Large-volume sample stacking-capillary electrophoresis used for the determination of 3-nitrotyrosine in rat urine

Large-volume sample stacking using the electroosmotic flow (EOF) pump technique has been investigated for the quantification of 3-nitrotyrosine in urine of diabetic rats. The best separation conditions for these highly complex samples were obtained using capillary electrophoresis (CE) in the reversed polarity mode (i.e., injecting at the cathode and detecting at the anode) using cetyltrimethylammonium bromide (CTAB) in the running buffer. The optimum CE separation conditions were achieved using a phosphate buffer prepared with 0.15M phosphoric acid and 0.5 mM CTAB adjusted to pH 6.4 with sodium hydroxide. In such CE conditions, the limit of detection (LOD) was 1.77 microM for 3-nitrotyrosine with normal injection mode, meanwhile with the large-volume sample stacking technique a more than 20-fold improvement was observed (i.e., LOD = 0.08 microM was obtained) without noticeable loss of resolution. This value allowed the detection of 3-nitrotyrosine in urine from diabetic rats. To our knowledge, this work is one of the few applications showing the great possibilities of these stacking procedures to analyse biological samples by CE.     

Determination of protein markers in human serum: Analysis of protein expression in toxic oil syndrome studies

Toxic oil syndrome (TOS) is a disease that appeared in Spain in 1981. It affected more than 20 000 people and produced over 300 deaths in the first 2 years. In this paper, a prospective study on the differences in gene expression in sera between a control versus a TOS-affected population, both originally exposed to the toxic oil, is presented. Differential protein expression was analyzed by two-dimensional electrophoresis (2-DE). Several problems related with serum analysis by 2-DE were addressed in order to improve protein detection in the gel images. Three new commercial systems for albumin depletion were tested to optimize the detection of minor proteins that can be obscured by the presence of a few families of high abundance proteins (albumin, immunoglobulins). Other factors, such as the use of nonionic reductants or the presence of thiourea in the gels, were also tested. From these optimized images, a group of 329 major gel spots was located, matched and compared in serum samples. Thirty-five of these protein spots were found to be under- or overexpressed in TOS patients (> three-fold increase or decrease). Proteins in the differential spots were identified by matrix-assisted laser desorption/ionization-time of flight peptide map fingerprinting and database search. Several haptoglobin isoforms were found to be differentially expressed, showing expression phenotypes that could be related with TOS affection. Haptoglobin phenotypes have been previously reported to have important biological and clinical consequences and have been described as risk factors for several diseases.     

20-HETE inotropic effects involve the activation of a nonselective cationic current in airway smooth muscle

20-Hydroxyeicosatetraenoic acid (20-HETE) controls several mechanisms such as vasoactivity, mitogenicity, and ion transport in various tissues. Our goal was to quantify the effects of 20-HETE on the electrophysiological properties of airway smooth muscle (ASM). Isometric tension measurements, performed on guinea pig ASM, showed that 20-HETE induced a dose-dependent inotropic effect with an EC50 value of 1.5 microM. This inotropic response was insensitive to GF-109203X, a PKC inhibitor. The sustained contraction, requiring Ca2+ entry, was partially blocked by either 100 microM Gd3+ or 1 microM nifedipine, revealing the involvement of noncapacitative Ca2+ entry and L-type Ca2+ channels, respectively. Microelectrode measurements showed that 3 microM 20-HETE depolarized the membrane potential in guinea pig ASM by 13 +/- 2mV(n = 7), as did 30 microM 1-oleoyl-2-acetyl-sn-glycerol. Depolarizing effects were also observed in the absence of epithelium. Patch-clamp recordings demonstrated that 1 microM 20-HETE activated a nonselective cationic inward current that may be supported by the activation of transient receptor potential channels. The presence of canonical transient receptor potential mRNA was confirmed by RT-PCR in guinea pig ASM cells.     

Fine needle aspiration cytology of neurothekeoma. A case report

BACKGROUND: Neurothekeoma (NT) is a rare, benign neoplasm of soft parts with a distinctive histologic appearance. To our knowledge, the cytologic findings have not been described before. We present a case of NT with the cytologic features on fine needle aspiration cytology (FNAC). CASE: A 54-year-old female presented with a circumscribed nodule in the left breast. The lesion was evaluated by FNAC. The smears showed an abundant, metachromatic, myxoid matrix with fusiform and epithelioid cells, some binucleated or multinucleated, loose or in groups and sometimes forming concentric whorls. The lesion was removed, and the diagnosis of NT was made after histopathologic study. CONCLUSION: NT is an extremely rare neoplasm in the mammary region. Fusiform and epithelioid cells arranged in concentric whorls in a myxoid tumor of soft tissue are a distinctive characteristic of this neoplasm and can suggest the diagnosis.     

