Purification and molecular cloning of a novel essential component of the apolipoprotein B mRNA editing enzyme-complex

Editing of apolipoprotein B (apoB) mRNA requires the catalytic component APOBEC-1 together with "auxiliary" proteins that have not been conclusively characterized so far. Here we report the purification of these additional components of the apoB mRNA editing enzyme-complex from rat liver and the cDNA cloning of the novel APOBEC-1-stimulating protein (ASP). Two proteins copurified into the final active fraction and were characterized by peptide sequencing and mass spectrometry: KSRP, a 75-kDa protein originally described as a splicing regulating factor, and ASP, a hitherto unknown 65-kDa protein. Separation of these two proteins resulted in a reduction of APOBEC-1-stimulating activity. ASP represents a novel type of RNA-binding protein and contains three single-stranded RNA-binding domains in the amino-terminal half and a putative double-stranded RNA-binding domain at the carboxyl terminus. Purified recombinant glutathione S-transferase (GST)-ASP, but not recombinant GST-KSRP, stimulated recombinant GST-APOBEC-1 to edit apoB RNA in vitro. These data demonstrate that ASP is the second essential component of the apoB mRNA editing enzyme-complex. In rat liver, ASP is apparently associated with KSRP, which may confer stability to the editing enzyme-complex with its substrate apoB RNA serving as an additional auxiliary component.     

Autocrine regulation of interleukin-8 production in human monocytes

Interleukin (IL)-8 is a C-X-C chemokine that plays an important role in acute inflammation through its G protein-coupled receptors CXCR1 and CXCR2. In this study, we investigated the role of IL-8 as an autocrine regulator of IL-8 production and the signaling mechanisms involved in human peripheral blood mononuclear cells (MNCs). Sepharose-immobilized IL-8 stimulated a sevenfold increase in IL-8 production within 2 h. IL-8 induced the expression of its own message, and IL-8 biosynthesis was inhibited by cycloheximide and actinomycin D, indicating de novo RNA and protein synthesis. In contrast to MNCs, polymorphonuclear neutrophils did not respond to the immobilized IL-8 with IL-8 production despite cell surface expression of CXCR1 and CXCR2. Melanoma growth-stimulatory activity/growth-related protein-alpha (MGSA/GROalpha), which binds CXCR2 but not CXCR1, was unable to either stimulate IL-8 secretion in MNCs or desensitize these cells to respond to immobilized IL-8. The involvement of mitogen-activated protein kinase (MAPK) in IL-8-induced IL-8 biosynthesis was suggested by the ability of PD-98059, an inhibitor of MAPK kinase, to block this function. Furthermore, IL-8 induced a significant increase in extracellular signal-regulated kinase 2 phosphorylation, whereas MGSA/GROalpha was much less effective. These findings support the role of IL-8 as an autocrine regulator of IL-8 production and suggest that this function is mediated by CXCR1 through activation of MAPK.     

Increase of anti-inflammatory cytokines in patients with esophageal cancer after perioperative treatment with G-CSF

Granulocyte colony-stimulating factor (G-CSF) has been shown to effectively stimulate granulopoiesis, in both neutropenic and in non-neutropenic patients. Recently, other effects of G-CSF on the immune system have attracted interest in treating non-neutropenic patients with a high risk of severe infection. In this phase II trial, we measured the effects of G-CSF on the serum cytokine levels in patients with esophageal cancer undergoing esophagectomy. Twenty subsequent patients (study group, 19 evaluable) received G-CSF (rhG-CSF, Filgrastim) at standard doses (300 microg or 480 microg) subcutaneously 2 days before and up to 7 days after surgery. G-CSF was well tolerated. Leukocytes increased from 7600/microl at study entry (day -2) to a maximum of 45 100/microl (day 6). In the study patients, we found a highly significant (P<0.001) postoperative increase of G-CSF, IL-1ra, sTNFRp55 and sTNFRp75 as compared with the baseline level. In contrast, IL-8 levels were decreased by a factor of 6.8; there were no changes in the very low TNF-alpha levels. The comparison of the study group with a control group of 21 cancer patients undergoing major surgery who were not treated with G-CSF showed significant differences in the serum levels of G-CSF, sTNFRp55, sTNFRp75, and IL-1ra, respectively. There was no infection in the study group up to 10 days after surgery as compared with 29.9% in a historical control group (P=0.008). Thus, the induction of anti-inflammatory cytokines and the downregulation of pro-inflammatory cytokines by G-CSF might be a promising adjuvant treatment of infectious complications in patients undergoing esophagectomy.     

