A transgenic mouse model for studying the lineage relationships and differentiation program of type II pneumocytes at various stages of lung development.

A pedigree of transgenic mice has been characterized that contains a H2-Kb/LacZ fusion gene that exhibits integration site-dependent expression from the earliest stages of lung development through adulthood. Histochemical and immunocytochemical studies indicate that the LacZ reporter appears throughout the pulmonary endoderm by embryonic day 11 (E11). A proximal-to-distal wave of extinction of transgene expression occurs during E13-14 that parallels the wave of cytodifferentiation of the pulmonary endoderm. By E16, the LacZ reporter is restricted to the distal portion of epithelial tubules and by birth to scattered cells located in alveoli. Crude epithelial cell suspensions were prepared from lungs harvested from E16 and 14 day postnatal transgenic mice, labeled with the fluorescent LacZ substrate fluorescein di-(beta-galactopyranoside), and the LacZ expressing population isolated by fluorescence-activated cell sorting. Electron microscopic, immunocytochemical and histochemical studies of this purified cell population establish that type II pneumocytes are the only cell lineage that support H2-Kb/LacZ expression in the mature postnatal lung. Fluorescence-activated cell sorting of E16 lung suspensions yielded a homogeneous population of cells that produced surfactant protein A, that could be maintained in cell culture, and that are likely precursors of adult type II pneumocytes. Together these studies indicate that (i) expression of the transgene in this pedigree of mice provides a marker for describing early differentiation of the pulmonary epithelium; (ii) the transgene may be useful as an enhancer trap to isolate cis-acting sequences that regulate gene transcription within this lineage; (iii) the LacZ reporter expression can be used to purify specific embryonic pulmonary epithelial cell populations; and (iv) primary cultures of these embryonic populations represent a potentially useful model system for analyzing the cellular components and signaling pathways necessary to support and complete passage through the type II pneumocyte differentiation program.

Expression of SV-40 T antigen in the small intestinal epithelium of transgenic mice results in proliferative changes in the crypt and reentry of villus-associated enterocytes into the cell cycle but has no apparent effect on cellular differentiation programs and does not cause neoplastic transformation.

The mouse intestinal epithelium represents a unique mammalian system for examining the relationship between cell division, commitment, and differentiation. Proliferation and differentiation are rapid, perpetual, and spatially well-organized processes that occur along the crypt-to-villus axis and involve clearly defined cell lineages derived from a common multipotent stem cell located near the base of each crypt. Nucleotides -1178 to +28 of the rat intestinal fatty acid binding protein gene were used to establish three pedigrees of transgenic mice that expressed SV-40 large T antigen (TAg) in epithelial cells situated in the uppermost portion of small intestinal crypts and in already committed, differentiating enterocytes as they exited these crypts and migrated up the villus. T antigen production was associated with increases in crypt cell proliferation but had no apparent effect on commitment to differentiate along enterocytic, enteroendocrine, or Paneth cell lineages. Single- and multilabel-immunocytochemical studies plus RNA blot hybridization analyses suggested that the differentiation programs of these lineages were similar in transgenic mice and their normal littermates. This included enterocytes which, based on the pattern of [3H]thymidine and 5-bromo-2'-deoxyuridine labeling and proliferating nuclear antigen expression, had reentered the cell cycle during their migration up the villus. The state of cellular differentiation and/or TAg production appeared to affect the nature of the cell cycle; analysis of the ratio of S-phase to M-phase cells (collected by metaphase arrest with vincristine) and of the intensities of labeling of nuclei by [3H]thymidine indicated that the duration of S phase was longer in differentiating, villus-associated enterocytes than in the less well-differentiated crypt epithelial cell population and that there may be a block at the G2/M boundary. Sustained increases in crypt and villus epithelial cell proliferation over a 9-mo period were not associated with the development of gut neoplasms--suggesting that tumorigenesis in the intestine may require that the initiated cell have many of the properties of the gut stem cell including functional anchorage.

Expression of a liver fatty acid binding protein/human decay-accelerating factor/HLA-B44 chimeric gene in transgenic mice.

