Fine structure of the branchial epithelium in the teleost Oreochromis niloticus

We have studied the gill epithelium of Oreochromis niloticus using transmission electron microscopy with the particular interested relationship between cell morphology and osmotic, immunoregulatory, or other non‐regulatory functions of the gill. Pavement cells covered the filament epithelium and lamellae of gills, with filament pavement cells showing distinct features from lamellar pavement cells. The superficial layer of the filament epithelium was formed by osmoregulatory elements, the columnar mitochondria‐rich, mucous and support cells, as well as by their precursors. Light mitochondria‐rich cells were located next to lamellae. They exhibited an apical crypt with microvilli and horizontal small dense rod‐like vesicles, sealed by tight junctions to pavement cells. Dark mitochondria‐rich cells had long dense rod‐like vesicles and a small apical opening sealed by tight junctions to pavement cells. The deep layer of the filament epithelium was formed by a network of undifferentiated cells, containing neuroepithelial and myoepithelial cells, macrophage and eosinophil‐like cells and their precursors, as well as precursors of mucous cells. The lateral‐basal surface was coated by myoepithelial cells and a basal lamina. The lamellar blood lacunae was lined by pillar cells and surrounded by a basal lamina and pericytes. The data presented here support the existence of two distinct types of pavement cells, mitochondria‐rich cells, and mitochondria‐rich cells precursors, a structural role for support cells, a common origin for pavement cells and support cells, a paracrine function for neuroepithelial cells in the superficial layer, and the control of the lamellar capillary base by endocrine and contractile cells. Data further suggest that the filament superficial layer is involved in gill osmoregulation, that may interact, through pale mitochondria‐rich cells, with the deep layer and lamellae, whereas the deep layer, through immune and neuroendocrine systems, acts in the regeneration and defense of the tissue. J. Morphol. 2010. © 2010 Wiley‐Liss, Inc.     

Oral exfoliative cytology in the diagnosis of paracoccidioidomycosis

OBJECTIVE: To assess the efficacy of conventional oral exfoliative cytology as a diagnostic tool in paracoccidioidomycosis. STUDY DESIGN: Cytologic smears and incisional biopsies were obtained from 10 patients with a clinical suspicion and oral manifestations of paracoccidioidomycosis. Cytologic smears and sections of the incisional biopsy underwent methenamine silver staining for fungi according to the Gomori-Grocott method. The dry glass slides were examined at 400 or 1,000 x magnification, and the presence and shape of yeasts of Paracoccidioides brasiliensis were investigated. RESULTS: Yeasts of the fungus P brasiliensis were clearly identified in cytologic smears and sections from incisional biopsies in all cases analyzed (100.0%). CONCLUSION: Cytology of oral samples proved an effective diagnostic method for the detection of paracoccidioidomycosis in humans.     

Characterisation of the last Fe-S cluster-binding subunit of Neurospora crassa complex I

We have cloned cDNAs encoding the last iron-sulphur protein of complex I from Neurospora crassa. The cDNA sequence contains an open reading frame that codes for a precursor polypeptide of 226 amino acid residues with a molecular mass of 24972 Da. Our results indicate that the mature protein belongs probably to the peripheral arm of complex I and is rather unstable when not assembled into the enzyme. The protein is highly homologous to the PSST subunit of bovine complex I, the most likely candidate to bind iron-sulphur cluster N-2. All the amino acid residues proposed to bind such a cluster are conserved in the fungal protein.     

Enantiomeric resolution of kielcorin derivatives by HPLC on polysaccharide stationary phases using multimodal elution

Analytical HPLC methods using carbamate chiral stationary phases of polysaccharide derivatives were developed for the enantiomeric resolution of five racemic mixtures of xanthonolignoids: rac-trans-kielcorin C, rac-cis-kielcorin C, rac-trans-kielcorin D, rac-trans-isokielcorin D, and rac-trans-kielcorin E. The separations were evaluated with the stationary phases cellulose tris-3,5-dimethylphenylcarbamate, amylose tris-3,5-dimethylphenylcarbamate, amylose tris-(S)-1-phenylethylcarbamate, and amylose tris-3,5-dimethoxyphenylcarbamate under normal, reversed-phase, and polar organic elution conditions. Chiral recognition of those chiral stationary phases, the influence of mobile phases on the enantiomers separation, and the effects of structural features of the solutes on the chiral discrimination observed are discussed. The best performance was achieved on an amylose tris-3,5-dimethylphenylcarbamate phase. Polar organic conditions gave shorter retention factors and better resolutions and were a valuable alternative to the alcohol-hexane or reversed-phase conditions.     

Isolation and characterization of eight pregnancy-associated glycoproteins present at high levels in the ovine placenta between day 60 and day 100 of gestation

Pregnancy-associated glycoproteins (PAG), structurally related to aspartic proteinases, are expressed in the outer epithelial cell layer (chorion/trophectoderm) of the ungulate placenta. The aim of the present study was to isolate as many PAG molecules as possible from placentae collected between day 60 and day 100 of gestation and to characterize their amino-terminal amino-acid sequences. Three heterologous radioimmunoassays were used to monitor PAG immunoreactivity throughout the isolation procedures. Sequential use of DEAE-cellulose, gel filtration, and CM ceramic chromatographies led to the isolation of several fractions rich in PAG immunoreactivity. The fractions with a large amount of proteins were also purified by chromatofocusing. The analysis of immunoreactive fractions by SDS-PAGE, Western blotting and two-dimensional electrophoresis followed by amino-terminal microsequencing on PVDF membranes allowed to identify eight different ovPAG with apparent molecular masses ranging from 55 to 66 kDa and isoelectric points from 4.0 to 6.8. The N-terminal sequences were determined and their comparison to those previously identified revealed that four of them are identical to those encoded by previously known cDNA, while the additional four sequences appear to be novel since they have not yet been described.     

