Cell death and lumen formation in spheroids of MCF‐7 cells

3D (three‐dimensional) cell culture permits a more integrated analysis of the relationship between cells, inserting them into a structure more closely resembling the cellular microenvironment in vivo. The development of in vitro parameters to approximate in vivo 3D cellular environments makes a less reductionist interpretation of cell biology possible. For breast cells, in vitro 3D culture has proven to be an important tool for the analysis of luminal morphogenesis. A greater understanding of this process is necessary because alterations in the lumen arrangement are associated with carcinogenesis. Following lumen formation in 3D cell culture using laser scanning confocal microscopy, we observed alterations in the arrangement of cytoskeletal components (F‐actin and microtubules) and increasing levels of cell death associated with lumen formation. The formation of a polarized monolayer facing the lumen was characterized through 3D reconstructions and the use of TEM (transmission electron microscopy), and this process was found to occur through the gradual clearing of cells from the medullary region of the spheroids. This process was associated with different types of cell death, such as apoptosis, autophagy and entosis. The present study showed that changes in the extracellular matrix associated with long periods of time in 3D cell culture lead to the formation of a lumen in MCF‐7 cell spheroids and that features of differentiation such as lumen and budding formation occur after long periods in 3D culture, even in the absence of exogenous extracellular compounds.     

Observations on the female internal reproductive organs of the brown howler monkey (Alouatta guariba clamitans)

Alouatta guariba clamitans (brown howler monkey) is an endemic primate from the southeastern Brazil tropical forests, classified as near threatened by the IUCN Red List 2007. The genus Alouatta is one of the most difficult New World monkeys to breed and rear in captivity. In this study we examined the macroscopic and histological aspects of the female genital tract of wild brown howler monkeys to provide baseline information for future reproduction research. The anatomical relationship between the vagina, uterus, broad ligament, oviducts and ovaries are those of a typical primate reproductive tract. The fundic portion of the uterus is globoid, the cervix is well developed, which confers to the uterus an elongated shape, and the vagina is a long flattened channel. Histological analysis conducted in females in the follicular phase revealed large quantities of interstitial luteinized tissue in the ovaries, a stratified nonkeratinized vaginal epithelium, lack of glands in the vaginal mucosa and simple tubular endometrial glands. The observed anatomical features should be considered in the adaptation and application of assisted reproductive techniques aimed at improving captive reproduction for species conservation. Am. J. Primatol. 71:145–152, 2009. © 2008 Wiley‐Liss, Inc.     

Effects of Bothrops asper snake venom on the expression of cyclooxygenases and production of prostaglandins by peritoneal leukocytes in vivo,and by isolated neutrophils and macrophages in vitro

In this study, the ability of Bothrops asper snake venom (BaV) to increase the production of prostaglandins PGE₂ and PGD₂ was assessed in a mouse model in vivo and in inflammatory cells in vitro. In addition, the expressions of COX-1 and COX-2 were assessed. BaV induced an increment in the in vivo synthesis of PGE₂ and PGD₂, together with an enhanced expression of COX-2, but not of COX-1. However, enzymatic activities of COX-1 and COX-2 were increased. Incubation of isolated macrophages and neutrophils with a sub-cytotoxic concentration of BaV in vitro resulted in increased release of PGE₂ and PGD₂ by macrophages and PGE₂ by neutrophils, concomitantly with an increment in the expression of COX-2, but not of COX-1 by both cell types. Our results demonstrate the ability of BaV to promote the expression of COX-2 and to induce the synthesis of proinflammatory prostaglandins. Macrophages and neutrophils may be important targets for this venom under in vivo situation.     

The Non-Benzodiazepine Anxiolytic Drug Etifoxine Causes a Rapid,Receptor-Independent Stimulation of Neurosteroid Biosynthesis

Neurosteroids can modulate the activity of the GABA_A receptors, and thus affect anxiety-like behaviors. The non-benzodiazepine anxiolytic compound etifoxine has been shown to increase neurosteroid concentrations in brain tissue but the mode of action of etifoxine on neurosteroid formation has not yet been elucidated. In the present study, we have thus investigated the effect and the mechanism of action of etifoxine on neurosteroid biosynthesis using the frog hypothalamus as an experimental model. Exposure of frog hypothalamic explants to graded concentrations of etifoxine produced a dose-dependent increase in the biosynthesis of 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone and tetrahydroprogesterone, associated with a decrease in the production of dihydroprogesterone. Time-course experiments revealed that a 15-min incubation of hypothalamic explants with etifoxine was sufficient to induce a robust increase in neurosteroid synthesis, suggesting that etifoxine activates steroidogenic enzymes at a post-translational level. Etifoxine-evoked neurosteroid biosynthesis was not affected by the central-type benzodiazepine (CBR) receptor antagonist flumazenil, the translocator protein (TSPO) antagonist PK11195 or the GABA_A receptor antagonist bicuculline. In addition, the stimulatory effects of etifoxine and the triakontatetraneuropeptide TTN, a TSPO agonist, were additive, indicating that these two compounds act through distinct mechanisms. Etifoxine also induced a rapid stimulation of neurosteroid biosynthesis from frog hypothalamus homogenates, a preparation in which membrane receptor signalling is disrupted. In conclusion, the present study demonstrates that etifoxine stimulates neurosteroid production through a membrane receptor-independent mechanism.     

