Purification of a cell-surface receptor for surfactant protein A

In the present report we have characterized the binding of surfactant protein A (SP-A) to bone marrow-derived macrophages, U937 cells, alveolar macrophages, and type II epithelial cells. The binding of SP-A to all cell types is Ca2+-dependent and trypsin-sensitive, but type II cells express distinct Ca2+-independent binding sites. The binding of SP-A to macrophages is independent of known cell surface carbohydrate-specific receptors and of glycoconjugate binding sites on the surface of the cells and is distinct from binding to C1q receptors. Based on ligand blot analysis, both type II cells and macrophages express a 210-kDa SP-A-binding protein. The 210-kDa protein was purified to apparent homogeneity from U937 macrophage membranes using affinity chromatography with noncovalently immobilized surfactant protein A, and was purified from rat lung by differential detergent and salt extraction of isolated rat lung membranes. Polyclonal antibodies against the rat lung SP-A-binding protein inhibit binding of SP-A to both type II cells and macrophages, indicating that the 210-kDa protein is expressed on the cell surface. The polyclonal antibodies also block the SP-A-mediated inhibition of phospholipid secretion by type II cells, indicating that the 210-kDa protein is a functional cell-surface receptor on type II cells. In a separate report we have determined that antibodies to the SP-A receptor block the SP-A-mediated uptake of Mycobacterium bovis, indicating that the macrophage SP-A receptor is involved in SP-A-mediated clearance of pathogens.     

Epistemological conflict: modern and non-modern frameworks for sustainability

A basic epistemological conflict is found to exist between modern and non-modern practitioners of sustainable development. These categories distinguish the ways professionals interpret or frame reality. The hypothesis developed is that this inconsistency, at least partially, explains the limited success that energy-efficiency research has realized in the prediction and control of climate change catalysed by the built environment. An analysis employs both historical and empirical methods to understand how the North American air-conditioning industry has framed, and subsequently regulated, the inseparable problems of human comfort and energy consumption. Historically, the dominant framework long-inhabited by moderns has constructed a unit-efficiency model of evaluation that is concerned with universal standardization and normal design. In the empirical analysis of the selected case, an emergent framework inhabited by non-moderns constructed a unit-efficacy model of evaluation concerned with local implementation and post-normal design. The two models came into conflict when designers applied code-required energy models and financing formulae based on unit-efficiency assumptions to a case of sustainable, affordable housing. The analysis concludes with seven findings designed to move building energy research and practice beyond the current epistemological divide.     

Adenovirus-mediated granulocyte-macrophage colony-stimulating factor improves lung pathology of pulmonary alveolar proteinosis in granulocyte-macrophage colony-stimulating factor-deficient mice

Mutation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene by homologous recombination causes progressive pulmonary alveolar proteinosis (PAP) in GM-CSF-deficient mice (GM-/-). The present study tested whether adenovirus-mediated expression of GM-CSF alters the progression of PAP in GM-/- mice. Adult mice were pretreated with an anti-T cell receptor (TCR) antibody to block T cell-mediated immune response, followed by intratracheal instillation of deltaE1-E3 replication-deficient adenovirus expressing mouse GM-CSF (Av1mGM). Mice were killed 1, 3, and 5 weeks after treatment to assess lungs for GM-CSF, surfactant protein B (SP-B), alveolar macrophage maturation, and type II cell proliferation. GM-CSF was detected in BAL fluid from GM-/- mice 1 week after Av1mGM treatment, and GM-CSF mRNA was detected by RT-PCR through 5 weeks. Five weeks after Av1mGM treatment, PAP was improved and SP-B decreased as assessed by ELISA and immunostaining. Increased numbers of alveolar macrophages stained with alpha-naphthyl acetate esterase (alpha-NAE) following treatment with Av1mGM. Local expression of GM-CSF with a recombinant adenovirus ameliorated PAP in the GM-/- mice in association with enhanced maturation of alveolar macrophages.     

