Phenoloxidase (PO) activity was examined in the tunic tissue of
Ciona intestinalis
following lipopolysaccharide (LPS) intratunic injection. Tunic homogenate supernatant (THS), assayed with the ...Dopa-MBTH reaction, displayed Ca
2+
-independent PO activity that was raised by LPS and further enhanced by proteases. Specific inhibitors (tropolone, phenylthiourea, diethylthiocarbamate) supported the specificity of the reaction. Assay with soybean trypsin inhibitor showed that, in the tunic, PO activation with trypsin was not significantly inhibited suggesting that proteases diverse from serine proteases were involved. In vivo experiments were carried out by injecting isosmotic medium or LPS, and THS was assayed for its PO activity. Analysis of variance of the time-course profiles showed that LPS was more effective in activating proPO. To disclose the PO response at the injured site, an assay with Dopa-MBTH was performed in vitro. Quinones were mainly contained in the tunic matrix enriched with inflammatory cells around the injection site. Microscopic observations and immunohistochemistry with anti-CinPO-2 antibodies showed granulocytes and unilocular refractile granulocytes containing PO, whereas few morula cells were stained. In THS zymograms (SDS-polyacrylamide gel electrophoresis), PO activity linked to 90-kDa and 120-kDa bands was observed as an effect of LPS injection, whereas the density of 170-kDa PO was weak. A third presumptive PO enzyme (CinPO-3) containing the CinPO-2 peptide was identified in the recent
Ciona
genome version. Presumably, LPS stimulated the production and dimerization (120 kDa) of CinPO-3 (66 kDa). Thus, the activated proPO system includes several POs that are distinguishable by size and that are contained and presumably released by tunic inflammatory cells and hemocytes of the pharynx bars.
The AMP-activated protein kinase (AMPK) is a heterotrimeric complex composed of a catalytic subunit (a) and two regulatory subunits (β and γ). Two isoforms of the catalytic subunit (αl and (α2) have ...been identified. We show here that the αl- and α2-containing complexes contribute approximately equally to total AMPK activity in rat liver. Furthermore, expression of al or a2 with β and Y in mammalian cells demonstrates that both complexes have equal specific activity measured with the SAMS peptide. Using variant peptides, however, we show that al and a2 exhibit slightly different substrate preferences, which suggest that the two isoforms could play different physiological roles within the cell.
Two-dimensional electrophoresis (2DE) is one of the most effective methods for the reliable separation of proteins in a single gel. In our proteome analyses of human chromosomes, we used two types of ...2DE: two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis (2D IEF/SDS-PAGE) and radical-free and highly reduced two-dimensional polyacrylamide gel electrophoresis (RFHR 2D PAGE) together with one-dimensional SDS-polyacrylamide gel electrophoresis (1DE). Experimental details of these gel electrophoresis procedures that have been shown to be effective for human proteome analyses are described in detail.
Animals spontaneously developing lupus-like autoimmune pathology (SLE) are very promising models to study the mechanisms of natural abzymes (Abzs) generation and their role in etiology and ...pathogenesis of autoimmune diseases, but Abzs from the sera of animals remain virtually unstudied. In this work, electrophoretically homogeneous IgGs were isolated from the sera of MRL/MpJ-
lpr mice. It was shown for the first time that amylase activity is an intrinsic property of antibodies (Abs) and their isolated heavy and light chains. Various markers of SLE pathology (proteinuria, enhanced concentration of anti-DNA Abs) increased with spontaneous development of SLE and especially after animal immunization, correlating with the increase in Abz relative amylase activity. The highest amylase activity was found in the sera Abs of healthy mice after delivery and at the beginning of lactation; this was not correlated with markers of mouse SLE but supports the idea that pregnancy could “activate” or “trigger” autoimmune-like manifestations and Abzs production in healthy mammals. The possible differences in mechanisms of Abzs production in lactating mice and animals developing SLE are discussed.
αB-crystallin, a low-molecular-weight heat shock protein (HSP), has binding sites on platelets. However, the exact role of αB-crystallin is not clarified. In this study, we investigated the effect of ...αB-crystallin on platelet granule secretion. αB-crystallin attenuated the adenosine diphosphate (ADP)-induced phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK) and p38 MAPK. The ADP-stimulated HSP27 phosphorylation was markedly reduced by αB-crystallin. αB-crystallin significantly suppressed the ADP-induced secretions of both platelet-derived growth factor (PDGF)-AB and serotonin. Therefore, our results strongly suggest that αB-crystallin extracellularly suppresses platelet granule secretion by inhibition of HSP27 phosphorylation via p44/p42 MAPK and p38 MAPK.
