Synovial fluid (SF) is capable of reflecting infectious, immunological, or inflammatory joint conditions in horses by altering its composition and appearance. Although plasma and SF compositions are ...quantitatively different, this latter compartment reflects changes in plasma macromolecules. Therefore, changes in serum immunoglobulin protein concentrations tend also to alter intracapsular levels. Therefore, it is necessary to know the physiological concentrations of proteins present in SF. The aim of this study was to determine the levels of total protein, albumin, transferrin, haptoglobin, α1-acid glycoprotein, ceruloplasmin, and immunoglobulins A and G in SF of six healthy horses. The synovial proteinogram was obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The SF proteins reached a maximum of 25% of serum concentrations, varying inversely with molecular weight of the protein, except for the ceruloplasmin.
Mitochondria-derived oxygen-free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O
2
•− generated by mitochondria under certain ...pathological conditions. In the mitochondrial electron transport chain, succinate–cytochrome
c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome
c. To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen-free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/
•OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron–H
2O
2-dependent Fenton reaction or O
2
•−-dependent Haber–Weiss mechanism. Direct EPR measurement of SCR at 77
K indicated the formation of a nonheme iron–NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce
•OH. In H9c2 cells, SCR-dependent oxygen-free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR-mediated oxygen-free radical production was also detected in cultured vascular endothelial cells.
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► Excess NO promotes the pro-oxidant activity of SCR to produce hydroxyl radical in vitro. ► SCR-mediated hydroxyl radical production depends on dinitroso iron intermediate formation at the S1 center. ► Excess NO induced by shear exposure or hypoxia/reoxygenation enhances oxygen-free radicals production by mitochondrial SCR in living cells.
The potential use of high contrast X-ray microtomography (XMT) for the reading of fragile historic documents without the need to physically unravel them is a new analytical imaging development in the ...field of conservation however, it is important to first assess if there is any evidence of change in the parchment structure during scanning by XMT. Modern and historic parchment samples were exposed to X-rays using the high contrast XMT equipment. Attenuated Total Reflection Fourier Transform Infra-red Spectroscopy (ATR-FTIR), SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Near-infrared Spectroscopy (NIR) and X-ray diffraction (XRD) were utilised to investigate whether there is any evidence for change to collagen within parchment samples after exposure to XMT. Results show that the inherent differences in the parchment structure due to the material source, production and storage appear to produce larger differences than that due to the exposure to XMT. This indicates that XMT may be a suitable technique for data recovery from parchment samples that cannot be unrolled.
Chitinase expression in microfilariae of the parasitic nematode Brugia malayi (B. malayi, Bm) is coincidental with the onset of their infectivity to mosquitoes. An antibody raised to Onchocerca ...volvulus (O. volvulus, Ov) infective-stage larval chitinase (Ov-CHI-1) was specifically reactive against B. malayi microfilarial chitinase and was used to study the localization of chitinase in B. malayi during microfilarial development and transmission to the insect vector. Immuno-electron microscopy (IEM) was used to demonstrate that the chitinase was confined to the inner body of the microfilariae and furthermore that chitinase was only present in sheathed microfilarial species, although the inner body is present in all species. Observation using the IEM implicates two distinct routes of chitinase secretion from the inner body, via either the pharyngeal thread, or during transmission of the microfilariae to the vector, contained in vesicle-like structures. Many morphological studies have described the structure of the inner body, but no function has been assigned to it as of yet. Although it has been commented that the cells surrounding the inner body and pharyngeal thread are those destined to become the intestine and pharynx and that the inner body represents a store of material. Our studies suggest that chitinase is one such product stored in the inner body and that it is secreted during the exsheathment of the microfilaria in the mosquito.
Poly(A)-specific ribonuclease (PARN), a member of the DEDD family, is a key enzyme involved in the deadenylation of mRNA in higher eukaryotic cells. In this research, it was found that Mg
2+ could ...protect PARN against thermal inactivation by increasing the midpoint of inactivation and decreasing the inactivation rate. This protective effect was unique to Mg
2+ in a concentration-dependent manner. However, the thermal unfolding and aggregation was promoted by the addition of Mg
2+ at high temperatures. These results revealed that Mg
2+ might have dual effects on PARN stability: protecting the active site but endangering the overall structural stability.
The study of the "proteomes" of human cells, tissues, and body fluids is a big challenge, and several highly sophisticated workflow approaches are pursued to achieve as comprehensive information as ...possible. Initially proteome analysis was exclusively based on the gel-based workflow, employing two-dimensional electrophoresis of protein extracts followed by mass spectrometry of the tryptic peptide digests of protein spots. Meanwhile several additional proteomics workflows are applied, which are mostly based on separation and analysis of tryptic peptides without separating the protein mixture. However, direct information on quantitative and qualitative changes of protein expressions can only be obtained by methods operating on the protein level, no other method can replace two-dimensional electrophoresis. In this review we compile the different techniques of high-resolution two-dimensional electrophoresis and their further developments to increase the degree of reliance of the method.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The plasma membrane H sup(+)-ATPase is activated by binding of 14-3-3 protein to the phosphorylated C terminus. Considering the large number of 14-3-3 and H sup(+)-ATPase isoforms in Arabidopsis (13 ...and 11 expressed genes, respectively), specificity in binding may exist between 14-3-3 and H sup(+)-ATPase isoforms. We now show that the H sup(+)-ATPase is the main target for 14-3-3 binding at the plasma membrane, and that all twelve 14-3-3 isoforms tested bind to the H sup(+)-ATPase in vitro. Using specific antibodies for nine of the 14-3-3 isoforms, we show that GF14epsilon, mu, lambda, omega, chi, phi, nu, and upsilon are present in leaves, but that isolated plasma membranes lack GF14chi, phi and upsilon. Northern blots using isoform-specific probes for all 14-3-3 and H sup(+)-ATPase isoforms showed that transcripts were present for most of the isoforms. Based on mRNA levels, GF14epsilon, mu, lambda and chi are highly expressed 14-3-3 isoforms, and AHA1, 3, and 11 highly expressed H sup(+)-ATPase isoforms in leaves. However, mass peptide finger-printing identified AHA1 and 2 with the highest score, and their presence could be confirmed by MS/MS. It may be calculated that under 'unstressed' conditions less than one percent of total 14-3-3 is attached to the H sup(+)-ATPase. However, during a condition requiring full activation of II" pumping, as induced here by the presence of the fungal toxin fusicoccin, several percent of total 14-3-3 may be engaged in activation of the H sup(+)-ATPase.
Glucose-regulated protein 58 (GRP58)-like immunoreactivity in rat liver obtained in the evening or after fasting underwent an electrophoretic band-shift, which disappeared after ...phosphatase-treatment. Since mass spectrometric analysis raised a possibility that Ser150 of GRP58 is phosphorylated, an antibody against the phosphoserine150 GRP58 was generated. Immunoreactivity to this antibody was increased in the evening and after fasting. Since GRP58 was shown to interact with signal transducer and activator of transduction 3 (STAT3), a leptin-related protein, the effect of leptin was examined. Immunoreactivity to the anti-phosphoGRP58 antibody was markedly elevated after the leptin injection, indicating that Ser150 of GRP58 is phosphorylated after fasting and leptin-treatment.
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