Preparation of a human lung purified plasma membrane fraction: Confirmation by enzyme markers,electron microscopy,and histamine H1 receptor binding

Summary A simple and rapid method of isolating plasma membranes from human peripheral lung tissue is described. The method involves homogenization of tissue in 0.25m sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Enzymatic and morphological characterization of the plasma membrane fraction revealed minimal contamination by nonplasma membrane fragments. The isolated plasma membranes showed an 18-fold purification of 5-nucleotidase activity compared to the original homogenate. Electronmicroscopic studies of the plasma membrane fraction revealed the presence of small membrane vesicles having a trilaminar membrane structure. To further examine the purity of the plasma membrane preparation, the binding of the H₁ receptor antagonist,³H pyrilamine, to the plasma membrane-enriched fraction was compared to the binding to crude membrane preparations. Both the plasma membrane-enriched fraction and the crude membrane preparation had similar K_d's for the histamine antagonist, but the plasma membrane-enriched fraction had a threefold greater binding capacity, reflecting the relative enrichment of plasma membranes of the preparation. Thus, a method has been developed for the isolation of plasma membranes from human peripheral lung which should provide material for a variety of biochemical and pharmacological studies.     

Acidic fibroblast growth factor stimulates adrenal chromaffin cells to proliferate and to extend neurites, but is not a long-term survival factor

Acidic fibroblast growth factor (aFGF) is a heparin-binding polypeptide that is a mitogen for endothelial cells and glial cells, as well as a differentiation factor for PC12 cells and certain neurons. We show here that aFGF is as potent as nerve growth factor (NGF) in stimulating both neuritic outgrowth and proliferation in adrenal chromaffin cells from young rats, but it fails to support long-term survival. Heparin strongly potentiates aFGF-dependent neuritic outgrowth but not aFGF-dependent proliferation. As is the case with NGF, phorbol myristate acetate depresses aFGF-induced cell division and increases the outgrowth of neurites. On the other hand, dexamethasone antagonizes neuritic outgrowth elicited by both NGF and aFGF but inhibits only proliferation induced by NGF. The effects of basic FGF (bFGF) are similar but not identical to those of aFGF. Thus the regulatory pathways controlled by aFGF, bFGF, and NGF are partially distinct.     

The HL-60 transforming sequence: a ras oncogene coexisting with altered myc genes in hematopoietic tumors

The oncogene of the HL-60 human promyelocytic leukemia cell line has been passed serially through NIH/3T3 mouse fibroblasts. Oncogene-specific probes prepared from the resulting tertiary transfectants by molecular cloning have been used to show that loss of the transfected oncogene from NIH/3T3 cells correlates with reversion to nontransformed morphology. Analysis of cells transfected by the oncogenes of other tumors and tumor cell lines indicates that the transforming gene of the HL-60 leukemia cell line is closely related to oncogenes of a Burkitt's lymphoma, an acute myelogenous leukemia, an adenocarcinoma of the colon, a neuroblastoma, and two sarcomas. This oncogene is distantly related to the viral oncogenes of Kirsten and Harvey sarcoma viruses. It has been termed N-ras. The active N-ras oncogene coexists with altered versions of the myc oncogene in the HL-60 and AW Ramos human tumors. This suggests a multistep mechanism involving both ras and myc genes in the creation of these tumors.     

Draft Genome Sequence of the Sulfobacillus thermosulfidooxidans Cutipay Strain,an Indigenous Bacterium Isolated from a Naturally Extreme Mining Environment in Northern Chile

Sulfobacillus thermosulfidooxidans strain Cutipay is a mixotrophic, acidophilic, moderately thermophilic bacterium isolated from mining environments of the north of Chile, making it an interesting subject for studying the bioleaching of copper. We introduce the draft genome sequence and annotation of this strain, which provide insights into its mechanisms for heavy metal resistance.     

Influence of the carbon and nitrogen sources on keratinase production by <Emphasis Type="Italic">Myrothecium verrucaria</Emphasis> in submerged and solid state cultures

Myrothecium verrucaria is a nondermatophytic filamentous fungus able to grow and to produce keratinase in submerged (93.0 ± 19 U/ml) and solid state (98.8 ± 7.9 U/ml) cultures in which poultry feather powder (PFP) is the only substrate. The purpose of the present work was to verify how different carbon and nitrogen sources can influence the production of keratinase by this fungus. Addition of carbohydrates, such as glucose and sucrose, caused only slight improvements in keratinase production, but the addition of starch caused a significant improvement (135.0 ± 25 U/ml). The highest levels of keratinase activity, however, were obtained by supplementing the PFP cultures with cassava bagasse, 168.0 ± 28 U/ml and 189.0 ± 26 U/ml in submerged and solid state cultures, respectively. Contrarily, the supplementation of PFP medium with organic or inorganic nitrogen sources, such as casein, soy bean protein, gelatin, ammonium nitrate and alanine, decreased the production of keratinase in both types of cultures (around 20 U/ml), showing that the production of keratinase by M. verrucaria is repressed by nitrogen sources. The results obtained in this work suggest that the association of the two residues PFP plus cassava bagasse could be an excellent option as a cheap culture medium for the production of keratinase in submerged and solid state cultures.     

Functional blockade of α<Subscript>5</Subscript>β<Subscript>1</Subscript> integrin induces scattering and genomic landscape remodeling of hepatic progenitor cells

Background  

Cell scattering is a physiological process executed by stem and progenitor cells during embryonic liver development and postnatal organ regeneration. Here, we investigated the genomic events occurring during this process induced by functional blockade of α₅β₁ integrin in liver progenitor cells.     

Development of a steroidogenic factor 1/Cre transgenic mouse line

The Cre-loxP strategy provides an approach to disrupt genes in specific tissues and/or cell types, circumventing lethality associated with global knockouts or secondary effects due to gene inactivation at other sites. A critical component is the development of transgenes that target Cre expression to specific cell types. Here, we describe the use of bacterial artificial chromosome (BAC) transgenesis to target Cre expression to tissues that express steroidogenic factor 1 (SF-1, officially designated Nr5a1). Consistent with the SF-1 expression pattern, the SF-1 BAC directed Cre expression to the somatic cells of the gonads, the adrenal cortex, the anterior pituitary, the spleen, and the ventromedial hypothalamic nucleus. This transgene provides a powerful tool to inactivate genes of interest in these tissues.