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Inhibition of airway epithelial sodium channel (ENaC) function enhances mucociliary clearance (MCC). ENaC is positively regulated by channel-activating proteases (CAPs), and CAP inhibitors are therefore predicted to be beneficial in diseases associated with impaired MCC. The aims of the present study were to 1) identify low-molecular-weight inhibitors of airway CAPs and 2) to establish whether such CAP inhibitors would translate into a negative regulation of ENaC function in vivo, with a consequent enhancement of MCC. To this end, camostat, a trypsin-like protease inhibitor, provided a potent (IC(50) approximately 50 nM) and prolonged attenuation of ENaC function in human airway epithelial cell models that was reversible upon the addition of excess trypsin. In primary human bronchial epithelial cells, a potency order of placental bikunin > camostat > 4-guanidinobenzoic acid 4-carboxymethyl-phenyl ester > aprotinin >> soybean trypsin inhibitor = alpha1-antitrypsin, was largely consistent with that observed for inhibition of prostasin, a molecular candidate for the airway CAP. In vivo, topical airway administration of camostat induced a potent and prolonged attenuation of ENaC activity in the guinea pig trachea (ED(50) = 3 microg/kg). When administered by aerosol inhalation in conscious sheep, camostat enhanced MCC out to at least 5 h after inhaled dosing. In summary, camostat attenuates ENaC function and enhances MCC, providing an opportunity for this approach toward the negative regulation of ENaC function to be tested therapeutically.

Low-polydispersity poly(vinyl oxazolone) has been prepared using nitroxide-mediated living free radical polymerization. Bulk homopolymerization of 2-vinyl-4,4-dimethyl-5-oxazolone (VDMO) in the presence of the α-hydrido alkoxyamine initiator 2 and the corresponding nitroxide 1 proceeds to high conversion with polydispersities of less than 1.10. Accurate molecular weight control and low polydispersities (~1.04−1.10) were obtained on statistical copolymerization with styrene. Well-defined reactive statistical copolymers with polydispersities ranging from 1.05 to 1.30 were also prepared by copolymerization of oxazolone-functional monomers with acrylates, acrylamides, and N-vinylamides. Reactive block copolymers were prepared by polymerization of VDMO from poly(n-butyl acrylate) starting blocks as well as by polymerization of styrene from poly(VDMO) starting blocks. New polymers were prepared from VDMO-containing polymers and copolymers by nucleophilic ring-opening of the pendant oxazolone rings with amines.

Dendrimer monolayers have been designed for use as positive resists for scanning probe lithography (SPL). Several new amphiphilic poly(benzyl ether) dendrimers with carboxylic acids at either the focal point or the “periphery” have been prepared. These can form ionically bound dendrimer monolayers that may serve as either positive or negative tone resists for SPL. The amphiphilic dendrimers self-assemble onto (3-aminopropyl)silanized Si(100) wafer surfaces to afford ultrathin films. The dendrimer monolayers were characterized by AFM, ellipsometry, and contact angle goniometry. Patterning a singly charged dendrimer monolayer results in the formation of positive tone holes ~35 nm in width. Similarly, patterning a multiply charged dendrimer monolayer in a direct-write manner with the scanning probe microscope results in the formation of negative tone oxide features ~80 nm in width.

Total poly (A)-containing messenger RNA [poly(A)-mRNAT] was isolated from seminal vesicles of adult male rats. By preparative 5%-20% linear sucrose gradient centrifugation in 1% SDS, a single peak was seen in the 11S region [poly(A)-mRNA11S]. Poly (A)-mRNAT and poly(A)-mRNA11S were translated in the wheat germ S-30 in vitro translation system. Laemmli-SDS-polyacrylamide slab gel electrophoresis of translation products of poly(A)-mRNAT revealed three major protein bands at approximately 18,500, 15,000, and 14,000 daltons. In vitro translation of poly(A)-mRNA11S across the 11S peak revealed partial purification of the mRNAs for the three predominant proteins. The lower molecular weight part of the 11S peak enriched for the mRNAs coding for the 15,000 and 14,000 dalton polypeptides, and the higher molecular weight side of the peak enriched for the 18,500 dalton mRNA. The highly abundant class of mRNA from rat seminal vesicle can be purified with one sucrose gradient centrifugation.

