Primary cell culture of human type II pneumonocytes: maintenance of a differentiated phenotype and transfection with recombinant adenoviruses

Studies of the regulation of surfactant lipoprotein metabolism and secretion and surfactant protein gene expression have been hampered by the lack of a cell culture system in which the phenotypic properties of type II cells are maintained. We have developed a primary culture system that facilitates the maintenance of a number of morphologic and biochemical properties of type II pneumonocytes for up to 2 wk. Cells were isolated by collagenase digestion of midgestation human fetal lung tissue that had been maintained in organ culture in the presence of dibutyryl cyclic AMP (Bt2cAMP) for 5 days. The isolated cells were enriched for epithelial components by treatment with DEAE-dextran, plated on an extracellular matrix (ECM) derived from Madin-Darby canine kidney (MDCK) cells, and incubated at an air/liquid interface in a minimal amount of culture medium containing Bt2cAMP. The cell cultures were comprised of islands of round epithelial-like cells containing numerous dense osmiophilic granules, surrounded by sparse spindle-shaped cells with the appearance of fibroblasts. Ultrastructural examination revealed that the osmiophilic granules had the appearance of lamellar bodies, the distinguishing feature of type II pneumonocytes. Additionally, the cultures maintained elevated levels of SP-A gene expression for up to 2 wk. The expression of mRNAs encoding SP-A, SP-B, and SP-C were regulated in the cultured cells by glucocorticoids and cyclic AMP in a manner similar to that observed in fetal lung tissue in organ culture. The differentiated phenotype was most apparent when the cells were cultured at an air/liquid interface. In order to utilize the cultured type II cells for study of the effects of overexpression of various proteins and for promoter analysis, it is of essence to transfect DNA constructs into these cells with high efficiency. Unfortunately, we found the cells to be refractory to efficient transfer of DNA using conventional methods (i.e., lipofection, electroporation, or calcium phosphate-mediated transfection). However, replication-defective recombinant human adenoviruses were found to provide a highly efficient means of introducing DNA into the type II pneumonocytes. Furthermore, we observed in type II cell-enriched cultures infected with recombinant adenoviruses containing the lacZ gene under control of a cytomegalovirus promoter, that beta-galactosidase was expressed uniformly in the islands of type II cells and surrounding fibroblasts. By contrast, in cultures infected with recombinant adenoviruses containing the human growth hormone (hGH) gene under control of the SP-A gene promoter and 5'-flanking region, hGH was expressed only in the type II cells. Thus, this culture system provides an excellent means for identifying genomic elements that mediate type II cell-specific gene expression.     

Loudness balance between acoustic and electric stimulation by a patient with a multichannel cochlear implant

Estimates of loudness balance were obtained for acoustically and electrically presented 250 Hz sine signals from a patient who uses the Ineraid multichannel cochlear implant. Acoustic and electric loudness matching was possible because the patient evidenced a 25 dB HL threshold at 250 Hz in his nonimplanted ear. The level of the electrical stimulus in microamperes required for a balance of loudness grew linearly with equal increments in decibels for the acoustic stimulus. These data, in concert with the very limited data from previous studies, provide a rationale for using a logarithmic transformation of acoustic to electric intensity in signal processors for cochlear implants.     

Alignment of fluorescent molecules vapour-deposited on to highly oriented PTFE substrates

The alignment of six fluorescent materials (p-terphenyl, p-quaterphenyl, p-quinquephenyl, diphenylstilbene, 1,4-bis(2-methylstyryl)benzene, and 1,4-bis-2-(5-phenyloxazolyl)benzene) grown from the vapour phase on to friction-deposited, highly oriented poly(tetrafluoroethylene) (PTFE) films was studied. The dichroic ratio of the fluorescent films produced was determined by polarized spectroscopic fluorescence measurements, and the crystallographic orientation of these materials was determined by transmission electron microscopy. The measured dichroic ratios ranged from 1.7 for diphenylstilbene to 5.0 for the p-quaterphenyl film. Electron diffraction patterns revealed that five of the fluorescent materials grew epitaxially on the single crystal-like PTFE films, the fluorescent crystals adopting particular orientations with respect to the crystallographic planes of PTFE. On the other hand, crystals of 1,4-bis-2-(5-phenyloxazolyl) benzene aligned in a fibre pattern, apparently by a grapho-epitaxial mechanism.     

Statistical studies of branched ester latex and paint properties

Vinyl versatate, butyl acrylate and 2-ethylhexyl acrylate were compared as comonomer, for colloid-stabilized vinyl acetate latex polymers to assess their impact on latex coating properties. Vinyl versatate afforded advantages in scrub resistance, gloss, hydrophobicity and higher glass transition temperatures. Butyl acrylate developed better wet adhesion efficiency, gel content and hiding efficiency. 2-Ethylhexyl acrylate offered performance comparable to butyl acrylate in hiding efficiency, lowering copolymer glass transition temperatures, and gloss development. Combinations of these monomers, in many instances, afforded a better balance of performance properties than individual monomers alone. In the subject study, a simplex-centroid design was utilized to statistically map polymer compositions for determining the effect of comonomer composition on latex and paint film properties. In addition to developing contour maps of polymer and paint properties as a function of composition, a technique for studying structure-property relationships is presented.     

Ligands with affinity to specific sequences of DNA base pairs. X. Synthesis and binding of netropsin analogs, containing a chelating copper ion peptide, with DNA

An analogue of netropsin has been synthesized consisting of two N-propylpyrrolcarboxamide units linked covalently to a copper-chelating tripeptide Gly-Gly-L-His by means of two and three glycine residues. Binding to DNA and synthetic polynucleotides of netropsin analogue containing three glycine residues between Gly-Gly-L-His tripeptide and the N-end of netropsin analogue (His-Nt) has been studied. It is shown that this netropsin analogue chelates a copper ion with 1:1 stoichiometry, similar to a free Gly-Gly-L-His peptide. It is found that this netropsin analogue occupies 3 to 4 base pairs upon binding to poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers, irrespective of whether it binds in Cu(2+)-ligated or unligated forms. Binding constants and binding site sizes have been calculated for netropsin analogue complexes with DNA, poly(dA).poly(dT) and poly[d(AT)].poly[d(AT)] polymers at the [Cu2+]/[His-Nt] ratio equal to 0 and 1.0. In the three-component system including His-Nt and Cu(2+)-His-Nt, cooperative effects are recognized which can be explained by heterodimer generation on interaction of His-Nt and Cu(2+)-His-Nt at adjacent binding sites.     

Applications of ultrashort pulse propagation in Bragg gratings forwavelength-division multiplexing and code-division multiple access

We discuss theoretical and experimental studies on the propagation of ultrashort pulses through fiber Bragg gratings. We also consider several applications in optical communications to be found by combining ultrashort pulses and fiber Bragg gratings: a multiwavelength source for wavelength-division-multiplexed systems and a means for implementing optical code-division multiple access     

Optical characterization of low-temperature-grown GaAs forultrafast all-optical switching devices

Low-temperature-grown GaAs (LT-GaAs) is a promising material for all-optical switching devices due to its outstanding optical characteristics. In this paper, we outline a simplified model we have developed to describe the dynamics of the carriers in this material. We also report the results of a series of measurements that we have performed to characterize the optical properties of the material. Specifically, we present the first measurements of the two-photon absorption coefficient and the refractive index changes as a function of the growth and annealing temperatures in LT-GaAs. Finally, we show how our model can be used to optimize the material for applications in all-optical switching