Keratocyte phenotype mediates proteoglycan structure: a role for fibroblasts in corneal fibrosis

In pathological corneas, accumulation of fibrotic extracellular matrix is characterized by proteoglycans with altered glycosaminoglycans that contribute to the reduced transparency of scarred tissue. During wound healing, keratocytes in the corneal stroma transdifferentiate into fibroblasts and myofibroblasts. In this study, molecular markers were developed to identify keratocyte, fibroblast, and myofibroblast phenotypes in primary cultures of corneal stromal cells and the structure of glycosaminoglycans secreted by these cells was characterized. Quiescent primary keratocytes expressed abundant protein and mRNA for keratocan and aldehyde dehydrogenase class 3 and secreted proteoglycans containing macromolecular keratan sulfate. Expression of these marker compounds was reduced in fibroblasts and also in transforming growth factor-beta-induced myofibroblasts, which expressed high levels of alpha-smooth muscle actin, biglycan, and the extra domain A (EDA or EIIIA) form of cellular fibronectin. Collagen types I and III mRNAs were elevated in both fibroblasts and in myofibroblasts. Expression of these molecular markers clearly distinguishes the phenotypic states of stromal cells in vitro. Glycosaminoglycans secreted by fibroblasts and myofibroblasts were qualitatively similar to and differed from those of keratocytes. Chondroitin/dermatan sulfate abundance, chain length, and sulfation were increased as keratocytes became fibroblasts and myofibroblasts. Fluorophore-assisted carbohydrate electrophoresis analysis demonstrated increased N-acetylgalactosamine sulfation at both 4- and 6-carbons. Hyaluronan, absent in keratocytes, was secreted by fibroblasts and myofibroblasts. Keratan sulfate biosynthesis, chain length, and sulfation were significantly reduced in both fibroblasts and myofibroblasts. The qualitatively similar expression of glycosaminoglycans shared by fibroblasts and myofibroblasts suggests a role for fibroblasts in deposition of non-transparent fibrotic tissue in pathological corneas.     

Human monocytes use Rac1, not Rac2, in the NADPH oxidase complex

Phagocyte NADPH oxidase is critical for defense against pathogens and contributes to inflammatory tissue injury. One component of the NADPH oxidase complex is the small GTP-binding protein Rac. There are two isoforms of Rac, and Rac2 is the predominant isoform in neutrophils and has been shown to be essential for NADPH oxidase activity. In primary human monocytes we report that in contrast to neutrophils, Rac1 is the predominantly expressed isoform. Upon monocyte activation by a variety of agents, we found that Rac1 dissociates from Rho GDP dissociation inhibitor (RhoGDI) and translocates to the membrane. We also found that Rac1 interacts with two other NADPH oxidase components, p67phox and p47phox, upon monocyte activation. These data indicate that Rac1, and not Rac2, is a component of the activated NADPH oxidase in monocytes. This finding suggests that it may be possible to selectively interfere with either monocyte or neutrophil NADPH oxidase activity, thereby selectively targeting chronic versus acute inflammatory processes.     

Mouse lymphoma thymidine kinase gene mutation assay: International Workshop on Genotoxicity Tests Workgroup report--Plymouth, UK 2002

The Mouse Lymphoma Assay (MLA) Workgroup of the International Workshop on Genotoxicity Tests (IWGT) met on June 28th and 29th, 2002, in Plymouth, England. This meeting of the MLA group was devoted to discussing the criteria for assay acceptance and appropriate approaches to data evaluation. Prior to the meeting, the group conducted an extensive analysis of data from both the microwell and soft agar versions of the assay. For the establishment of criteria for assay acceptance, 10 laboratories (6 using the microwell method and 4 using soft agar) provided data on their background mutant frequencies, plating efficiencies of the negative/vehicle control, cell suspension growth, and positive control mutant frequencies. Using the distribution curves generated from this data, the Workgroup reached consensus on the range of values that should be used to determine whether an individual experiment is acceptable. In order to establish appropriate approaches for data evaluation, the group used a number of statistical methods to evaluate approximately 400 experimental data sets from 10 laboratories entered into a database created for the earlier MLA Workshop held in New Orleans [Environ. Mol. Mutagen. 40 (2002) 292]. While the Workgroup could not, during this meeting, make a final recommendation for the evaluation of data, a general strategy was developed and the Workgroup members agreed to evaluate this new proposed approach using their own laboratory data. This evaluation should lead to a consensus global approach for data evaluation in the near future.     

Use of Real-Time Quantitative PCR for the Analysis of φLC3 Prophage Stability in Lactococci

Bacteriophages are a common and constant threat to proper milk fermentation. It has become evident that lysogeny is widespread in lactic acid bacteria, and in this work the temperate lactococcal bacteriophage LC3 was used as a model to study prophage stability in lactococci. The stability was analyzed in six LC3 lysogenic Lactococcus lactis subsp. cremoris host strains when they were growing at 15 and 30°C. In order to perform these analyses, a real-time PCR assay was developed. The stability of the LC3 prophage was found to vary with the growth phase of its host L. lactis IMN-C1814, in which the induction rate increased during the exponential growth phase and reached a maximum level when the strain was entering the stationary phase. The maximum spontaneous induction frequency of the LC3 prophage varied between 0.32 and 9.1% (28-fold) in the six lysogenic strains. No correlation was observed between growth rates of the host cells and the spontaneous prophage induction frequencies. Furthermore, the level of extrachromosomal phage DNA after induction of the prophage varied between the strains (1.9 to 390%), and the estimated burst sizes varied up to eightfold. These results show that the host cells have a significant impact on the lytic and lysogenic life styles of temperate bacteriophages. The present study shows the power of the real-time PCR technique in the analysis of temperate phage biology and will be useful in work to reveal the impact of temperate phages and lysogenic bacteria in various ecological fields.     