A Versatile Click-Compatible Monolignol Probe to Study Lignin Deposition in Plant Cell Walls

Lignin plays important structural and functional roles in plants by forming a hydrophobic matrix in secondary cell walls that enhances mechanical strength and resists microbial decay. While the importance of the lignin matrix is well documented and the biosynthetic pathways for monolignols are known, the process by which lignin precursors or monolignols are transported and polymerized to form this matrix remains a subject of considerable debate. In this study, we have synthesized and tested an analog of coniferyl alcohol that has been modified to contain an ethynyl group at the C-3 position. This modification enables fluorescent tagging and imaging of this molecule after its incorporation into plant tissue by click chemistry-assisted covalent labeling with a fluorescent azide dye, and confers a distinct Raman signature that could be used for Raman imaging. We found that this monolignol analog is incorporated into in vitro-polymerized dehydrogenation polymer (DHP) lignin and into root epidermal cell walls of 4-day-old Arabidopsis seedlings. Incorporation of the analog in stem sections of 6-week-old Arabidopsis thaliana plants and labeling with an Alexa-594 azide dye revealed the precise locations of new lignin polymerization. Results from this study indicate that this molecule can provide high-resolution localization of lignification during plant cell wall maturation and lignin matrix assembly.     

Upregulation of miR21 and Repression of Grhl3 by Leptin Mediates Sinusoidal Endothelial Injury in Experimental Nonalcoholic Steatohepatitis

Sinusoidal endothelial dysfunction (SED) has been found to be an early event in nonalcoholic steatohepatitis (NASH) progression but the molecular mechanisms underlying its causation remains elusive. We hypothesized that adipokine leptin worsens sinusoidal injury by decreasing functionally active nitric oxide synthase 3 (NOS)3 via miR21. Using rodent models of NASH, and transgenic mice lacking leptin and leptin receptor, results showed that hyperleptinemia caused a 4–5 fold upregulation of hepatic miR21 as assessed by qRTPCR. The upregulation of miR21 led to a time-dependent repression of its target protein Grhl3 levels as shown by western blot analyses. NOS3-p/NOS3 ratio which is controlled by Grhl3 was significantly decreased in NASH models. SED markers ICAM-1, VEGFR-2, and E-selectin as assessed by immunofluorescence microscopy were significantly up regulated in the progressive phases of NASH. Lack of leptin or its receptor in vivo, reversed the upregulation of miR21 and restored the levels of Grhl3 and NOS3-p/NOS3 ratio coupled with decreased SED dysfunction markers. Interestingly, leptin supplementation in mice lacking leptin, significantly enhanced miR21 levels, decreased Grhl3 repression and NOS3 phosphorylation. Leptin supplementation in isolated primary endothelial cells, Kupffer cells and stellate cells showed increased mir21 expression in stellate cells while sinusoidal injury was significantly higher in all cell types. Finally miR21 KO mice showed increased NOS3-p/NOS3 ratio and reversed SED markers in the rodent models of NASH. The experimental results described here show a close association of leptin-induced miR21 in aiding sinusoidal injury in NASH.     

Quantitative NMR spectroscopy of biologically active substances and excipients

Biologically active ingredients and excipients are the essentials of a drug formulation, such as a tablet, dragee, solution, etc. Quality control of such substances thus plays a pivotal role in the production process of pharmaceutical drugs. Since these agents often exhibit complex structures, consist of multiple components, or lack of a chromophore, traditional means of characterization are often not feasible. Furthermore, substances of small molecular weight or strong polar character generally exhibit poor chromatographic properties, thus, conventional procedures such as high-performance liquid chromatography are often not applicable. Instead, quantitative nuclear magnetic resonance (qNMR) spectroscopy has emerged as an alternative or orthogonal method in drug analysis. In this review, we elaborate on the application of qNMR to three important classes of biological substances, namely polysaccharides, amino acids, and lipids, and demonstrate the benefits of this modern tool in contrast to traditional techniques.     

Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, <Emphasis Type="Italic">in vitro</Emphasis>effects on cartilage metabolism and intra-articular levels after injection

Introduction  

Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration.