In vitro and in vivo growth and casein gene expression of mouse mammary tumor epithelial cells in response to hormones

Cells from autochthonous mouse mammary carcinomas which display estrogen-independent growth _vivo were studied for their hormonal responses in primary culture. A culture system employing insulin-supplemented, serum-free medium and basement membrane Matrigel as a substratum was used to cultivate tumor cells. The cells did not exhibit in vitro estrogenor prolactin-dependent growth. Primary tumors still displayed a constitutional expression of α-, β-, and γ-casein mRNAs. These messages were dramatically reduced during the culture period. However, seven to eightfold increases in α- and β-casein mRNAs were inducible in the 5-day cultures by treatment with prolactin and hydrocortisone. If the hormones were present through a 2-week culture period, the levels of α-, β-, and γ-casein mRNAs in the cells were maintained and displayed in a time-dependent increase with a peak at 10–14 days. The accumulation of β-casein mRNA in vitro did not require DNA synthesis. Administration of prolactin directly into the growing tumors in vivo could also enhance β-casein mRNA levels in the tumor cells. Morphological studies of the cells cultured in the presence of prolactin and hydrocortisone did not reveal visible changes compared with those without hormonal treatment. Transplantation of tumor cells cultured in the presence or absence of hormones resulted in the development of tumors in mice at approximately the same time. The current studies suggest that the autochthonous mammary tumor cells, independent of estrogen for cell growth, were still inducible for casein gene expression in vitro and in vivo by appropriate hormones. The induction and maintenance of casein messages by a single hormonal treatment did not appear to correlate with morphology and DNA synthesis of cells in vitro or with tumor-producing capacities in vivo.     

Human small cell lung cancer NYH cells resistant to the bisdioxopiperazine ICRF-187 exhibit a functional dominant Tyr165Ser mutation in the Walker A ATP binding site of topoisomerase II alpha

Bisdioxopiperazine anti-cancer agents are catalytic inhibitors of topoisomerase II which by unknown means lock the enzyme in a closed clamp form and inhibit its ATPase activity. In order to demarcate a putative pharmacophore, we here describe a novel Tyr165Ser mutation in the enzyme's Walker A ATP binding site leading to specific bisdioxopiperazine resistance when transformed into a temperature-conditional yeast system. The Tyr165Ser mutation differed from a previously described Arg162Gln by being heterozygous and by purified Tyr165Ser enzyme being drug-resistant in a kinetoplast DNA decatenation enzymatic assay. This suggested dominant nature of Tyr165Ser was supported by co-transformation studies in yeast of plasmids carrying wild type and mutant genes. These results enable a model of the bisdioxopiperazine pharmacophore using the proposed asymmetric ATP hydrolysis of the enzyme.     

The Response to Stimulation in Neurons and Astrocytes

Neurochemical Research - The brain uses mainly glucose as fuel with an index of glucose to oxygen utilization close to 6, the maximal index if all glucose was completely oxidized. However, this...     

A Combination of Proteomic Approaches Identifies A Panel of Circulating Extracellular Vesicle Proteins Related to the Risk of Suffering Cardiovascular Disease in Obese Patients

Plasma‐derived extracellular vesicles (EVs) have been extensively described as putative biomarkers in different diseases. Interestingly, increased levels of EVs subpopulations are well known to associate with obesity. The goal of this study is to identify EVs‐derived biomarkers in plasma from obese patients in order to predict the development of pathological events associated with obesity. Samples are obtained from 22 obese patients and their lean‐matched controls are divided into two cohorts: one for a 2D fluorescence difference gel electrophoresis (2D‐DIGE)‐based study, and the other one for a label free LC–MS/MS‐based approach. EVs are isolated following a serial ultracentrifugation protocol. Twenty‐two and 23 differentially regulated features are detected from 2D‐DIGE and label free LC–MS/MS, respectively; most of them involve in the coagulation and complement cascades. Remarkably, there is an upregulation of complement C4, complement C3, and fibrinogen in obese patients following both approaches, the latter two also validated by 2D‐western‐blotting in an independent cohort. These results correlate with a proinflammatory and prothrombotic state of those individuals. On the other hand, a downregulation of adiponectin leading to an increased risk of suffering cardiovascular diseases has been shown. The results suggest the relevance of plasma‐derived‐EVs proteins as a source of potential biomarkers for the development of atherothrombotic events in obesity.     