Many studies demonstrated that cancer sera contain antibodies which react with autologous cellular antigens generally known as tumor-associated antigens (TAAs). In our laboratories, the approach used ...in the identification of TAAs has involved initially examining the sera of cancer patients using extracts of tissue culture cells as source of antigens in Western blotting and by indirect immunofluorescence on whole cells. With these two techniques, we identify sera which have high-titer fluorescent staining or strong signals to cell extracts on Western blotting and subsequently use these sera as probes in immunoscreening cDNA expression libraries, and also in proteomic approaches to isolate and identify targeted antigens which might potentially be involved in malignant transformation. In this manner, several novel TAAs including HCC1, p62, p90, and others have been identified. In extension of these studies, we evaluate the sensitivity and specificity of different antigen-antibody systems as markers in cancer in order to develop "tumor-associated antigen array" systems for cancer diagnosis, cancer prediction, and for following the response of patients to treatment.
The HBx protein of hepatitis B virus is involved in deregulation of cell cycle and development of hepatocellular carcinoma. Since c-Myc also plays an important role in cell proliferation and tumor ...development, we studied its regulation by HBx in a human hepatoma cell line. Co-expression of HBx and c-Myc resulted in increased stability of intracellular c-Myc. HBx blocked the ubiquitination of Myc through a direct interaction with the F box region of Skp2 and destabilization of the SCF
Skp2 complex. We suggest that sustained presence of c-Myc combined with mitogenic activity inherent to HBx may be associated with cell cycle deregulation and transformation.
Casein-maltodextrin glyco-conjugates were prepared using an economical, food-grade process based on the Amadori re-arrangement of the Maillard reaction.
The resultant glyco-conjugates were slightly ...yellow in colour and the degree of discolouration was dependent on heating time. Formation of glyco-conjugates was demonstrated by determining the reduction of free amino-groups by the
O-phthaldialdehyde (OPA) assay and sugar reducing ends by gas chromatography-mass spectrometry (GC-MS). Increases in molecular weight were monitored by SDS-polyacrylamide gel electrophoresis (PAGE) and were in agreement with those predicted for the conjugation of casein monomers with malto-oligosaccharides of average DP 7–10. 2D-Urea-SDS-PAGE demonstrated that both protein and saccharide components co-migrate, indicating that covalent bonds were upon heating. This resulted in increases in mass-to-charge ratio of the materials, which suggested decreases in pI. These observed chemical and physical changes were reconciled with previously documented improvements in emulsifying properties.
Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubule and the kinetochore. Our recent ultrastructural studies demonstrated a dynamic distribution of ...TTK, from the kinetochore to the centrosome, as cell enters into anaphase. Here, we show that a centrosomal protein TACC2 is phosphorylated in mitosis by TTK signaling pathway. TACC2 was pulled down by wild type TTK but not kinase death mutant, suggesting the potential phosphorylation‐mediated interaction between these two proteins. Our immunofluorescence studies revealed that both TTK and TACC2 are located to the centrosome. Interestingly, expression of kinase death mutant of TTK eliminated the centrosomal localization of TACC2 but not other centrosomal proteins such as γ‐tubulin and NuMA, a phenotype seen in TTK‐depleted cells. In these centrosomal TACC2‐liberated cells, chromosomes were lagging and mis‐aligned. In addition, the distance between two centrosomes was markedly reduced, suggesting that centrosomal TACC2 is required for mitotic spindle maintenance. The inter‐relationship between TTK and TACC2 established here provides new avenue to study centrosome and spindle dynamics underlying cell divisional control.
Erodium (L.) L’Hér. with 75 species is the third largest genus in Geraniaceae. Erodium is distributed in all continents but it shows a great diversity in the Mediterranean region. In Flora Iranica , ...Schonbeck-Temesy (1970), 15 species, four subsections and three subspecies have been mentioned for Erodium in Iran. Electrophoretic patterns of seed storage proteins in the genus Erodium has not been studied extensively. In this study for the first time seed protein electrophoretic patterns of seven Erodium species from different regions of Iran have been investigated. Protein extracts of seeds were obtained and protein concentration was determined by the Bradford method and analyzed with SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Results were evaluated by multivariate analysis methods including cluster analysis and factor analysis. In the electrophoretic pattern of seed storage proteins, generally, 48 specific bands with molecular weight between 13-102 kDa were detected. Electrophoretic data confirmed sub-generic classification of Erodium (to Plumosa and Erodium sections). Species relationships are discussed.
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