Surface airway epithelium is frequently injured by noxious inhaled agents, epithelial wound repair may be an important process by which the epithelial barrier integrity is maintained. To evaluate the role of surface airway cells in the wound repair process, we developed an in vitro wounding model of human nasal epithelial respiratory cells in primary culture. Circular wounds were made in the epithelial cell culture by detaching, with a glass capillary, approximately 50 cells from the collagen matrix. Video microscopy and electron microscopy observations demonstrated the contribution of two main events during the repair process: the spreading of the cells at the edge of the wounded surface, and the migration of epithelial cell sheets. Complete wound closure occurred within 5 to 8 h. The inhibition of wound repair by cytoskeleton inhibitors or cellular protein synthesis inhibitors suggested that these factors are involved in the wound repair process of surface airway epithelium.

The GASA gene family previously identified in Arabidopsis belongs to a wide-spread class of genes found in mono- and dicotyledonous plants, all structurally related to the original GA-regulated GAST1 gene from tomato. They encode small peptides (97 to 112 residues) of unknown function sharing a 60 amino acid conserved C-terminal domain comprising twelve conserved cysteine residues which define a pattern not related to other known cysteine-rich motifs. Northern blot hybridization analysis revealed sequential expression of three genes during flowering, silique development and seed germination. GASA4 transcripts were detected in flower buds. GASA1 transcripts markedly accumulated in siliques, about five days after pollination, and correlated with the peak of GA biosynthesis at this stage of silique development. GASA3 transcripts accumulated at the end of the maturation stage of the silique, and transcripts were still present in dry seeds but degraded rapidly during imbibition. In addition, the GASA4 gene was again actively transcribed after germination and this expression was shown to be dependent on the presence of GAs in GA-deficient mutants. Immunoblot analysis confirmed the presence of the GASA4 gene product in flower buds, seedlings and roots. We focused on the GASA4 gene and characterized its expression. The upstream region (-890 to +128) was fused to the GUS reporter gene. GASA4/GUS expression was detected in transgenic Arabidopsis primarily in all meristematic regions, including vegetative, inflorescence and floral meristems, as well as primary and lateral root tips. In a GA-deficient background (ga1-3), GUS activity in the vegetative meristem was detected only in the presence of supplied GA. In root and flower meristems, basal GUS activity was slightly enhanced by exogenous GA. Interestingly, GA strongly inhibit GUS activity in expanding cotyledons and leaves in ga1-3 mutants supplied with exogenous GAs, as well as in the wild type. The GA-dependent meristem-specific expression pattern suggests that the GASA4 protein plays a role in dividing cells rather than in elongating cells.

Human nasal polyps in outgrowth culture were used to study the Pseudomonas aeruginosa adhesion to respiratory cells. By scanning electron microscopy, P. aeruginosa were seen associated with ciliated cells, but by transmission electron microscopy, bacteria were never seen at the interciliary spaces or attached along cilia, but were identified trapped at the extremities of cilia, usually as bacterial aggregates. A fibronectin-containing fibrillar material was seen associated with aggregated bacteria. By time-lapse video microscopy, bacteria were seen to aggregate in the culture medium following their addition to the culture wells. Progressively, these aggregates were trapped by cilia or attached to migrating cells of a lower cell layer that protruded beneath the upper layer cells, at the outgrowth periphery. P. aeruginosa adhesion to these lower cell layer migrating cells was significantly higher than to ciliated or nonciliated cells of the upper cell layer. Migrating cells were intensely labeled by the complexes Con A and arachis hypogea agglutinin (PNA)-FITC, in contrast to the other cells. The percentage of PNA-labeled cells with attached bacteria was significantly higher than that without bacteria. These results suggest that changes of cell surface glycoconjugates related with cell migration may favor P. aeruginosa adhesion to respiratory cells. (J. Clin.