Ethylene and growth control in the fringed waterlily (Nymphoides peltata): Stimulation of cell division and interaction with buoyant tension in petioles

The effect of ethylene on petiole growth of the Fringed Waterlily (Nymphoides peltata (S.G. Gmelin) O. Kuntze) changes during leaf ontogeny. During early development (before expansion of laminae), ethylene causes an increase in both cell number and cell size; later in development, promotion of rapid cell expansion is the dominant effect. The early effects may contribute to the accommodation of new leaves to water columns of different depth. The later effects on cell expansion only are shown to contribute to the rapid accommodation of floating leaves when changes in water level submerge the laminae. This kind of accommodation results from an interaction between accumulated ethylene, which increases wall extensibility, and the tension in petioles due to natural buoyancy which, it is suggested, supplements the driving force for cell expansion. Cell age (position) within a petiole and age of the whole petiole influence the growth response to ethylene alone and the amount of extra growth produced by applying tension when ethylene is present. In young petioles, apical cells are highly sensitive to ethylene and tension causes little further growth; older cells in both immature and mature petioles show little response to ethylene unless the petiole is under tension. Young (but not mature) petioles respond slowly to applied tension even in the absence of ethylene. It is concluded that as cells age the driving force for expansion limits increasingly their capacity to respond to the wall-loosening effects of ethylene. Dual sensitivity to ethylene and buoyant tension facilitates rapid accommodation responses but sensitivity of young petioles to tension alone may exclude Nymphoides from habitats where current velocity is appreciable.     

Absence of DICER in Monocytes and Its Regulation by HIV-1

MicroRNAs (miRNAs) are a class of small RNA molecules that function to control gene expression and restrict viral replication in host cells. The production of miRNAs is believed to be dependent upon the DICER enzyme. Available evidence suggests that in T lymphocytes, HIV-1 can both suppress and co-opt the host''s miRNA pathway for its own benefit. In this study, we examined the state of miRNA production in monocytes and macrophages as well as the consequences of viral infection upon the production of miRNA. Monocytes in general express low amounts of miRNA-related proteins, and DICER in particular could not be detected until after monocytes were differentiated into macrophages. In the case where HIV-1 was present prior to differentiation, the expression of DICER was suppressed. MicroRNA chip results for RNA isolated from transfected and treated cells indicated that a drop in miRNA production coincided with DICER protein suppression in macrophages. We found that the expression of DICER in monocytes is restricted by miR-106a, but HIV-1 suppressed DICER expression via the viral gene Vpr. Additionally, analysis of miRNA expression in monocytes and macrophages revealed evidence that some miRNAs can be processed by both DICER and PIWIL4. Results presented here have implications for both the pathology of viral infections in macrophages and the biogenesis of miRNAs. First, HIV-1 suppresses the expression and function of DICER in macrophages via a previously unknown mechanism. Second, the presence of miRNAs in monocytes lacking DICER indicates that some miRNAs can be generated by proteins other than DICER.     

The initial step in human immunodeficiency virus type 1 GagProPol processing can be regulated by reversible oxidation

Background

Maturation of human immunodeficiency virus type 1 (HIV-1) occurs upon activation of HIV-1 protease embedded within GagProPol precursors and cleavage of Gag and GagProPol polyproteins. Although reversible oxidation can regulate mature protease activity as well as retrovirus maturation, it is possible that the effects of oxidation on viral maturation are mediated in whole, or part, through effects on the initial intramolecular cleavage event of GagProPol. In order assess the effect of reversible oxidation on this event, we developed a system to isolate the first step in protease activation involving GagProPol.

Methodology/Principal Findings

To determine if oxidation influences this step, we created a GagProPol plasmid construct (pGPfs-1C) that encoded mutations at all cleavage sites except p2/NC, the initial cleavage site in GagProPol. pGPfs-1C was used in an in vitro translation assay to observe the behavior of this initial step without interference from subsequent processing events. Diamide, a sulfhydral oxidizing agent, inhibited processing at p2/NC by >60% for pGPfs-1C and was readily reversed with the reductant, dithiothreitol. The ability to regulate processing by reversible oxidation was lost when the cysteines of the embedded protease were mutated to alanine. Unlike mature protease, which requires only oxidation of cys95 for inhibition, both cysteines of the embedded protease contributed to this inhibition.

