Vacuolar apical compartment (VAC) in breast carcinoma cell lines (MCF-7 and T47D): failure of the cell-cell regulated exocytosis mechanism of apical membrane

Abstract. We have previously shown that an integral plasma membrane glycoprotein (AP2) is highly polarized to the apical domain in confluent Madin-Darby canine kidney (MDCK) epithelial cells. However, when the monolayers are prevented from forming intercellular contacts, approximately 60% of the AP2 cellular content is stored in the intracellular vacuolar apical compartment (VAC). In the current work we found that AP2 was present in the non-tumorigenic human mammary epithelial cell line MCF-10A. in the breast carcinoma cell lines MCF-7 and T47D, and in breast ductal carcinomas in vivo. By radioimmunoassay, an intracellular Compartment of AP2 was identified in the mammary cell lines in culture. In MCF-10A, this compartment behaved as in MDCK cells; namely it was observed only when the cells cannot form cell-cell contacts. However, in the carcinoma cell lines MCF-7 and T47D, a significant AP2 intracellular compartment was observed also under conditions permissive for the formation of intercellular contacts. These results were confirmed by immunofluorescence and immunoelectron microscopy experiments that showed VACs in MCF-7 and T47D, even in cells with extensive intercellular contacts. In MCF-7 cells, the addition of serum caused a partial decrease of the AP2 intracellular compartment. The exocytosis of VACs occurred towards the center of multi-cellular groups, forming intercellular lumens, similar to those transiently observed in MDCK cells and to structures described by others during embryo development. Altogether, these results suggest that VAC exocytosis is controlled by cell-cell contact signalling, which may be defective in carcinoma cells.     

Determination of phenylbutazone and oxyphenbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography-mass spectrometry

A method is described for the qualiitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deproteinized with acetonitrile and 20 gml were injected directly into the HPLC system equipped with a UV detector and LiChrospher RP-18 column. The mobile phase used was 0.01 M acetic acid in methanol (45:55, v/v). The limit of detection was 0.5 μg/ml for phenylbutazone and oxyphenbutazone and the limit of quantitation was 1.0 μg/ml for both drugs. Using the proposed method it was possible to quantify phenylbutazone up to 30 h and oxyphenbutazone up to 39 h after administration.     

Prophylactic and curative effects of Bacopa monniera in gastric ulcer models.

Bacopa monniera Wettst. (BM, syn. Herpestis monniera L; Scrophulariaceae), is an Ayurvedic drug used as a rasayana. Its fresh juice was earlier reported to have significant antiulcerogenic activity. In continuation, methanolic extract of BM (BME) standardized to bacoside-A content (percentage-38.0 ± 0.9), when given in the dose of 10–50 mg/kg, twice daily for 5 days, showed dose-dependent anti-ulcerogenic on various gastric ulcer models induced by ethanol, aspirin, 2 h cold restraint stress and 4 h pylorus ligation. BME in the dose of 20 mg/kg, given for 10 days, twice daily showed healing effects against 50% acetic acid-induced gastric ulcers. Further work was done to investigate the possible mechanisms of its action by studying its effect on various mucosal offensive acid-pepsin secretion and defensive factors like mucin secretion, mucosal cell shedding, cell proliferation and antioxidant activity in rats. BME 20 mg/kg showed no effect on acid-pepsin secretion, increased mucin secretion, while it decreased cell shedding with no effect on cell proliferation. BME showed significant antioxidant effect per se and in stressed animals. Thus, the gastric prophylactic and curative effects of BME may be due to its predominant effect on mucosal defensive factors.     

Production of rhamnolipids in solid-state cultivation: Characterization,downstream processing and application in the cleaning of contaminated soils

Pseudomonas aeruginosa UFPEDA 614 produced a rhamnolipid biosurfactant when grown on sugarcane bagasse impregnated with a solution containing glycerol. Biosurfactant levels reached 40 g of rhamnolipid per kilogram of dry initial substrate after 12 days. On the basis of the volume of liquid used, the biosurfactant levels were similar to those obtained in submerged liquid culture of a medium identical to the impregnating solution. The properties of the biosurfactant were very similar to those obtained with rhamnolipids produced in submerged culture, with a critical micelle concentration of 46.8 mg/L and an emulsification index at 24 h of over 90% against gasoline. The surface properties were maintained after autoclaving of the fermented solids, meaning that it is possible to minimize safety risks by killing the producing organism with a heat treatment of the solids prior to product extraction. The biosurfactant was used in the washing of soils contaminated with gasoline. An aqueous biosurfactant solution was 3.2-fold more efficient than water in leaching organic material from the soil, demonstrating the viability of application of rhamnolipids in the bioremediation of soils contaminated with gasoline.     

Profiling the changes in signaling pathways in ascorbic acid/β‐glycerophosphate‐induced osteoblastic differentiation

Despite numerous reports on the ability of ascorbic acid and β‐glycerophosphate (AA/β‐GP) to induce osteoblast differentiation, little is known about the molecular mechanisms involved in this phenomenon. In this work, we used a peptide array containing specific consensus sequences (potential substrates) for protein kinases and traditional biochemical techniques to examine the signaling pathways modulated during AA/β‐GP‐induced osteoblast differentiation. The kinomic profile obtained after 7 days of treatment with AA/β‐GP identified 18 kinase substrates with significantly enhanced or reduced phosphorylation. Peptide substrates for Akt, PI3K, PKC, BCR, ABL, PRKG1, PAK1, PAK2, ERK1, ERBB2, and SYK showed a considerable reduction in phosphorylation, whereas enhanced phosphorylation was observed in substrates for CHKB, CHKA, PKA, FAK, ATM, PKA, and VEGFR‐1. These findings confirm the potential usefulness of peptide microarrays for identifying kinases known to be involved in bone development in vivo and in vitro and show that this technique can be used to investigate kinases whose function in osteoblastic differentiation is poorly understood. J. Cell. Biochem. 112: 71–77, 2011. © 2010 Wiley‐Liss, Inc.     