The intestinal epithelium is characterized by the rapid and continuous renewal of its four principal cell types and by its ability to establish and maintain remarkably complex spatial differentiation along its crypt-to-villus and duodenal-to-colonic axes. We have previously used transgenic mice containing liver fatty acid binding protein/human growth hormone (L-FABP/hGH) fusion genes to analyze the molecular mechanisms responsible for encoding positional information in this epithelium. Because these studies could not distinguish whether cis-acting sequences in the L-FABP promoter or hGH structural gene were responsible for the observed cellular and regional patterns of transgene transcription in the gut, a second model fusion gene has now been constructed. It consists of nucleotides -596 to +21 of rat L-FABP linked to a cDNA encoding a chimeric protein, human decay-accelerating factor (DAF, minus the site of attachment of its COOH-terminal glycophospholipid anchor), coupled to the transmembrane (TM) and cytoplasmic domains of human HLA-B44. RNA blot hybridization and immunocytochemical analyses revealed that the cell-specific and region-specific expressions of DAF-TM and hGH in adult mice appear identical along both axes of the gut, indicating that cis-acting elements contained within the 5' nontranscribed region of the L-FABP gene rather than in the reporter are largely responsible for these observed patterns of transgene expression. Unlike pre-hGH, a prototypical secreted protein, DAF-TM is a membrane protein. The ability to direct its expression along the length of both axes of the gut provides an opportunity to analyze in vivo the sorting pathways of membrane-associated proteins in normal epithelial cells as a function of their location and differentiation. Light microscopic studies indicate that DAF-TM is targeted to the basolateral and apical surfaces of villus-associated enterocytes.

Biochemical and immunohistochemical evidence for selective expression of novel epithelial lipoxygenases.

Human tracheal epithelial cells contain an arachidonate 15-lipoxygenase, while the same cells from animals (including bovine, ovine, canine, porcine) cells express a 12-lipoxygenase. The epithelial 12-lipoxygenase is antigenically related to the leukocyte 12-lipoxygenase but is biochemically distinct from platelet and leukocyte forms of the enzyme, in that it is more efficient at metabolizing a wider array of fatty acid substrates. We have suggested that this lipoxygenase heterogeneity may provide a basis for different functional roles for the enzyme in different cell types. In addition, animal epithelial 12-lipoxygenase and human epithelial 15-lipoxygenase are antigenically related and have similar but distinct distributions in the lung. Our findings might suggest that the species diversity for epithelial lipoxygenases represents molecular divergence within a family of closely related genes with perhaps closely related functions.

Identification of a novel arachidonate 12-lipoxygenase in bovine tracheal epithelial cells distinct from leukocyte and platelet forms of the enzyme.

We examined the characteristics of an arachidonate 12-lipoxygenase in bovine tracheal epithelial cells in relation to the enzyme expressed in leukocytes and platelets. Homogenous preparations of intact or disrupted tracheal epithelial cells metabolized arachidonic acid predominantly to (12S)-hydroxyeicosatetraenoic acid, and subcellular fractionation by differential centrifugation demonstrated that the 12-lipoxygenase activity was localized predominantly to the 100,000 x g supernatant (cytosol fraction). Analysis of cytosolic enzymatic activity for pH dependence (maximum activity at pH 7.4-8.0), divalent cation effects (no dependence on cations), and kinetic characteristics (lag phase elimination by addition of hydroperoxide) exhibited similarity to leukocyte and platelet 12-lipoxygenases. Immunoprecipitation experiments demonstrated that the epithelial 12-lipoxygenase reacted with a monoclonal antibody (lox-2) directed against leukocyte 12-lipoxygenase but not with an antibody (HPLO-3) against the platelet enzyme. Immunoaffinity chromatography of the epithelial 100,000 x g supernatant fraction using lox-2 linked to Affi-Prep 10 yielded a single predominant protein band (Mr = 72,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis identical in apparent mass to the bovine leukocyte lipoxygenase. Western blotting using a polyclonal antibody to leukocyte 12-lipoxygenase showed peroxidase staining of the same 72-kDa protein band. Activity assays of the purified enzymes demonstrated that substrate specificity for the epithelial 12-lipoxygenase was similar to that of the leukocyte enzyme, but the epithelial enzyme more efficiently converted 18-carbon fatty acids to the corresponding monohydroxylated conjugated dienes. We conclude that bovine tracheal epithelial cells express a 12-lipoxygenase that has immunological reactivity similar to leukocyte and distinct from platelet 12-lipoxygenase and possesses substrate specificity distinct from both enzymes. We further suggest that lipoxygenase heterogeneity may provide a basis for different functional roles for the enzyme in different cell types.

