Human blood plasma advanced oxidation protein products (AOPP) correlates with fibrinogen levels

In 1996 a novel oxidative stress biomarker, referred to as advanced oxidation protein products (AOPP) was detected in the plasma of chronic uremic patients. The aim of the present studies was to find out that which plasma fraction(s) is responsible for AOPP reactivity. Thermal treatment of pooled samples of human citrate-plasma or EDTA-plasma at 50 degrees C resulted in a rapid and parallel loss of fibrinogen concentration and AOPP reactivity. On the basis of time course and t1/2 values following thermal treatment, AOPP was indistinguishable from fibrinogen. There was a statistically significant (p < 0.0001) correlation between levels of blood plasma fibrinogen and AOPP in patients (n = 61) with various peripheral vascular or cardiovascular diseases. There was also a significant (p < 0.0001) relationship between plasma levels of fibrinogen and molar AOPP/fibrinogen ratio indicating that higher fibrinogen concentrations were associated with more oxidatively transformed groups on the molecule. Results of the present studies suggest that post-translationally modified fibrinogen is a key molecule responsible for human plasma AOPP reactivity. It remains to be elucidated what is the pathophysiological significance of the post-translationally modified fibrinogen in the inflammation-associated events of atherosclerosis, in platelet aggregation, and as a cardiovascular risk biomarker.     

Functional and topological analysis of phosphatidylcholine synthase from Sinorhizobium meliloti

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (<20% of wild type activity) of Pcs activity. Six of these essential residues were subjected to further mutagenesis studies replacing them by amino acids with similar properties or size. A topological analysis of sinorhizobial Pcs showed the presence of eight transmembrane helices, with the C- and N-terminus located in the cytoplasm. The majority of the conserved residues is predicted to be either located within the cytoplasmic loops or on the cytoplasmic side of the membrane which can be expected for an enzyme using one membrane-associated and one soluble substrate.     

Cdc42 activity regulates hematopoietic stem cell aging and rejuvenation

1. Download : Download high-res image (312KB)
2. Download : Download full-size image

Highlights? Constitutively increasing Cdc42 activity causes aging of young HSCs ? Elevated Cdc42 activity correlates with a loss of cell polarity in aged HSCs ? Inhibition of Cdc42 activity rejuvenates aged LT-HSC function ? Cdc42 inhibition restores the level and spatial distribution of AcH4K16     

Acute heat stress prior to downhill running may enhance skeletal muscle remodeling

Heat shock proteins (HSPs) are chaperones that are known to have important roles in facilitating protein synthesis, protein assembly and cellular protection. While HSPs are known to be induced by damaging exercise, little is known about how HSPs actually mediate skeletal muscle adaption to exercise. The purpose of this study was to determine the effects of a heat shock pretreatment and the ensuing increase in HSP expression on early remodeling and signaling (2 and 48 h) events of the soleus (Sol) muscle following a bout of downhill running. Male Wistar rats (10 weeks old) were randomly assigned to control, eccentric exercise (EE; downhill running) or heat shock + eccentric exercise (HS; 41°C for 20 min, 48 h prior to exercise) groups. Markers of muscle damage, muscle regeneration and intracellular signaling were assessed. The phosphorylation (p) of HSP25, Akt, p70s6k, ERK1/2 and JNK proteins was also performed. As expected, following exercise the EE group had increased creatine kinase (CK; 2 h) and mononuclear cell infiltration (48 h) compared to controls. The EE group had an increase in p-HSP25, but there was no change in HSP72 expression, total protein concentration, or neonatal MHC content. Additionally, the EE group had increased p-p70s6k, p-ERK1/2, and p-JNK (2 h) compared to controls; however no changes in p-Akt were seen. In contrast, the HS group had reduced CK (2 h) and mononuclear cell infiltration (48 h) compared to EE. Moreover, the HS group had increased HSP72 content (2 and 48 h), total protein concentration (48 h), neonatal MHC content (2 and 48 h), p-HSP25 and p-p70s6k (2 h). Lastly, the HS group had reduced p-Akt (48 h) and p-ERK1/2 (2 h). These data suggest that heat shock pretreatment and/or the ensuing HSP72 response may protect against muscle damage, and enhance increases in total protein and neonatal MHC content following exercise. These changes appear to be independent of Akt and MAPK signaling pathways.     

Targeted glycomics by selected reaction monitoring for highly sensitive glycan compositional analysis

The development of glycomics increasingly requires the detection and quantification of large numbers of glycans, which is only partially achieved by current glycomics approaches. Taking advantage of selected reaction monitoring to enhance both sensitivity and selectivity, we report here a strategy termed targeted glycomics that enables highly sensitive and consistent identification and quantification of diverse glycans across multiple samples at the same time. In this proof-of-principle study, we validated the method by analyzing global N-glycans expressed in different systems: single proteins, cancer cells, and serum samples. A dynamic range of three orders of magnitude was obtained for the detection of all five glycans released from ribonuclease B. The limit of detection of 80 attomole for Man(9) GlcNAc(2) demonstrated the excellent sensitivity of the method. The capability of the strategy to identify diverse glycans was demonstrated by identification and detection of 162 different glycans and isomers from pancreatic cancer cells. The sensitivity of the method was illustrated further by the ability to detect eight glycans from 250 cancer cells and five glycans released from 100 cancer cells. In serum obtained from rabbits fed control diet or diet enriched with 2% cholesterol, differences to 42 glycans were accurately measured and this indicates that this strategy might find use in studies of biomarker discovery and validation.     

