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.     

Relative positions of some epitopes on carcinoembryonic antigen

Summary To investigate whether anti-(carcinoembryonic antigen) monoclonal antibodies (mAb) react with single or repeated epitopes, sandwich radioimmunoassays in homologous and heterologous combinations were performed. Four mAb (I-27, I-47, II-17 and to some degree II-16) gave homologous binding while two mAb (I-38S1 and II-10) did not. Taken together with previous immunoprecipitation studies we conclude that all these mAb except II-10 react with repeated epitopes. The relative positions of the epitopes recognized by these mAb and of three additional mAb (II-6, II-7 and CB-CEA-1) were investigated using a plate antibody competition test with enzyme-labelled carcinoembryonic antigen (CEA). mAb I-38S1, II-6, II-7, II-10, II-16 and CB-CEA-1 were mutually cross-reactive, and were classified as belonging to one epitope group. mAb I-27 and I-47 fell outside this group and did not interfere with the binding of CEA conjugate to mAb II-17 either. They therefore represent a second epitope group. mAb II-17 showed no interference with the binding of CEA to any of the other mAb and must therefore represent a third epitope group. The slopes of the plate antibody competition curves were used for calculation of a correlation matrix, which in turn was used to depict the relative positions of the epitopes recognized by the mAb in the large group.     

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.     

Use of gas chromatography-mass spectrometry/solid phase microextraction for the identification of MVOCs from moldy building materials

Gas chromatography-mass spectrometry/solid phase microextraction (GC-MS/SPME) was applied to identify microbial volatile organic compounds (MVOCs) in water-damaged, mold-infested building materials (gypsum board papers (n=2), mineral wool, and masonite) and in cultivated molds (Aspergillus penicillioides, Stachybotrys chartarum, and Chaetomium globosum). Three SPME fibers (65-microm PDMS-DVB, 75-microm Carboxen-PDMS, and 70-microm Carbowax-stableflex) designed for automated injection were used of which the latter showed best performance. A number of previously reported MVOCs were detected both in the building materials and the cultivated molds. In addition, methyl benzoate was identified both in the S. chartarum and A. penicillioides cultures and in the building materials. SPME combined with GC-MS may be a useful method for the determination of MVOCs emitted from mold-infested building materials.     

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...     

Population genetic structure and connectivity of the seagrass Thalassia hemprichii in the Western Indian Ocean is influenced by predominant ocean currents

This study is the first large‐scale genetic population study of a widespread climax species of seagrass, Thalassia hemprichii, in the Western Indian Ocean (WIO). The aim was to understand genetic population structure and connectivity of T. hemprichii in relation to hydrodynamic features. We genotyped 205 individual seagrass shoots from 11 sites across the WIO, spanning over a distance of ~2,700 km, with twelve microsatellite markers. Seagrass shoots were sampled in Kenya, Tanzania (mainland and Zanzibar), Mozambique, and Madagascar: 4–26°S and 33–48°E. We assessed clonality and visualized genetic diversity and genetic population differentiation. We used Bayesian clustering approaches (TESS) to trace spatial ancestry of populations and used directional migration rates (DivMigrate) to identify sources of gene flow. We identified four genetically differentiated groups: (a) samples from the Zanzibar channel; (b) Mozambique; (c) Madagascar; and (d) the east coast of Zanzibar and Kenya. Significant pairwise population genetic differentiation was found among many sites. Isolation by distance was detected for the estimated magnitude of divergence (D_EST), but the three predominant ocean current systems (i.e., East African Coastal Current, North East Madagascar Current, and the South Equatorial Current) also determine genetic connectivity and genetic structure. Directional migration rates indicate that Madagascar acts as an important source population. Overall, clonality was moderate to high with large differences among sampling sites, indicating relatively low, but spatially variable sexual reproduction rates. The strongest genetic break was identified for three sites in the Zanzibar channel. Although isolation by distance is present, this study suggests that the three regionally predominant ocean current systems (i.e., East African Coastal Current, North East Madagascar Current, and the South Equatorial Current) rather than distance determine genetic connectivity and structure of T. hemprichii in the WIO. If the goal is to maintain genetic connectivity of T. hemprichii within the WIO, conservation planning and implementation of marine protection should be considered at the regional scale—across national borders.     

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.