Decreased surfactant protein A in patients with bacterial pneumonia

Abnormalities have been previously noted in the lipid content of the lavage fluid of patients with bacterial pneumonia. In order to determine if these changes were also seen in surfactant apoproteins, we studied levels of surfactant protein A (SP-A) in patients with bacterial pneumonia. Patients without human immunodeficiency virus who were being evaluated for pulmonary infiltrates underwent bronchoscopy with bronchoalveolar lavage (BAL). Twenty-two patients with pneumonia, 12 caused by gram-positive organisms (Gm+ PNEU) and 10 caused by gram-negative organisms (Gm- PNEU), were compared with 10 patients with idiopathic pulmonary fibrosis (IPF) and 11 control subjects (CON). The percentage of neutrophils in the BAL was significantly higher in the patients with IPF and the pneumonia groups than in the control group (CON: mean, 1; range, 0 to 3. IPF: mean, 26; range, 13 to 42). Gm+ PNEU: mean, 33; range, 8 to 99. Gm- PNEU: mean, 64; range, 10 to 92; p < 0.0001). The amount of SP-A in the BAL fluid was similar for the CON and the IPF groups (CON: mean, 15; range, 5.75 to 26.5 micrograms/ml BAL. IPF: mean, 18.4; range, 6.49 to 45.64 micrograms/ml), whereas both pneumonia groups had significantly less SP-A (Gm- PNEU: mean, 5.54; range, 0.58 to 12.7. G+ PNEU: mean, 1.93; range, 0.47 to 6.74; p < 0.001). There was significantly less SP-A in the Gm+ PNEU group than in the Gm- PNEU group (p < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-4 enhances pulmonary clearance of Pseudomonas aeruginosa

To determine the effects of interleukin-4 (IL-4) on bacterial clearance from the mouse lung, transgenic mice expressing IL-4 in respiratory epithelial cells under the control of the Clara cell secretory protein promoter (CCSP-IL-4 mice) were infected intratracheally with Pseudomonas aeruginosa. Survival of CCSP-IL-4 mice following bacterial administration was markedly improved compared with that of control mice. While bacteria proliferated in lungs of wild-type mice, a rapid reduction in the number of bacteria was observed in the IL-4 mice as early as 6 h postinfection. Similarly, intranasal administration of IL-4 enhanced bacterial clearance from the lungs of wild-type mice. While acute and chronic IL-4 increased the numbers of neutrophils in bronchoalveolar lavage fluid, bacterial infection was associated with acute neutrophilic pulmonary infiltration, and this response was similar in the presence or absence of IL-4. Local administration or expression of IL-4 in the mouse lung enhanced pulmonary clearance of P. aeruginosa in vivo and decreased mortality following infection.     

Intraamniotic administration of an adenoviral vector for gene transfer to fetal sheep and mouse tissues

Replication-deficient adenoviruses have been used to transfer various genes of interest to mammalian tissues in vivo. Effective gene therapy for inborn genetic defects presenting with significant morbidity and mortality at birth will require correction of the defect prenatally. To test the hypothesis that intra-amniotically administered adenovirus transfers gene expression to fetal tissues, replication-deficient human type 5 adenovirus carrying the lacZ gene which encodes nuclear-targeted bacterial beta-galactosidase (Av1LacZ4) was instilled into the amniotic cavity of fetal sheep (10(10) to 1.5 x 10(11) pfu) and fetal mice (10(9) pfu) at 0.8 term gestation. Amniotic membranes and gastrointestinal and respiratory tract tissues were harvested after 3 d, bacterial beta-galactosidase activity was determined by 5-bromo-4-chloro-3-indoyl-beta-D-galactopyranoside (X-gal) enzyme-histochemistry, and tissue integrity was assessed in sections stained with hematoxylin and eosin. Bacterial beta-galactosidase activity was abundant in amniotic membranes and present in lower levels in esophagus, stomach, and small intestine as well as in conducting airways and pulmonary alveoli. To determine whether gene transfer by intraamniotic injection of adenovirus was dose-dependent, Av1Luc1, an adenoviral vector carrying the gene for luciferase (10(5)-10(9) pfu), was injected intraamniotically into fetal mice at 0.8 term gestation. Luciferase activity measured after 3 d in tissue homogenates of Av1Luc1-treated fetal mice revealed a linear dose response in amniotic membranes and gastrointestinal and respiratory tract organs. Intraamniotic administration of an adenoviral gene vector leads to expression of the transferred gene in amniotic membranes as well as in fetal gastrointestinal and respiratory tract tissues in a dose-dependent manner.     

