José Luna

Plasmid-Encoded Toxin of Enteroaggregative Escherichia coli is Internalized by Epithelial Cells

ABSTRACT We have previously described a 104-kDa protein termed Pet (for plasmid-encoded toxin) secreted by some strains of enteroaggregativeEscherichia coli (EAEC). Through an unknown mechanism, this toxin (i) raises transepithelial short-circuit current (Isc) and decreases the electrical resistance of rat jejunum mounted in the Ussing chamber, (ii) causes cytoskeletal alterations in HEp-2 cells and HT29/C1 cells, and (iii) is required for histopathologic effects of EAEC on human intestinal mucosa. Pet is a member of the autotransporter class of secreted proteins and together with Tsh, EspP, EspC, ShMu, and SepA proteins comprises the SPATE subfamily. Here, we show that Pet is internalized by HEp-2 cells and that internalization appears to be required for the induction of cytopathic effects. Evidence supporting Pet internalization includes the facts that (i) the effects of Pet on epithelial cells were inhibited by brefeldin A, which interferes with various steps of intracellular vesicular transport; (ii) immunoblots using anti-Pet antibodies detected Pet in the cytoplasmic fraction of intoxicated HEp-2 cells; (iii) Pet was detected inside HEp-2 cells by confocal microscopy; and (iv) a mutant in the passenger domain cleavage site, which prevents Pet release from the bacterial outer membrane, did not produce cytopathic effects on epithelial cells, whereas the release of mutant Pet from the outer membrane with trypsin yielded active toxin. We have also shown that the Pet serine protease motif is required to produce cytopathic effects but not for Pet secretion. Our results suggest an intracellular mode of action for the Pet protease and are consistent with we our recent report suggesting an intracellular mode of action for Pet (J. M. Villaseca, F. Navarro-Garcı́a, G. Mendoza-Hernández, J. P. Nataro, A. Cravioto, and C. Eslava, Infect. Immun. 68:5920–5927, 2000).

Biochemical and histochemical analysis of 71 kDa dystrophin isoform (Dp71f) in rat brain.

Dp71 is a member of the dystrophin family and the most abundant dmd gene product in the brain. In the present study, we focused on a short dystrophin transcript named Dp71f, which is alternatively spliced when exon 78 is absent The topographic localization of this protein in the encephalon has not been properly described yet, nor its cellular or subcellular localization, and even less its functions. Dp71f was found to be a cytoplasmic 70 kDa protein and localized in all encephalon regions studied. Double labeling using specific markers for various cell types confirmed Dp71f distribution in the cytoplasm of all cell types studied. Labeling was more conspicuous near the nucleus and diminished towards the periphery of cells. In some cases, we observed cells that were positive for actin and Dp71f in regions corresponding to lamellipodia-like structures. Dp71f and Dp71d isoforms were differently distributed. Our study is the first specific and unambiguous description of the topography and cellular localization patterns of Dp71f in brain, suggesting that Dp71f is a ubiquitous protein.

Use of microscopical techniques in the study of human chondrocytes from osteoarthritic cartilage: An overview

Several microscopical techniques, such as high resolution light microscopy, Normaski microscopy, laser confocal and transmission electron microscopy, were used in a correlative morphological study of human osteoarthritic (OA) cartilage. Emphasis was made on the characterization of chondrocytes heterogeneity observed in this tissue. Novel findings were assessed in the morphological and immunocytological study of the chondrocytes organized in aggregates or “clones” typical of this degenerative disease, consisting of the modification of certain elements of the cytoskeleton that influence changes in the cell shape. Also, the presence of cilia and centrioles found in certain cells raised the question if chondrocytes are able to move and regroup as an alternative mechanism to mitosis in the formation of cell clusters or “clones.” The presence of two types of secretory chondrocytes was observed and discussed.