Anomalous features of EMT during keratinocyte transformation

During the evolution of epithelial cancers, cells often lose their characteristic features and acquire a mesenchymal phenotype, in a process known as epithelial-mesenchymal transition (EMT). In the present study we followed early stages of keratinocyte transformation by HPV16, and observed diverse cellular changes, associated with EMT. We compared primary keratinocytes with early and late passages of HF1 cells, a cell line of HPV16-transformed keratinocytes. We have previously shown that during the progression from the normal cells to early HF1 cells, immortalization is acquired, while in the progression to late HF1, cells become anchorage independent. We show here that during the transition from the normal state to late HF1 cells, there is a progressive reduction in cytokeratin expression, desmosome formation, adherens junctions and focal adhesions, ultimately leading to poorly adhesive phenotype, which is associated with anchorage-independence. Surprisingly, unlike "conventional EMT", these changes are associated with reduced Rac1-dependent cell migration. We monitored reduced Rac1-dependent migration also in the cervical cancer cell line SiHa. Therefore we can conclude that up to the stage of tumor formation migratory activity is eliminated.     

Binding of retinoic acid by the inhibitory serpin protein C inhibitor.

The serpin superfamily includes inhibitors of serine proteases and noninhibitory members with other functions (e.g. the hormone precursor angiotensinogen and the hormone carriers corticosteroid-binding globulin and thyroxine-binding globulin). It is not known whether inhibitory serpins have additional, noninhibitory functions. We studied binding of (3)H-labeled hydrophobic hormones (estradiol, progesterone, testosterone, cortisol, aldosterone, and all-trans-retinoic acid) to the inhibitory serpins antithrombin III, heparin cofactor II, plasminogen activator inhibitor-1, and protein C inhibitor (PCI). All-trans-[(3)H]retinoic acid bound in a specific dose-dependent and time-dependent way to PCI (apparent K(d) = 2.43 microm, 0.8 binding sites per molecule of PCI). We did not observe binding of other hormones to serpins. Intact and protease-cleaved PCI bound retinoic acid equally well, and retinoic acid did not influence inhibition of tissue kallikrein by PCI. Gel filtration confirmed binding of retinoic acid to PCI in purified systems and suggested that PCI may also function as a retinoic acid-binding protein in seminal plasma. Therefore, our present data, together with the fact that PCI is abundantly expressed in tissues requiring retinoic acid for differentiation processes (e.g. the male reproductive tract, epithelia in various organs), suggest an additional biological role for PCI as a retinoic acid-binding and/or delivering serpin.     

Toward a PKB inhibitor: modification of a selective PKA inhibitor by rational design

Protein kinase B/Akt (PKB) is an anti-apoptotic protein kinase that has strongly elevated activity in human malignancies. We therefore initiated a program to develop PKB inhibitors, "Aktstatins". We screened about 500 compounds for PKB inhibitors, using a radioactive assay and an ELISA assay that we established for this purpose. These compounds were produced as combinatorial libraries, designed using the structure of the selective PKA inhibitor H-89 as a starting point. We have identified a successful lead compound, which inhibits PKB activity in vitro and in cells overexpressing active PKB. The new compound shows reversed selectivity to H-89: In contrast to H-89, which inhibits PKA 70 times better than PKB, the new compound, NL-71-101, inhibits PKB 2.4-fold better than PKA. The new compound, but not H-89, induces apoptosis in tumor cells in which PKB is amplified. We have identified structural features in NL-71-101 that are significant for the specificity and that can be used for future development and optimization of PKB inhibitors.     

Bulk Flow Revisited: Transport of a Soluble Protein in the Secretory Pathway

The C-terminal domain, Cp, of the Semliki Forest virus capsid protein, known for its rapid, efficient and chaperone-independent folding, was used to measure bulk fluid flow in the secretory pathway of Chinese hamster ovary cells. Being small, nonglycosylated, soluble and cytoplasmic in origin, Cp was not likely to interact with lectins, cargo receptors and retention factors. Using pulse-chase analysis, we observed that translocation into the endoplasmic reticulum resulted in rapid and efficient folding and transport of the newly synthesized Cp protein to the extracellular medium. The first Cp molecules were secreted 15 min after synthesis, which is the fastest transport of a protein so far recorded in mammalian cells. The rate constant of secretion was 1.2% per min, which amounts to an estimated bulk flow rate of about 155 coat protein II (COPII) vesicles per second. Transport was independent of expression level, and blocked by CI-976, brefeldin A and ATP depletion indicating that it depended on COPII vesicle formation, and followed the classical secretory pathway. In polarized Madin-Darby canine kidney cells, the secretion rate was similar but occurred mainly apically. The results demonstrated that fluid flow in the secretory pathway is fast, and can therefore play a significant role in the secretion of soluble secretory products.