Calcium uptake and binding by membrane fractions of human placenta: ATP-dependent calcium accumulation

An ATP-dependent calcium (Ca2+) sequestration activity was demonstrated in membrane vesicles prepared from the human term placenta. Microsomal and brush border membrane fractions accumulated Ca2+ within a vesicular space by a saturable process requiring Mg2+ and ATP. The "uptake" activity was enriched six-fold in a microsomal membrane fraction and was only 1.5-fold enriched in purified brush border membranes compared to the activity present in the filtered homogenate. Mitochondrial inhibitors such as azide and oligomycin did not inhibit Ca2+ uptake in these preparations. The process was temperature dependent and displayed Michaelis-Menten-like kinetics with respect to free Ca2+ concentrations. At 30 degrees C, the Vmax was 1.05 nmole/mg/min; Km = 74 nM for free Ca2+ in the microsomal fraction. Oxalate and phosphate enhanced uptake in both fractions. Ca2+ uptake activity was not associated with Ca2+-stimulated ATPase, alkaline phosphatase, or other brush border markers during cell fractionation. The characteristics of the Ca2+ uptake process contrasted sharply with those of Ca2+-stimulated ATPase, and a Ca2+-stimulated, Mg2+-dependent ATPase activity could not be identified in these membrane vesicle preparations.     

Sex difference in the control of scent-marking behavior in the Mongolian gerbil (Meriones unguiculatus).

Investigated the importance of the gonadal hormones in the control of the scent-marking behavior of 51 adult female Mongolian gerbils. Gonadectomy did not lead to the decrease in marking which had been expected from previous studies with males. The 2nd experiment was a longitudinal study of the development of scent marking in 36 female Ss. Ss were ovariectomized or given sham operations at 22 days of age. Gonadal hormones were not required for the development of marking. In both experiments morphological measures of the ventral sebaceous scent gland and the presence of the vaginal orifice were affected by gonadectomy. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Surfactant protein A (SP-A) gene targeted mice

Mice lacking surfactant protein A (SP-A) mRNA and protein in vivo were generated using gene targeting techniques. SP-A (-/-) mice have normal levels of SP-B, SP-C and SP-D mRNA and protein and survive and breed normally in vivarium conditions. Phospholipid composition, secretion and clearance, and incorporation of phospholipid precursors are normal in the SP-A (-/-) mice. Lungs of SP-A (-/-) mice have markedly decreased tubular myelin figures and clear Group B streptococci and Pseudomonas aeruginosa less efficiently than SP-A wild type mice. These studies of SP-A (-/-) mice demonstrate that SP-A has an important role in the innate immune system of the lung in vivo.     

Influence of photoperiod and social environment on sexual maturation in female deer mice (Peromyscus maniculatus bairdii).

Tested the reproductive response of young female deer mice to either a long or short photoperiod combined with the presence of adult males, adult females, or social isolation. 266 Ss were reared on either a long photoperiod (15 hrs light, 9 hrs dark) or on short days (8 hrs light, 16 hrs dark) from birth. Beginning at weaning, females were housed with an adult male, with an adult female, or in social isolation. In Exp I, vaginas opened more slowly in Ss on short days than in those on long days. Vaginal introitus was also retarded in Ss reared with an adult female in comparison with Ss reared in isolation. When examined at 37 days of age, Ss reared with an adult male had larger uteri than those reared alone; uteri were also larger in long-day than in short-day Ss. In Exp II, Ss were killed at 30 days of age; again, uterine growth was stimulated by exposure to either long days or an adult male. As was previously demonstrated for male deer mice, sexual maturation in females is regulated by photoperiod and social cues. Heterospecific social stimuli accelerate maturation in individuals that otherwise would be inhibited by having been reared on a short photoperiod. (27 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)