Determination of Parameters for the Supercritical Extraction of Antioxidant Compounds from Green Propolis Using Carbon Dioxide and Ethanol as Co-Solvent

The aim of this study was to determine the best processing conditions to extract Brazilian green propolis using a supercritical extraction technology. For this purpose, the influence of different parameters was evaluated such as S/F (solvent mass in relation to solute mass), percentage of co-solvent (1 and 2% ethanol), temperature (40 and 50°C) and pressure (250, 350 and 400 bar) using supercritical carbon dioxide. The Global Yield Isotherms (GYIs) were obtained through the evaluation of the yield, and the chemical composition of the extracts was also obtained in relation to the total phenolic compounds, flavonoids, antioxidant activity and 3,5-diprenyl-4-hydroxicinnamic acid (Artepillin C) and acid 4-hydroxycinnamic (p-coumaric acid). The best results were identified at 50°C, 350 bar, 1% ethanol (co-solvent) and S/F of 110. These conditions, a content of 8.93±0.01 and 0.40±0.05 g/100 g of Artepillin C and p-coumaric acid, respectively, were identified indicating the efficiency of the extraction process. Despite of low yield of the process, the extracts obtained had high contents of relevant compounds, proving the viability of the process to obtain green propolis extracts with important biological applications due to the extracts composition.     

Pre-Training Reversible Inactivation of the Basal Amygdala (BA) Disrupts Contextual,but Not Auditory,Fear Conditioning,in Rats

The basolateral amygdala complex (BLA), including the lateral (LA), basal (BA) and accessory basal (AB) nuclei, is involved in acquisition of contextual and auditory fear conditioning. The BA is one of the main targets for hippocampal information, a brain structure critical for contextual learning, which integrates several discrete stimuli into a single configural representation. Congruent with the hodology, selective neurotoxic damage to the BA results in impairments in contextual, but not auditory, fear conditioning, similarly to the behavioral impairments found after hippocampal damage. This study evaluated the effects of muscimol-induced reversible inactivation of the BA during a simultaneous contextual and auditory fear conditioning training on later fear responses to both the context and the tone, tested separately, without muscimol administration. As compared to control rats micro-infused with vehicle, subjects micro-infused with muscimol before training exhibited, during testing without muscimol, significant reduction of freezing responses to the conditioned context, but not to the conditioned tone. Therefore, reversible inactivation of the BA during training impaired contextual, but not auditory fear conditioning, thus confirming and extending similar behavioral observations following selective neurotoxic damage to the BA and, in addition, revealing that this effect is not related to the lack of a functional BA during testing.     

Cryoprotectants and Extreme Freeze Tolerance in a Subarctic Population of the Wood Frog

Wood frogs (Rana sylvatica) exhibit marked geographic variation in freeze tolerance, with subarctic populations tolerating experimental freezing to temperatures at least 10-13 degrees Celsius below the lethal limits for conspecifics from more temperate locales. We determined how seasonal responses enhance the cryoprotectant system in these northern frogs, and also investigated their physiological responses to somatic freezing at extreme temperatures. Alaskan frogs collected in late summer had plasma urea levels near 10 μmol ml^-1, but this level rose during preparation for winter to 85.5 ± 2.9 μmol ml^-1 (mean ± SEM) in frogs that remained fully hydrated, and to 186.9 ± 12.4 μmol ml^-1 in frogs held under a restricted moisture regime. An osmolality gap indicated that the plasma of winter-conditioned frogs contained an as yet unidentified osmolyte(s) that contributed about 75 mOsmol kg^-1 to total osmotic pressure. Experimental freezing to –8°C, either directly or following three cycles of freezing/thawing between –4 and 0°C, or –16°C increased the liver’s synthesis of glucose and, to a lesser extent, urea. Concomitantly, organs shed up to one-half (skeletal muscle) or two-thirds (liver) of their water, with cryoprotectant in the remaining fluid reaching concentrations as high as 0.2 and 2.1 M, respectively. Freeze/thaw cycling, which was readily survived by winter-conditioned frogs, greatly increased hepatic glycogenolysis and delivery of glucose (but not urea) to skeletal muscle. We conclude that cryoprotectant accrual in anticipation of and in response to freezing have been greatly enhanced and contribute to extreme freeze tolerance in northern R. sylvatica.     

Intravoxel Incoherent Motion Diffusion Weighted MR Imaging at 3.0 T: Assessment of Steatohepatitis and Fibrosis Compared with Liver Biopsy in Type 2 Diabetic Patients

ObjectiveTo evaluate the capability of intravoxel incoherent motion (IVIM) diffusion-weighted imaging (DWI) to assess steatohepatitis and fibrosis determined by histopathology in type 2 diabetic patients.MethodsFifty-nine type 2 diabetic patients (49 women, 10 men; mean age, 54 ± 9 years) were submitted to liver biopsy for the evaluation of non-alcoholic fatty liver disease (NAFLD) and underwent DWI on a 3.0T MR system using 10 b values. Institutional approval and patient consent were obtained. Pure molecular-based (D), perfusion-related (D*), and vascular fraction (f) were calculated using a double exponential model and least squares curve fitting. D, D*, and f were compared between patients with and without steatohepatitis and between patients with and without fibrosis. The variables were compared by using the Ranksum test and Student t-test.ResultsSteatohepatitis was observed in 22 patients and fibrosis in 16 patients. A lower D median (0.70 s/mm² vs. 0.83 s/mm², p<0.05) and a lower D* median (34.39 s/mm² vs. 45.23 s/mm², p<0.05) were observed among those with steatohepatitis. A lower D median (0.70 s/mm² vs. 0.82 s/mm², p<0.05) and a lower D* median (35.01 s/mm² vs. 44.76 s/mm², p=0.05) were also observed among those with fibrosis.ConclusionIVIM-DWI has the potential to aid in the characterization of steatohepatitis and fibrosis.