Regulated expression of osteopontin in the peri-implantation rabbit uterus

Blastocyst attachment to the lining of the mammalian uterus during early implantation involves the initial apposition of the trophoblast to the uterine epithelial surface. Osteopontin (OPN) is a glycoprotein component of the extracellular matrix that is secreted by the glandular epithelium of mammalian uteri at the time of implantation. This protein is recognized by several members of the integrin family and promotes cell-cell attachment and adhesion. In the present study, rabbit uteri were examined using Northern and in situ hybridization to evaluate the temporal and spatial distribution of OPN mRNA during early pregnancy. Northern blot analysis demonstrated a dramatic increase in OPN expression on Days 4-7 of pregnancy, corresponding to the rise in circulating progesterone and the time of initial embryo attachment in this species. In situ hybridization analysis revealed OPN mRNA expression on Day 6.75 of pregnancy, which was most prominent on endometrial epithelium. Using immunofluorescence, OPN protein was present on the glandular epithelium on Day 6.75 of pregnancy, but was absent on blastocysts. Further, no expression of OPN mRNA or protein was found in the nonpregnant endometrium. Induction of endometrial OPN expression was observed in unmated rabbits treated with progesterone alone and was prevented by cotreatment with the antiprogestin ZK137.316. Estradiol-17beta had no effect on OPN expression by itself, and estrogen priming was not necessary to demonstrate the stimulatory effect of progesterone. In The rabbit uterus, as in other mammalian species studied, OPN is expressed in a stage-specific manner by the endometrial glands during the peri-implantation period and is regulated by progesterone.     

Karyotype evolution in South American subterranean rodents Ctenomys magellanicus (Rodentia: Octodontidae): chromosome rearrangements and (TTAGGG)n telomeric sequence localization in 2n=34 and 2n=36 chromosomal forms

Ctenomys is the most numerous genus of South American subterranean rodents and one of the most karyotypically diverse clades of mammals known. Ctenomys magellanicus is the southernmost species of the group and the only one living in Isla Grande de Tierra del Fuego (Argentina). This species presents two chromosomal forms, i.e. 2n=34, and 2n=36 (FN=68). Recent studies suggest that genetic divergence between both karyotypic forms resulted from a chromosomal speciation process. In order to identify the chromosomal rearrangement involved in the process of karyotype evolution in this species, we used chromosome banding techniques and fluorescence in situ hybridization with a telomeric probe to metaphase chromosomes of the two chromosomal forms of Ctenomys magellanicus. Chromosome analysis of Giemsa-stained and G-banding preparations showed that Cm34 and Cm36 karyotypes differ in one rearrangement involving chromosomes A9 from Cm34 and B12 and B17 from Cm36. In addition FISH analysis showed that all of the chromosomes from both chromosomal forms exhibit a telomeric-only distribution pattern of the (TTAGGG)n sequence, indicating that none of the chromosomal forms of Ctenomys magellanicus has true telocentric chromosomes. Our results suggest that a chromosome fission event would have occurred during the process of karyotype evolution in this species.     

Beta-helix model for the filamentous haemagglutinin adhesin of Bordetella pertussis and related bacterial secretory proteins

Bordetella pertussis establishes infection by attaching to epithelial cells of the respiratory tract. One of its adhesins is filamentous haemagglutinin (FHA), a 500-A-long secreted protein that is rich in beta-structure and contains two regions, R1 and R2, of tandem 19-residue repeats. Two models have been proposed in which the central shaft is (i) a hairpin made up of a pairing of two long antiparallel beta-sheets; or (ii) a beta-helix in which the polypeptide chain is coiled to form three long parallel beta-sheets. We have analysed a truncated variant of FHA by electron microscopy (negative staining, shadowing and scanning transmission electron microscopy of unstained specimens): these observations support the latter model. Further support comes from detailed sequence analysis and molecular modelling studies. We applied a profile search method to the sequences adjacent to and between R1 and R2 and found additional "covert" copies of the same motifs that may be recognized in overt form in the R1 and R2 sequence repeats. Their total number is sufficient to support the tenet of the beta-helix model that the shaft domain--a 350 A rod--should consist of a continuous run of these motifs, apart from loop inserts. The N-terminus, which does not contain such repeats, was found to be weakly homologous to cyclodextrin transferase, a protein of known immunoglobulin-like structure. Drawing on crystal structures of known beta-helical proteins, we developed structural models of the coil motifs putatively formed by the R1 and R2 repeats. Finally, we applied the same profile search method to the sequence database and found several other proteins--all large secreted proteins of bacterial provenance--that have similar repeats and probably also similar structures.