The rat seminal vesicle secretion IV (SVS IV) gene was isolated from a lambda Charon 4A library. The SVS IV gene transcription unit was found to be on one 3.3-kilobase (kb) EcoRI fragment. Restriction mapping and DNA sequence analysis demonstrated that the entire length of the SVS IV transcription unit is 1,930 base pairs (bp) and contains two introns. The 3.3-kb EcoRI fragment contains 144 bp of 5'-flanking region. At -113 bp from the presumed transcription initiation site an interesting structure with perfect dyad symmetry is noted. In another lambda clone, a 3.5-kb EcoRI fragment was isolated that contains the SVS IV gene and was shown to be identical to the 3.3-kb EcoRI fragment except for 180 bp of DNA in the second intron. The extra DNA consists of several (8-10) 20-bp tandem repeats flanked on each side by seven or eight copies of this same 20-bp repeat. Fisher X Sprague-Dawley hybrid rats, which contain both the EcoRI 3.5-kb form and the 3.3-kb form of the SVS IV gene, were crossed with each other. Analysis of the F1 generation demonstrated that the presence or absence of the 180-bp intronic insertion in the SVS IV gene defines an allelic difference. This report also presents the DNA sequence of the transcription unit and flanking regions of the SVS IV gene.

Poly(amidoamine) (PAMAM) dendrimers, generations 1−6, were peripherally modified with cationically substituted naphthalene diimide groups. Several monomeric diimides were also prepared as models. The structure and loading of the dendrimers were determined by vis, IR, and NMR spectroscopies, elemental analysis, and coulometric analysis. In general it was possible to obtain loadings greater than 70% even from generation 6 dendrimer where there are 192 amine groups to be substituted. These polymers and monomers were reduced using sodium dithionite or electrochemically with one electron per imide group converting each imide into its anion radical. Near-infrared (NIR) spectroscopy showed that in D2O or formamide solutions the anion radicals aggregated into π-dimers and π-stacks. Cyclic voltammograms are interpreted in terms of anion radical aggregation and precipitation of the reduced dendrimers. The CV and NIR results for the various dendrimer generations were quite similar. These results allow insight into the possibilities and limitations of dendrimers to provide a scaffold for intramolecular aggregation.

To exert their effects, glucocorticoid receptor complexes interact selectively with DNA sequences known as glucocorticoid regulatory elements. We have studied the interaction between human glucocorticoid receptors and mouse mammary tumor virus (MMTV) DNA by means of a procedure that permits analysis after immobilization of the receptor on nitrocellulose. Proteins from crude cytosolic or nuclear extracts were electrophoresed on NaDodSO4/PAGE gels, soaked in a urea buffer to remove NaDodSO4, transferred to nitrocellulose, and probed with nick-translated MMTV [32P]DNA in a 5% nonfat dry milk buffer, which minimizes nonselective DNA-protein interactions. We present evidence that MMTV [32P]DNA interacts selectively with the glucocorticoid receptor. These data include comigration of [3H]dexamethasone mesylate-labeled band and bound MMTV [32P]DNA on gel electrophoresis systems; localization of DNA-binding activity in the cytosol of cells incubated with steroid at 0 degrees C and in the nucleus and cytosol of cells incubated at 37 degrees C; binding of the MMTV DNA to highly purified receptor; and absence of MMTV DNA binding activity in extracts from cells whose receptor has been down-regulated. Furthermore, glucocorticoid receptors analyzed under these conditions exhibit selective binding to DNA fragments that contain glucocorticoid regulatory elements.

As part of its mixtures program, the Agency for Toxic Substances and Disease Registry (ATSDR) supports in vitro and limited in vivo toxicity testing to further our understanding of the toxicity and health effects of chemical mixtures. There are increasing concerns that environmental chemicals adversely affect the health of humans and wildlife. These concerns have been augmented by the realization that exposure to chemicals often occurs to mixtures of these chemicals that may exhibit complex synergistic or antagonistic interactions. To address such concerns, we have conducted two studies with techniques that are being used increasingly in experimental toxicology. In the first study, six organochlorine pesticides (4,4'-DDT, 4,4'-DDD, 4,4'-DDE, aldrin, dieldrin, or endrin) were selected from the ATSDR Comprehensive Environmental Response, Compensation and Liability Act of 1980 (or Superfund) priority list and tested for their ability to modulate transcriptional activation of an estrogen-responsive reporter gene in transfected HeLa cells. In these assays. HeLa cells cotransfected with an expression vector encoding estrogen receptor and an estrogen-responsive chloramphenicol acetyltransferase (CAT) reporter plasmid were dosed with and without selected environmental chemicals either individually or in defined combinations. Estradiol consistently elicited 10- to 23-fold dose-dependent inductions in this assay. By contrast, all six of the organochlorine pesticides showed no detectable dose-related response when tested either individually or in binary combinations. Thus, these chemicals as binary mixtures do not exhibit any additional estrogenicity at the levels tested in these assays. In the second study, arsenic [As(V)], cadmium [Cd(II)], chromium [Cr(III, VI)], and lead [Pb(II)] were tested in a commercially developed assay system, CAT-Tox (L), to identify metal-responsive promoters and to determine whether the pattern of gene expression changed with a mixture of... [ABSTRACT FROM AUTHOR]