profile in this mission was equivalent to that of the currently planned Space Station, and provided an opportunity to investigate variations in count rate and dose equivalent rate depending on altitude, longitude, and latitude in detail. Particle count rate and dose equivalent rate were mapped geographically during the mission. Based on the map of count rate, an analysis was made by dividing whole region into three regions: South Atlantic Anomaly (SAA) region, high latitude region and other regions. The averaged absorbed dose rate during the mission was 39.3 microGy/day for a LET range of 3.5-6000 keV/micrometer. The corresponding average dose equivalent rates during the mission are estimated to be 293 microSv/day with quality factors from International Commission on Radiological Protection (ICRP)-Pub. 60 and 270 microSv/day with quality factors from ICRP-Pub. 26. The effective quality factors for ICRP-Pub. 60 and 26 are 7.45 and 6.88, respectively. From the present data for particles of LET > 3.5keV/micrometer, we conclude that the average dose equivalent rate is dominated by the contribution of galactic cosmic ray (GCR) particles. The dose-detector depth dependence was also investigated.