Combination of two-dimensional electrophoresis and shotgun peptide sequencing in comparative proteomics

Two-dimensional electrophoresis (2-DE) and shotgun peptide sequencing are the two major technologies to compare the expression profile of proteins, which is also referred to as comparative proteomics or quantitative proteomics. Although the methodologies, such as difference gel electrophoresis for 2-DE and isotope-coded affinity tags for shotgun peptide sequencing, have made rapid progress, these two approaches have their own strengths and weaknesses. Therefore, the combination of the two methodologies is beneficial for the purpose of better comparative proteomics, especially in comprehensive coverage of the proteome and protein information such as post-translational modifications.     

IL‐17 induces osteoclastogenesis from human monocytes alone in the absence of osteoblasts,which is potently inhibited by anti‐TNF‐α antibody: A novel mechanism of osteoclastogenesis by IL‐17

IL‐17 is a proinflammatory cytokine crucial for osteoclastic bone resorption in the presence of osteoblasts or synoviocytes in rheumatoid arthritis. However, the role of IL‐17 in osteoclastogenesis from human monocytes alone remains unclear. Here, we investigated the role of IL‐17 in osteoclastogenesis from human monocytes alone and the direct effect of infliximab on the osteoclastogenesis induced by IL‐17. Human peripheral blood mononuclear cells (PBMC) were cultured for 3 days with M‐CSF. After non‐adherent cells were removed, IL‐17 was added with either infliximab or osteoprotegerin (OPG). Seven days later, adherent cells were stained for vitronectin receptor. On the other hand, CD11b‐positive monocytes purified from PBMC were also cultured and stained as described above. CD11b‐positive cells were cultured with TNF‐α and receptor activator of NF‐κB ligand (RANKL). In the cultures of both adherent cells and CD11b‐positive cells, IL‐17 dose‐dependently induced osteoclastogenesis in the absence of soluble‐RANKL. OPG or infliximab inhibited IL‐17‐induced osteoclastogenesis. Interestingly, in the culture of CD11b‐positive cells, the osteoclastogenesis was more potently inhibited by infliximab than by OPG. TNF‐α and RANKL synergistically induced osteoclastogenesis. The present study clearly demonstrated the novel mechanism by which IL‐17 directly induces osteoclastogenesis from human monocytes alone. In addition, infliximab potently inhibits the osteoclastogenesis directly induced by IL‐17. J. Cell. Biochem. 108: 947–955, 2009. © 2009 Wiley‐Liss, Inc.     

Dynamic modeling and simulation of continuous airlift bioreactors

For dynamic behaviors of continuous airlift bioreactors, a mathematical model based on a tanks-in-series model with backflow has been developed. The equations describing the dynamics of airlift bioreactors are material balances for micro-organism, substrate, dissolved oxygen and oxygen in gas-phase and heat balances. Non-ideal mixing of liquid and gas phases is taken into account using a tanks-in-series model with backflow. The batch operation, startup operation and the consequence of plant failure were simulated and the effects of design and operating parameters for an airlift bioreactor on its dynamic behaviors were discussed. The concentration profiles of micro-organism, substrate, dissolved oxygen and oxygen in gas-phase and the temperature profile in an airlift bioreactors and their dynamics were obtained. The computational results indicate that the transients of a chemostat in the case of bubble column bioreactor are slower compared with those in the case of airlift bioreactor. The proposed simulator is more precise as compared with models published previously in the literature and therefore provides more reliable and rational examination of continuous airlift bioreactor performance.     