Cloning and characterization of the MAL11 gene encoding a high-affinity maltose transporter from Torulaspora delbrueckii

The transport and regulation of maltose utilization by Torulaspora delbrueckii, one of the most abundant non-Saccharomyces species present in home-made corn and rye bread dough, has been investigated. A DNA fragment containing the MAL11 gene from T. delbrueckii (TdMAL11) was isolated by complementation cloning in Saccharomyces cerevisiae. DNA sequence analysis revealed the presence of an open reading frame (ORF) of 1884 bp, encoding a 627-amino acid membrane protein, which displays high homology to other yeast maltose transporters. Upstream of TdMAL11, the DNA insert contained a partial ORF (TdMAL12) on the opposite strand, which showed high similarity to the S. cerevisiae MAL12 gene. Sequence analysis, Northern blot and transport measurements indicated that TdMAL11 expression is regulated by the carbon source. Attempts to disrupt TdMAL11 revealed the presence of two functional MAL loci. Disruption of a single copy decreased the V(max) of maltose transport, but not the K(m), whereas the double disruption abolished the uptake of this sugar in T. delbrueckii.     

Myosin-X provides a motor-based link between integrins and the cytoskeleton

Unconventional myosins are actin-based motors with a growing number of attributed functions. Interestingly, it has been proposed that integrins are transported by unidentified myosins to facilitate cellular remodelling. Here we present an interaction between the unconventional myosin-X (Myo10) FERM (band 4.1/ezrin/radixin/moesin) domain and an NPXY motif within beta-integrin cytoplasmic domains. Importantly, knock-down of Myo10 by short interfering RNA impaired integrin function in cell adhesion, whereas overexpression of Myo10 stimulated the formation and elongation of filopodia in an integrin-dependent manner and relocalized integrins together with Myo10 to the tips of filopodia. This integrin relocalization and filopodia elongation did not occur with Myo10 mutants deficient in integrin binding or with a beta(1)-integrin point mutant deficient in Myo10 binding. Taken together, these results indicate that Myo10-mediated relocalization of integrins might serve to form adhesive structures and thereby promote filopodial extension.     

The effect of cations on the amidase activity of human tissue kallikrein: 1-linear competitive inhibition by sodium, potassium, calcium and magnesium. 2-linear mixed inhibition by aluminium

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide by human tissue kallikrein (hK1) was studied in the absence and in the presence of increasing concentrations of the following chloride salts: sodium, potassium, calcium, magnesium and aluminium. The data indicate that the inhibition of hK1 by sodium, potassium, calcium and magnesium is linear competitive and that divalent cations are more potent inhibitors of hK1 than univalent cations. However the inhibition of hK1 by aluminium cation is linear mixed, with the cation being able to bind to both the free enzyme and the ES complex. This cation was the best hK1 inhibitor. Aluminium is not a physiological cation, but is a known neurotoxicant for animals and humans. The neurotoxic actions of aluminium may relate to neuro-degenerative diseases.     

Effects of 'casoparan', a peptide isolated from casein hydrolysates with mastoparan-like properties

Casein, a protein found in milk of several species, is divided into different chains from 19 to 25 kDa. Casein is also considered as a source of amino acids and generating peptides with biological activities such as opiate, immunostimulating, antibacterial, peptidase inhibitors, among others. In this work, Sephadex G-10 chromatography followed by high-performance liquid chromatography isolation purified NZCase TT, an industrial culture media for tetanus toxin production. In the first step, four pools were isolated and tested in different assays: isolated smooth muscle assay (guinea pig ileum, rat uterus), phagocytosis in vitro of opsonized sheep red blood cells, and hydrogen peroxide (H2O2) release from mouse peritoneal macrophages. Pool III was the main active pool being able to potentiate bradykinin action in guinea pig ileum, stimulating phagocitic activity by resident macrophages and increasing H2O2 release from macrophages previously activated with bacille Calmette Guérin. Using mass spectra the primary structure of the main peptide from pool III was obtained--INKKI, which corresponds to beta-casein fragment 26-30. The immunostimulating action is probably related to a direct action in macrophage cells.     

Relationships among murine CD11c(high) dendritic cell subsets as revealed by baseline gene expression patterns

The functional relationships and properties of different subtypes of dendritic cells (DC) remain largely undefined. To better characterize these cells, we used global gene analysis to determine gene expression patterns among murine CD11c(high) DC subsets. CD4(+), CD8alpha(+), and CD8alpha(-) CD4(-) (double negative (DN)) DC were purified from spleens of normal C57/BL6 mice and analyzed using Affymetrix microarrays. The CD4(+) and CD8alpha(+) DC subsets showed distinct basal expression profiles differing by >200 individual genes. These included known DC subset markers as well as previously unrecognized, differentially expressed CD Ags such as CD1d, CD5, CD22, and CD72. Flow cytometric analysis confirmed differential expression in nine of nine cases, thereby validating the microarray analysis. Interestingly, the microarray expression profiles for DN cells strongly resembled those of CD4(+) DC, differing from them by <25 genes. This suggests that CD4(+) and DN DC are closely related phylogenetically, whereas CD8alpha(+) DC represent a more distant lineage, supporting the historical distinction between CD8alpha(+) and CD8alpha(-) DC. However, staining patterns revealed that in contrast to CD4(+) DC, the DN subset is heterogeneous and comprises at least two subpopulations. Gene Ontology and literature mining analyses of genes expressed differentially among DC subsets indicated strong associations with immune response parameters as well as cell differentiation and signaling. Such associations offer clues to possible unique functions of the CD11c(high) DC subsets that to date have been difficult to define as rigid distinctions.