Transgenic overexpression of interleukin (IL)-10 in the lung causes mucus metaplasia,tissue inflammation,and airway remodeling via IL-13-dependent and -independent pathways

To address the complex chronic effector properties of interleukin (IL)-10, we generated transgenic mice in which IL-10 was overexpressed in the lung. In these mice, IL-10 inhibited endotoxin-induced tumor necrosis factor production and neutrophil accumulation. IL-10 also caused mucus metaplasia, B and T cell-rich inflammation, and subepithelial fibrosis and augmented the levels of mRNA encoding Gob-5, mucins, and IL-13. In mice bred to have null mutations of IL-13, IL-4R(alpha), or STAT-6, transgenic IL-10 did not induce mucus metaplasia but did induce inflammation and fibrosis. IL-10 was also a critical mucin regulator of virus-induced mucus metaplasia. Thus, IL-10, although inhibiting lipopolysaccharide-induced inflammation, also causes mucus metaplasia, tissue inflammation, and airway fibrosis. These responses are mediated by multiple mechanisms with mucus metaplasia being dependent on and the inflammation and fibrosis being independent of an IL-13/IL-4R(alpha)/STAT-6 activation pathway.     

A label-free optical technique for detecting small molecule interactions

A novel approach for the label-free detection of molecular interactions is presented in which a colorimetric resonant grating is used as a surface binding platform. The grating, when illuminated with white light, is designed to reflect only a single wavelength. When molecules are attached to the surface, the reflected wavelength (color) is shifted due to the change of the optical path of light that is coupled into the grating. By linking receptor molecules to the grating surface, complementary binding molecules can be detected without the use of any kind of fluorescent probe or radioactive label. The detection technique is capable of detecting the addition and removal of small molecules as they interact with receptor molecules on the sensor surface or enzymes in the solution surrounding the sensor. Two assays are presented to exemplify the detection of small molecule interactions with the biosensor. First, an avidin receptor layer is used to detect 244 Da biotin binding. Second, a protease assay is performed in which a 136 Da p-nitroanilide (pNA) moeity is cleaved from an immobilized substrate. Because the sensor structure can be embedded in the plastic surfaces of microtiter plates or the glass surfaces of microarray slides, it is expected that this technology will be most useful in applications where large numbers of biomolecular interactions are measured in parallel, particularly when molecular labels will alter or inhibit the functionality of the molecules under study. Screening of pharmaceutical compound libraries with protein targets, and microarray screening of protein-protein interactions for proteomics are examples of applications that require the sensitivity and throughput afforded by this approach.     

CLEISTOGAMY IN SALPIGLOSSIS SINUATA

Cleistogamy in Salpiglossis sinuata L. (Solanaceae) involves a series of developmental events that includes germination of pollen tetrads en masse inside the anther, penetration of pollen tubes through the anther wall toward the stigma which is in contact with the anther, growth of pollen tubes down the style, and, finally, fertilization. This occurs within 6 days from the first appearance of the flower as compared to 10–12 days for normal anthesis of chasmogamous flowers. Coincident with these events in cleistogamous flowers are lack of corolla development and reduced size of androecium and gynoecium.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

A pH-dependent conformational change in the coat protein subunits from potato virus X.

Both the circular dichroism and fluorescence spectra of the dissociated coat protein subunits from potato virus X changed substantially over the pH range 8 to 4, irreversible changes resulted below pH 4, with tyrosyl and tryptophanyl residues affected most. The titration curves show a pKa of about 5.6 and do not require cooperative interactions between the coat protein subunits, thus they are in marked contrast to titrations of tobacco mosaic virus A-protein. The spectra of the intact virus were little changed between pH 8 and 4 and suggested that the coat protein was locked into a conformation similar to that of the subunits in solution at pH 7. It is proposed that the pH induced conformational change is responsible for determining the acidic branch of the pH profile for reconstitution of potato virus X from its dissociated coat protein subunits and RNA.     

Giemsa-based cytological assessment of area,shape and nucleus:cytoplasm ratio of goblet cells of rabbit bulbar conjunctiva

Goblet cells were visualized in impression cytology specimens from bulbar conjunctiva of the rabbit eye using Giemsa staining. Highly magnified images were used to generate outlines of the goblet cells and their characteristic eccentric nuclei. Using sets of 10 cells from 15 cytology specimens, I found that the longest dimension of the goblet cells averaged 16.7 ± 2.3 μm, the shortest dimension averaged 14.4 ± 1.8 μm and the nucleus averaged 6.3 ± 0.8 μm. The goblet cells were ellipsoid in shape and the longest:shortest cell dimension ratio averaged 1.169 ± 0.091. The goblet cell areas ranged from 108 to 338 μm² (average 193 ± 50 μm²). The area could be predicted reliably from the longest and shortest dimensions (r² = 0.903). The areas of goblet cell nuclei were 15–58 μm² (average 33 ± μm²) and the nucleus:cytoplasm area fraction was predictably greater in smaller goblet cells and less in the larger goblet cells (Spearman correlation = 0.817). The nuclei were estimated to occupy an average of 9.5% of the cell volume. The differences in size, shape and nucleus:cytoplasm ratio may reflect differences in goblet cell maturation.     

The C10/CCL6 chemokine and CCR1 play critical roles in the pathogenesis of IL-13-induced inflammation and remodeling

IL-13 is a potent stimulator of inflammation and tissue remodeling that plays a key role in the pathogenesis of a wide variety of human disorders. To further understand these responses, studies were undertaken to define the role(s) of the chemokine C10/CCL6 in the pathogenesis of IL-13-induced alterations in the murine lung. IL-13 was a very potent stimulator of C10/CCL6 mRNA and protein, and IL-13-induced inflammation, alveolar remodeling, and compliance alterations were markedly ameliorated after C10/CCL6 neutralization. Treatment with anti-C10/CCL6 decreased the levels of mRNA encoding matrix metalloproteinase-2 (MMP-2), MMP-9, and tissue inhibitor of metalloproteinase-4 (TIMP-4) in lungs from wild-type mice. C10/CCL6 neutralization also decreased the ability of IL-13 to stimulate the production of monocyte chemoattractant protein-1, macrophage inflammatory protein-1alpha, MMP-2, MMP-9, and cathepsins-K, -L, and -S and the ability of IL-13 to inhibit alpha1-antitrypsin. In accord with these findings, a targeted null mutation of CCR1, a putative C10/CCL6 receptor, also decreased IL-13-induced inflammation and alveolar remodeling and caused alterations in chemokines, proteases, and antiproteases comparable to those seen after C10/CCL6 neutralization. These C10/CCL6 and CCR1 manipulations did not alter the production of transgenic IL-13. These studies demonstrate that IL-13 is a potent stimulator of C10/CCL6 and highlight the importance of C10/CCL6 and signaling via CCR1 in the pathogenesis of the IL-13-induced pulmonary phenotype. They also describe a C10/CCL6 target gene cascade in which C10/CCL6 induction is required for optimal IL-13 stimulation of selected chemokines (monocyte chemoattractant protein-1 and MIP-1alpha) and proteases (MMP-2, MMP-9, and cathepsins-K, -L, and -S) and the inhibition of alpha1-antitrypsin.