Arachidonate 12-lipoxygenase and cyclooxygenase: PGE isomerase are predominant pathways for oxygenation in bovine tracheal epithelial cells.

The capacity of bovine tracheal epithelial cells to convert arachidonic acid to oxygenation products with potential biologic activity was studied in homogeneous preparations of isolated cells. Purified epithelial cell suspensions were incubated with radiolabeled arachidonic acid, and oxygenated metabolites were identified using high-pressure liquid chromatography and gas chromatography-mass spectrometry. The cells released predominantly two products during incubation with 0.3 to 150 microM arachidonic acid for 1 to 60 min at 37 degrees C: prostaglandin E2 (PGE2) and 12-hydroxyeicosatetraenoic acid (12-HETE). Concentration-response curves for the two products yielded half-maximal effects at 2 and 45 microM arachidonic acid, respectively. Stereochemical analysis by chiral-phase high-pressure liquid chromatography demonstrated that the epithelial 12-HETE consisted exclusively of the 12(S) isomer, providing supporting evidence that it was derived from an arachidonate 12-lipoxygenase. Epithelial cells prelabeled with arachidonic acid and incubated with 5 microM A23187 to stimulate endogenous arachidonic acid metabolism also released two predominant products with the chromatographic properties of PGE2 and 12-HETE. The findings demonstrate that bovine tracheal epithelial cells express both a cyclooxygenase:PGE isomerase and a 12-lipoxygenase pathway and therefore implicate this pathway as a new source of epithelial cell mediators.

Heterogeneity of cellular expression of arachidonate 15-lipoxygenase: implications for biological activity.

Comparison of relative arachidonate 15-lipoxygenase activities in different types of human cell reveals striking differences in levels of product generation. Among available human cell types, the tracheal epithelial cell and the eosinophil have markedly higher levels of activity than other cell types with reported activity. Theories of functional significance for this enzymatic pathway will need to account for the selective expression of high levels of activity in a highly limited number of cell types.

Uptake, release and novel species-dependent oxygenation of arachidonic acid in human and animal airway epithelial cells.

To determine identities of mediators and mechanisms for their release from pulmonary airway epithelial cells, we examined the capacities of epithelial cells from human, dog and sheep airways to incorporate, release and oxygenate arachidonic acid. Purified cell suspensions were incubated with radiolabeled arachidonic acid and/or ionophore A23187; fatty acid esterification and hydrolysis were traced chromatographically, and oxygenated metabolites were identified using high-pressure liquid chromatography and mass-spectrometry. In each species, cellular uptake of 10 nM arachidonic acid was concentrated in the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine fractions, and subsequent incubation with 5 microM A23187 caused release of 10-12% of the radiolabeled pool selectively from phosphatidylcholine and phosphatidylinositol. By contrast, the products of arachidonic acid oxygenation were species-dependent and in the case of human cells were also novel: A23187-stimulated human epithelial cells converted arachidonic acid predominantly to 15-hydroxyeicosatetraenoic acid (15-HETE) and two distinct 8,15-diols in addition to prostaglandin (PG) E2 and PGF2 alpha. Cell incubation with exogenous arachidonic acid (2.0-300 microM) led to progressively larger amounts of 15-HETE and the dihydroxy, epoxyhydroxy and keto acids characteristic of arachidonate 15-lipoxygenase. Both dog and sheep cells converted exogenous or endogenous arachidonic acid to low levels of 5-lipoxygenase products, including leukotriene B4 without significant 15-lipoxygenase activity. In the cyclooxygenase series, sheep cells selectively released PGE2, while dog cells generated predominantly PGD2. The findings demonstrate that stereotyped esterification and phospholipase activities are expressed at uniform levels among airway epithelial cells from these species, but pathways for oxygenating arachidonic acid allow mediator diversity depending greatly on species and little on arachidonic acid presentation.