Collagen receptors mediate early events in the attachment of epithelial (MDCK) cells

Summary Madin-Darby canine kidney (MDCK) cells kept in suspension culture for 12–15 hr displayed high-affinity binding sites for¹²⁵I-lathyritic (soluble) collagen (120,000/cell,K _D =30nm) and preferred collagens types I and IV over laminin or fibronectin as substrates during the first hour of attachment. On the other hand, after 4 hr, attachment to all four substrates was equally efficient. Upon challenge with a collagen substrate, the high-affinity sites were rapidly recruited on it (T1/2=6 min). Their occupancy by soluble collagen triggered the exocytosis of a second large population of low-affinity collagen binding sites that included laminin and seems to be involved in a second cell-attachment mechanism. These results are compatible with a twostep model of MDCK cell attachment to the substrate: first, via high-affinity collagen binding sites, and second, via laminin of cellular origin.     

The apparent absence of serotonin-mediated vascular thermogenesis in perfused rat hindlimb may result from vascular shunting

Vasoconstriction by norepinephrine, angiotensin II and vasopressin in the constant-flow perfused rat hindlimb is associated with increased oxygen uptake and has given rise to the concept of vascular thermogenesis. In the present study serotonin (5-hydroxytryptamine, 5HT) was found to inhibit oxygen uptake by up to 40% in a dose dependent manner whilst inducing vasoconstriction in this model, whereas norepinephrine increased oxygen consumption by up to 100% during vasoconstriction. This contrasted with the perfused isolated rat mesenteric artery arcade in which serotonin stimulated oxygen uptake by up to 130% in association with vasoconstriction in a dose dependent manner similar to the previously described norepinephrine induced vascular thermogenesis in this arterial preparation. In both perfusion systems, changes in pressure and oxygen uptake mediated by serotonin were completely blocked by ketanserin. These results and evidence from dye washout studies suggest that serotonin-mediated vascular thermogenesis, if it occurs in the constant-flow hindlimb, is masked by vascular shunting.     

Adaptive foraging of leaf‐cutter ants to spatiotemporal changes in resource availability in Neotropical savannas

1. Generalist herbivores feed on a wide and diverse set of species, but fine‐scale foraging patterns may be affected by the interplay between the quality, quantity and spatial distribution of host plants. 2. The foraging patterns of a prevalent Neotropical herbivore, the leaf‐cutter ant Atta laevigata, in the Brazilian Cerrado savannas were examined in order to determine if patterns observed are in concert with central‐place foraging predictions. 3. The results showed that A. laevigata acts as a polyphagous but highly selective herbivore, with ant attacks often resulting in partial defoliation of less‐preferred species and full defoliation of preferred ones. It was found, for the first time, that there is a strong and positive relationship between the relative attack frequency on plants from preferred species and foraging distance to the nest. This suggests a balance between the quality of plant resources harvested and costs involved in their transportation. It was also observed that colonies focused their harvest on preferred species in months with low availability of young leaves. Consequently, high herbivory rate was more frequent in plants attacked far away from the nest and in dry months. 4. These assessments highlight the fact that Atta colonies may become more selective as foraging distance to the nest increases and in response to fluctuations in the availability of palatable resources throughout the year. The results also show some dissimilarities in the foraging behaviours of A. laevigata when compared with other locations, suggesting that widely distributed herbivores may modify foraging strategies across their geographic range.     

AVPR1b variation and the emergence of adaptive phenotypes in Platyrrhini primates

Platyrrhini (New World monkeys, NWm) are a group of primates characterized by behavioral and reproductive traits that are otherwise uncommon among primates, including social monogamy, direct paternal care, and twin births. As a consequence, the study of Platyrrhine primates is an invaluable tool for the discovery of the genetic repertoire underlying these taxon‐specific traits. Recently, high conservation of vasopressin (AVP) sequence, in contrast with high variability of oxytocin (OXT), has been described in NWm. AVP and OXT functions are possible due to interaction with their receptors: AVPR1a, AVPR1b, AVPR2, and OXTR; and the variability in this system is associated with the traits mentioned above. Understanding the variability in the receptors is thus fundamental to understand the function and evolution of the system as a whole. Here we describe the variability of AVPR1b coding region in 20 NWm species, which is well‐known to influence behavioral traits such as aggression, anxiety, and stress control in placental mammals. Our results indicate that 4% of AVPR1b sites may be under positive selection and a significant number of sites under relaxed selective constraint. Considering the known role of AVPR1b, we suggest that some of the changes described here for the Platyrrhini may be a part of the genetic repertoire connected with the complex network of neuroendocrine mechanisms of AVP–OXT system in the modulation of the HPA axis. Thus, these changes may have promoted the emergence of social behaviors such as direct paternal care in socially monogamous species that are also characterized by small body size and twin births.