Altered glycosylation pattern of proteins in Alzheimer disease

Abstract. Post-translational modifications due to glycosylation of proteins in human brains from patients with Alzheimer disease (AD) were analyzed using lectin histochemistry. Results indicate a significant increase in the production of Oglycosylated (containing Galβ1,3GalNAcα1,0 Ser/Thr or GalNAcα1,0 Ser/Thr) proteins in neuritic plaques and neurofibrillary tangles which are the major histopathological hallmarks of AD brains. These alterations were determined by positive labelling with lectins obtained from Amaranthus leucocarpus(ALL) and Macrobrachium rosenbergii (MRL) respectively. Immunohistochemistry indicated that the lectin-staining labelled specifically both neurofibrillary tangles and neuritic plaques. In contrast, lectins labelling was restricted to microvessels in normal control brains. These results provide evidence that modifications of the specific glycosylation patterns are closely related with the presence of the hallmark lesions of this disease, suggesting that an abnormal enzymatic processing of proteins may be an early event in the neuronal degeneration which characterises AD

Regional and temporal progression of reactive astrocytosis in the brain of the myelin mutant taiep rat.

Reactive astrocytosis in taiep rats was shown by glial fibrillary acidic protein (GFAP) immunoreactivity measured by means of enzyme-linked immunosorbent assay and indirect immunofluorescence. Increased GFAP immunoreactivity was first observed in the brainstem of 15-day-old taiep rats and was widespread throughout all brain regions at 6 months of age. Characteristically, astrocytes were hypertrophic and displayed strong GFAP fluorescence. The pattern of these reactive cells may correlate with the process of dysmyelination in the taiep rat.

Dystrophin isoform Dp71 is present in lamellipodia and focal complexes in human astrocytoma cells U-373 MG

Dp71 is the most abundant product of the dmd gene in the brain. There are at least 2 isoforms derived from alternative splicing of exon 78 (Dp71d, which contains exon 78 and Dp71f, the spliced isoform) but the precise localization and function of each isoform is still unknown. In the present study, we demonstrate by RT-PCR that the Dp71f isoform is present in an astrocytoma cell line U-373 MG, and its subcellular localization was determined in the cytoplasm, particularly in perinuclear areas, with lower amounts towards the periphery but increasing in the leader borders of lamellipodia and focal complexes. Double labeling indirect immunofluorescence showed that Dp71f colocalized with actin-like beta-dystroglycan and beta-1 integrin. We also demonstrated by triple labeling that Dp71f was colocalized with actin and two members of integrin complexes, alpha-actinin and vinculin, in focal complexes. Ventral plasma membranes were enriched and in those containing focal complex proteins, we found colocalization of Dp71f, actin and vinculin. It is concluded that U-373 MG cells express Dp71f as part of lamellipodia and focal complex proteins, and possibly connected via distroglycan complexes to integrin complexes.

Identification of latexin by a proteomic analysis in rat normal articular cartilage

BackgroundOsteoarthritis (OA) is characterized by degeneration of articular cartilage. Animal models of OA induced are a widely used tool in the study of the pathogenesis of disease. Several proteomic techniques for selective extraction of proteins have provided protein profiles of chondrocytes and secretory patterns in normal and osteoarthritic cartilage, including the discovery of new and promising biomarkers. In this proteomic analysis to study several proteins from rat normal articular cartilage, two-dimensional electrophoresis and mass spectrometry (MS) were used. Interestingly, latexin (LXN) was found. Using an immunohistochemical technique, it was possible to determine its localization within the chondrocytes from normal and osteoarthritic articular cartilage.ResultsIn this study, 147 proteins were visualized, and 47 proteins were identified by MS. A significant proportion of proteins are involved in metabolic processes and energy (32%), as well as participating in different biological functions including structural organization (19%), signal transduction and molecular signaling (11%), redox homeostasis (9%), transcription and protein synthesis (6%), and transport (6%). The identified proteins were assigned to one or more subcellular compartments.Among the identified proteins, we found some proteins already recognized in other studies such as OA-associated proteins. Interestingly, we identified LXN, an inhibitor of mammalian carboxypeptidases, which had not been described in articular cartilage. Immunolabeling assays for LXN showed a granular distribution pattern in the cytoplasm of most chondrocytes of the middle, deep and calcified zones of normal articular cartilage as well as in subchondral bone. In osteoarthritic cartilage, LXN was observed in superficial and deep zones.ConclusionsThis study provides the first proteomic analysis of normal articular cartilage of rat. We identified LXN, whose location was demonstrated by immunolabeling in the chondrocytes from the middle, deep and calcified zones of normal articular cartilage, and superficial and deep zones of osteoarthritic cartilage.