Anaphylaxis to intravenous furosemide.

A patient with long-standing hypertension developed urticaria, angioedema, and hypotension within 5 minutes after the intravenous administration of furosemide. Immediate hypersensitivity was documented by positive skin tests to furosemide as well as to related sulfonamide-based drugs. This is the first finding of an anaphylactic reaction to furosemide and underscores the need to consider such adverse reactions when patients who are sensitive to other sulfonamide-containing drugs are being treated.

A prospective study of lung water measurements during patient management in an intensive care unit.

We prospectively evaluated a protocol that included extravascular thermal volume (ETV) as a measure of extravascular lung water (EVLW) instead of pulmonary artery wedge pressure (Ppaw) measurements to guide the hemodynamic management of 48 critically ill patients. Patients were randomized to either a protocol management (PM), or to a routine management (RM) group. In the RM group, EVLW measurements were unknown to the primary care physicians. The 2 groups were similar with respect to age, gender, and severity of illness. In patients with initially high EVLW, EVLW fell to a greater extent in PM than in RM patients (18 +/- 5 versus 4 +/- 8% decrease, p less than 0.05). This difference was even greater in patients with heart failure. No adverse effects on oxygenation or renal function occurred in following the protocol. Mortality for the groups as a whole was similar, but was significantly better (p less than 0.05) for PM patients with initially high EVLW and normal Ppaw (predominantly patients with sepsis or the adult respiratory distress syndrome). For both groups, patients with an initial EVLW greater than 14 ml/kg had a significantly greater mortality than did those with a lesser amount of EVLW: 13 of 15 (87%) versus 13 of 32 (41%), p less than 0.05. We conclude that management based on a protocol using EVLW measurements is safe, may hasten the resolution of pulmonary edema, and may lead to improved outcome in some critically ill patients.

Speract. Purification and characterization of a peptide associated with eggs that activates spermatozoa.

A low molecular weight peptide (speract) associated with sea urchin eggs has been purified to apparent homogeneity by charcoal adsorption, DEAE-Sephacel chromatography, Bio-Gel P-2 filtration, and Dowex AG 50W-X4 chromatography. Gametes from 5000 female sea urchins were required for the isolation of approximately 9 mg of the peptide. The isolated peptide is homogenous based on [3H]acetic anhydride labeling, gel filtration, and reverse phase high pressure liquid chromatography. Speract is composed entirely of neutral and acidic amino acids with glycine as the major component, and it appears to have a blocked NH2 terminus based on its insensitivity to leucine aminopeptidase, its failure to react with dansyl chloride, and its chromatographic behavior on strong cation exchange resins. Speract is a potent stimulator of sea urchin sperm oxygen consumption, causing significant increases of sperm respiration rates at concentrations as low as 10(-12) M and producing 20-fold increases of oxygen consumption at maximal concentrations of 10(-8) M. Sperm cyclic GMP and cyclic AMP concentrations are also increased by speract, but concentrations of at least 10(-10) M and 10(-9) M are required for half-maximal elevations, respectively. The peptide, purified from Strongylocentrotus purpuratus eggs, also cross-reacts with spermatozoa from Lytechnis pictus sea urchins, suggesting that speract does not show species specificity. These results represent the first report of the purification of a peptide associated with eggs that may affect spermatozoa under natural conditions.

Purification and characterization of calmodulin from sea urchin spermatozoa.

Calmodulin was purified to apparent homogeneity from sea urchin spermatozoa by heat-treatment at 85 degrees C, ammonium sulphate precipitation at pH 4.2, DEAE-Sephacel chromatography and gel filtration on Sephadex G-100. Approximately 8.3 micrograms calmodulin were recovered per 10(10) sperm cells. The sperm calmodulin had an apparent molecular weight of 17 800. The purified calmodulin activated calmodulin-deficient phosphodiesterase from pig coronary arteries, with half-maximal activation occurring at approximately 40 ng calmodulin/ml. Trifluoperazine also inhibited the sperm calmodulin activity. These results demonstrate that calmodulin is present in high amounts in sea urchin spermatozoa, and that it is essentially the same as the calmodulin isolated from various other tissues.