Retinoic acid binds to the C2-domain of protein kinase C(alpha)

Protein kinase C(alpha) (PKC(alpha)) is a key enzyme regulating the physiology of cells and their growth, differentiation, and apoptosis. PKC activity is known to be modulated by all-trans retinoic acid (atRA), although neither the action mechanism nor even the possible binding to PKCs has been established. Crystals of the C2-domain of PKC(alpha), a regulatory module in the protein that binds Ca(2+) and acidic phospholipids, have now been obtained by cocrystallization with atRA. The crystal structure, refined at 2.0 A resolution, shows that RA binds to the C2-domain in two locations coincident with the two binding sites previously reported for acidic phospholipids. The first binding site corresponds to the Ca(2+)-binding pocket, where Ca(2+) ions mediate the interactions of atRA with the protein, as they do with acidic phospholipids. The second binding site corresponds to the conserved lysine-rich cluster localized in beta-strands three and four. These observations are strongly supported by [(3)H]-atRA-binding experiments combined with site-directed mutagenesis. Wild-type C2-domain binds 2 mol of atRA per mol of protein, while the rate reduces to one in the case of C2-domain variants, in which mutations affect either Ca(2+) coordination or the integrity of the lysine-rich cluster site. Competition between atRA and acidic phospholipids to bind to PKC is a possible mechanism for modulating PKC(alpha) activity.     

The Klebsiella pneumoniae wabG gene: role in biosynthesis of the core lipopolysaccharide and virulence

To determine the function of the wabG gene in the biosynthesis of the core lipopolysaccharide (LPS) of Klebsiella pneumoniae, we constructed wabG nonpolar mutants. Data obtained from the comparative chemical and structural analysis of LPS samples obtained from the wild type, the mutant strain, and the complemented mutant demonstrated that the wabG gene is involved in attachment to alpha-L-glycero-D-manno-heptopyranose II (L,D-HeppII) at the O-3 position of an alpha-D-galactopyranosyluronic acid (alpha-D-GalAp) residue. K. pneumoniae nonpolar wabG mutants were devoid of the cell-attached capsular polysaccharide but were still able to produce capsular polysaccharide. Similar results were obtained with K. pneumoniae nonpolar waaC and waaF mutants, which produce shorter LPS core molecules than do wabG mutants. Other outer core K. pneumoniae nonpolar mutants in the waa gene cluster were encapsulated. K. pneumoniae waaC, waaF, and wabG mutants were avirulent when tested in different animal models. Furthermore, these mutants were more sensitive to some hydrophobic compounds than the wild-type strains. All these characteristics were rescued by reintroduction of the waaC, waaF, and wabG genes from K. pneumoniae.     

Mouse novel Ly9: a new member of the expanding CD150 (SLAM) family of leukocyte cell-surface receptors

Human CS1, also known as novel Ly9, 19A24, or CRACC, is a member of the immunoglobulin gene superfamily (IgSF) expressed on natural killer cells and other leukocytes. Here we describe the cloning of the mouse homologue of this gene. The mouse novel Ly9 gene is shown to encode a transmembrane protein composed of two extracellular immunoglobulin-like domains, a transmembrane region and an 88-amino acid cytoplasmic domain. Mouse novel Ly9 is structurally similar to the extracellular domains of CD84 and CD229 (Ly9). Both mouse and human novel Ly9 genes mapped close to the CD229gene in a region where other members of the CD150 family have also been mapped, and analysis of their genomic sequences showed that they have an identical intron/exon organization. Northern blot analysis revealed that the expression of mouse and human novel Ly9 was predominantly restricted to hematopoietic tissues, with the exception of testis. Here we show that SAP (SH2D1A), an adapter protein responsible for the X-linked lymphoproliferative disease, binds to the phosphorylated cytoplasmic tail of human but not mouse novel Ly9. Taken together, these data indicate that mouse novel Ly9 is a new member of the expanding CD150 family of cell surface receptors.     

Correlation between electrical activity and intracellular Ca2+ oscillations in GH3 rat anterior pituitary cells

Simultaneous measurements of electrical activity and intracellular Ca(2+) levels were performed in perforated-patch current-clamped individual GH3 cells. Both in cells showing brief (<100 ms) and long action potentials (APs), we found a good correlation between the averaged intracellular Ca2+ concentration ([Ca2+]i) and AP frequency, but not between the mean [Ca2+]i and AP duration. Nevertheless, the magnitude of spontaneous Ca2+ oscillations was highly dependent on the size and duration of the APs. The decay of the Ca2+ transients was not slowed when the size of the oscillations was varied either spontaneously or after elongation of the AP with the K+ channel blocker tetraethyl ammonium. Furthermore, the recovery from Ca2+ loads similar to those induced by the APs was slightly retarded after treatment of the cells with intracellular store Ca2+-ATPase inhibitors. Among previous results showing that caffeine-induced [Ca2+]i increases are secondary to electrical activity enhancements in GH3 cells, these data indicate that the Ca2+ entry triggered via APs is the primary determinant of the [Ca2+]i variations, and that Ca2+-induced Ca2+ release has a minor contribution to Ca2+ oscillations recorded during spontaneous activity. They also point to modulation of electrical activity patterns as a crucial factor regulating spontaneous [Ca2+]i signalling, and hence pituitary cell functions in response to physiological secretagogues.