Epidermal growth factor induces an increase in cell adhesion and an arrangement of actin skeleton in stress fibres in pituitary cultured cells from infantile rats but not adult rats

The rat anterior pituitary gland undergoes changes in its cyto‐architecture during the second and third weeks of postnatal life. However, little is known about the factors that regulate these tissue conformational changes. The epidermal growth factor (EGF) is one of the growth factors that are synthesized by the pituitary gland, and almost all of the pituitary cells have EGF receptors (EGFR). In addition to the effects of the EGF on mitosis and differentiation, this growth factor can modulate cell adhesion, cell migration, and cytoskeletal organization. In this study we focussed our attention in examining the effects of EGF on the adhesion of cells to the extracellular matrix and on the actin cytoskeletal arrangement of pituitary cells from infantile and adult rats. Our results show that in infantile cells the EGF induces cell adhesion with increase in cell surface area. The arrangement of actin‐F in infantile EGF‐treated cells was in stress fibers and vinculin acquired a striped shape at the membrane border, suggesting the assembly of focal adhesion contacts. In contrast, in adult pituitary cells EGF does not induce any change in cell adhesion, and the cells maintain a rounded shape with an arrangement of actin‐F in thin cortical bands even though, immuno‐localization of the EGFR was observed in adult cells cultured in defined medium. We also looked for the EGFR in membrane preparations from infantile and adult pituitaries, and a marked difference in membrane EGFR was observed between them, the infantile pituitaries showing a significantly higher amount. Our results suggest that in infantile cells EGF induces the assembly of focal adhesion contacts, and that in adult cells the receptor of this growth factor is uncoupled of the signaling pathway by which a rearrangement of actin cytoskeleton occurs.

P4-156: Thiazin red is a sensitive and accurate marker for the fast diagnosis of Alzheimer's disease in nonfixed brain tissue in touch imprints preparations

with AD-type pathology but cognition within normal limits for age. The lack of clinical symptoms in such cases has been hypothesized to relate to “cognitive reserve”. The association of pAD with demographic, clinical, genetic, and other comorbid pathological features remains unknown. Few detailed neuropathological studies of pAD exist. Methods: The University of Kentucky Alzheimer’s Disease Center follows a large group of cognitively normal subjects that agree to undergo extensive longitudinal annual clinical evaluations and consent to brain autopsy at death. Since 1989, 153 cognitively normal subjects have come to autopsy from this group. Detailed neuropathological assessment includes presence of AD, cerebrovascular disease (CVD), and Lewy body disease (LBD) pathology. Results: All subjects were cognitively normal on last examination before death (mean time from last examination to death one year). pAD in this group was defined by NIA-Reagan intermediate or high-likelihood of AD without clinical symptoms (n 31, 20%). Other classifications included pathologically normal (NIA-Reagan low or no-likelihood of AD, n 87), -amyloid predominant pathology (CERAD probable or definite, Braak stage II or lower, n 13), neurofibrillary tangle predominant pathology (CERAD possible or no, Braak stage III or higher, n 23). No differences were seen for gender, education, MMSE, or ApoE 2 allele frequency between groups in this analysis. Age was lower in the pathologically normal group (p 0.0005, t-test), and ApoE 4 allele frequency was higher in both pAD and -amyloid predominant cases (p 0.01, 2 test). The presence of amyloid angiopathy was associated with preclinical AD (p 0.002, Kruskal-Wallis ANOVA). All other measures of CVD (large vessel or lacunar infarcts, atherosclerosis, arteriosclerosis, microvascular disease, hemorrhagic infarcts) did not differ between groups. Likewise, LBD pathology did not differ between groups (p 0.62, Kruskal-Wallis ANOVA). Conclusions: AD-type neuropathology is seen relatively often in cognitively normal elderly persons. Both advanced age and ApoE 4 status are associated with pAD (as they are with AD). The lack of association of pAD with CVD or LBD suggests that AD develops independently of these pathological processes, at least in the early preclinical stages of disease.