Conclusions/Significance

We developed a system that can be used to study the first step in the cascade of HIV-1 GagProPol processing and show that reversible oxidation of cysteines of HIV-1 protease embedded in GagProPol can block this initial GagProPol autoprocessing. This type of regulation may be broadly applied to the majority of retroviruses.     

Spores morphology and synangia in neotropical fern species of Marattia (Marattiaceae)

The Marattiaceae are represented by a small family of four to six genera that bear esporogenous structures of two types: sorus with free eusporangia in Angiopteris and Archangiopteris, and indurated synangium in Christensenia, Danaea and Marattia. Marattia is a pantropical genus of about eight to ten species in the paleotropic and seven to eight species in the neotropic. In order to describe the spores and sinangia morphology, this study analyzed the shape of the receptacles, and the position of the synangia, and evaluated the spores with SEM, of seven neotropical species of the genus Marattia: M. alata, M. cicutifolia, M. excavata, M. interposita, M. laevis, M. laxa y M. weinmanniifolia from several collections. The receptacles were fully developed in M. cicutifolia and M. laevis, and scarcely overelevated in the rest of the species. The synangium was ellipsoidal and had intramarginal to supramedial position in the laminae. The spores of Marattia were elliptic. Among the taxa, only monolete spores were found, with no trilete, aborted or deformed spores. The laesura was linear and reached about two of the total length of the spore. The perispore appears as a continuous thin layer deposited on the exospore according to its ornamentation in M. cicutifolia and M. laevis. It is smooth in M. alata, rugate in M. excavata and pustulate-rugate in two species: M. interposita and M. laxa. The exospore is echinate in M. cicutifolia and M. laevis and pustulate in the other species. In M. weinmannifolia spores produced by the same sinangium may have different ornamentation types. We concluded that, while the presence of ellipsoidal and superficial synangia and monolete spores aperture were generic traits, the micro and macro-ornamentation types of the perispores and exospores vary at specific level. Besides, macro-ornamentation can be bulliform (pustulate), a combination of bulliform and muriform types (pustulate-rugate), muriform (rugate-retate) and stelliform (echinate); finally, granular micro-ornamentation can be seen frecuently in perispores.     

Down-regulation of E-cadherin in human bronchial epithelial cells leads to epidermal growth factor receptor-dependent Th2 cell-promoting activity

Airway epithelial cells are well-known producers of thymus- and activation-regulated chemokine (TARC), a Th2 cell-attracting chemokine that may play an important role in the development of allergic airway inflammation. However, the mechanism responsible for up-regulation of TARC in allergy is still unknown. In the asthmatic airways, loss of expression of the cell-cell contact molecule E-cadherin and reduced epithelial barrier function has been observed, which may be the result of an inadequate repair response. Because E-cadherin also suppressed multiple signaling pathways, we studied whether disruption of E-cadherin-mediated cell contact may contribute to increased proallergic activity of epithelial cells, e.g., production of the chemokine TARC. We down-regulated E-cadherin in bronchial epithelial cells by small interference RNA and studied effects on electrical resistance, signaling pathways, and TARC expression (by electric cell-substrate impedance sensing, immunodetection, immunofluorescent staining, and real-time PCR). Small interference RNA silencing of E-cadherin resulted in loss of E-cadherin-mediated junctions, enhanced phosphorylation of epidermal growth factor receptor (EGFR), and the downstream targets MEK/ERK-1/2 and p38 MAPK, finally resulting in up-regulation of TARC as well as thymic stromal lymphopoietin expression. The use of specific inhibitors revealed that the effect on TARC is mediated by EGFR-dependent activation of the MAPK pathways. In contrast to TARC, expression of the Th1/Treg cell-attracting chemokine RANTES was unaffected by E-cadherin down-regulation. In summary, we show that loss of E-cadherin-mediated epithelial cell-cell contact by damaging stimuli, e.g., allergens, may result in reduced suppression of EGFR-dependent signaling pathways and subsequent induction of Th2 cell-attracting molecule TARC. Thus, disruption of intercellular epithelial contacts may specifically promote Th2 cell recruitment in allergic asthma.