The sequence presented -4877 nucleotides-includes the mRNA and protein coding regions, the introns, the 5' and 3' flanking DNA of the gene encoding the protein P25. P25 is the 25000 Dalton silk protein which binds through disulfide bonds to the 370.000 D "large fibroin" of Bombyx mori. The developmental regulation of of expression of its gene, with regard to that of the fibroin gene, has been described previously (1,2).The sequence of the P25 gene is derived from the DNA of the ox ori Japanese strain 703. The transcription start is labelled +1 and the 3' sANA end is at nucleotide +3485 (e), but may occur at any of the three following A residues. The CAAT sequence (-90,-87). the TATA sequence (-32,-29) and the AATAAA polyadenylation signal (+3461-3466) are underlined. Exon/intron boundaries are marked by arrows. The translation stop codon is labelled at nucleotides +2995-2997. The mature RNEA length is 1173 nucleotides and the protein is 220 aminoacids long.

Human surface respiratory epithelial (HSRE) cells from nasal polyps have been cultured within collagen lattices in a serum-free defined medium. Cell growth observed over a period of 12 days showed a population doubling time of 36 h. Under these culture conditions, we observed a contraction of the lattices. Phase-contrast light microscopy and transmission electron microscopy demonstrated that the HSRE cells formed tubular ductlike structures. Lumens formed by HSRE cells were surrounded by cuboidal-shaped polarized cells with numerous ciliated cells, secretory cells, and undifferentiated cells. Epidermal growth factor (EGF) was observed to stimulate the tubule formation and the contraction of the lattices. Videomicroscopic observations and analysis of the ciliary beating frequency (CBF) demonstrated that the cilia were homogeneously distributed on the whole apical surface of the ciliated cells and that their movement was well coordinated, with a CBF similar to that observed in outgrowth cells from cultured human nasal and tracheal epithelia. Immunofluorescent staining of basement membrane components synthesized and secreted by cells revealed the presence of type III collagen around the tubules. Type IV collagen and laminin were present in the cytoplasm and at the periphery of the cells. The biotin-streptavidin-gold immunocytochemical technique with monoclonal anti-mucin antibody showed intracellular localization of mucins in secretory granules of the secretory cells. With the use of substrate gel electrophoresis polyacrylamide gels impregnated with gelatin, collagenase activity was detected in the conditioned medium of the cultured HSRE cells. These results suggest that both three-dimensional collagen gel and soluble factors such as EGF regulate tubule formation by HSRE cells. Moreover, the capacity of the epithelial cells to contract the gel suggests they may be involved in the wound healing process.

We have cloned a large portion of the P25 gene of Bombyx mori encoding the 25,000 dalton polypeptide which associates with fibroin to constitute the major silk protein. Its structure has been investigated by restriction mapping R-loop analysis, S1 nuclease protection experiments and nucleotide sequencing of the region spanning the 5' end of the gene and its flanking DNA. This has permitted a comparative sequence analysis of the DNA from the P25 and fibroin genes. The genes demonstrate no relatedness in their coding regions but they exhibit large blocks of sequence homology in their 5' flanking regions. In particular, the DNA upstream of the P25 gene possesses a sequence very similar to a region of fibroin 5' flanking DNA that is known to possess transcription modulation signals. The functional significance of these homologous regions is discussed with regard to the highly coordinated expression of these two genes.

The growth, differentiation, ciliary beating pattern and frequency of human respiratory ciliated cells in primary culture were studied by scanning and transmission electron microscopy and by videomicroscopy. The epithelial cells were obtained as outgrowth from explants of adult nasal polyps. When the explants were grown on type-I and type-IV collagen substrates in a standard serum-free, hormone-supplemented medium, a high percentage of ciliated cells (range 29 +/- 5% to 37 +/- 6%) was present within 2 days of culture. After 5 days of culture, the percentage of ciliated cells near the explant was 51 +/- 5%. Most of the cultured ciliated cells (85%) were characterized by individual cilia showing a coordinated movement during the beat cycle and a beating frequency (13.3 +/- 1.3 Hz) similar to that reported in vivo. In the other 15% of the ciliated cells, the dyskinetic cilia were aggregated into clumps and characterized by a rigid and planar bending movement and a lower (P less than 0.01) beating frequency (10.7 +/- 1.4 Hz). It is suggested that the latter type of cell, already described during fetal development, might be an intermediate type of ciliated cell which appears temporarily during the surface respiratory epithelial differentiation.

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