Docosahexaenoic acid induces ERK1/2 activation and neuritogenesis via intracellular reactive oxygen species production in human neuroblastoma SH-SY5Y cells

Docosahexaenoic acid (22: 6n-3; DHA) is a long chain polyunsaturated fatty acid that exists highly enriched in fish oil, and it is one of the low molecular weight food chemicals which can pass a blood brain barrier. A preliminary survey of several fatty acids for expression of growth-associated protein-43 (GAP-43), a marker of axonal growth, identified DHA as one of the most potent inducers. The human neuroblastoma SH-SY5Y cells exposed to DHA showed significant and dose-dependent increases in the percentage of cells with longer neurites. To elucidate signaling mechanisms involved in DHA-enhanced basal neuritogenesis, we examined the role of extracellular signal-regulated kinase (ERK)1/2 and intracellular reactive oxygen species (ROS) production using SH-SY5Y cells. From immunoblotting experiments, we observed that DHA induced the ROS production, protein tyrosine phosphatase inhibition, mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) phosphorylation, and sequentially ERK1/2 phosphorylation, the last of which was significantly reduced by MEK inhibitor U0126. Both antioxidants and MEK inhibitor affected DHA-induced GAP-43 expression, whereas the specific PI3K inhibitor LY294002 did not. We found that total protein tyrosine phosphatase activity was also downregulated by DHA treatment, which was counteracted by antioxidant pretreatment. These results suggest that the ROS-dependent ERK pathway, rather than PI3K, plays an important role during DHA-enhanced neurite outgrowth.     

A specific peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligand,pioglitazone, ameliorates gastric mucosal damage induced by ischemia and reperfusion in rats

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, has recently been implicated as a regulator of cellular proliferation and inflammatory responses. The aim of the present study was to investigate the effects of pioglitazone on ischemia-reperfusion (I/R)-induced gastric mucosal injury in rats. Gastric ischemia was induced for 30 min by applying a small vascular clamp to the celiac artery and reperfusion was produced by removal of the clamp in male Sprague-Dawley rats treated with and without pioglitazone. Pioglitazone was given to the rats intraperitoneally 2 h before the vascular clamping. The area of gastric mucosal erosion (erosion index) significantly increased from mean basal levels after 60 min of reperfusion. This erosion index was significantly inhibited by pretreatment with pioglitazone in a dose-dependent manner. The concentration of thiobarbituric acid reactive substances (TBARS) and myeloperoxidase (MPO) activity in the gastric mucosa were both significantly increased after I/R, and pretreatment with pioglitazone significantly reduced these increases. The contents of both mucosal TNF-alpha and CINC-2beta in the I/R group were significantly increased compared with the levels in the sham-operated group. These increases in TNF-alpha and CINC-2beta were significantly inhibited by pretreatment with pioglitazone at a dose of 10 mg/kg. The results of the present study indicate that pioglitazone inhibited lipid peroxidation and reduced development of the gastric mucosal inflammation induced by I/R in rats. This investigation suggests that pioglitazone has potential as a new therapeutic agent for reperfusion injury.     

Oral Factors Affecting Titanium Elution and Corrosion: An In Vitro Study Using Simulated Body Fluid

Objectives

Ti, which is biocompatible and resistant to corrosion, is widely used for dental implants, particularly in patients allergic to other materials. However, numerous studies have reported on Ti allergy and the in vitro corrosion of Ti. This study investigated the conditions that promote the elution of Ti ions from Ti implants.

Methods

Specimens of commercially pure Ti, pure nickel, a magnetic alloy, and a gold alloy were tested. Each specimen was immersed in a simulated body fluid (SBF) whose pH value was controlled (2.0, 3.0, 5.0, 7.4, and 9.0) using either hydrochloric or lactic acid. The parameters investigated were the following: duration of immersion, pH of the SBF, contact with a dissimilar metal, and mechanical stimulus. The amounts of Ti ions eluted were measured using a polarized Zeeman atomic absorption spectrophotometer.

Results

Eluted Ti ions were detected after 24 h (pH of 2.0 and 3.0) and after 48 h (pH of 9.0). However, even after 4 weeks, eluted Ti ions were not detected in SBF solutions with pH values of 5.0 and 7.4. Ti elution was affected by immersion time, pH, acid type, mechanical stimulus, and contact with a dissimilar metal. Elution of Ti ions in a Candida albicans culture medium was observed after 72 h.

Significance

Elution of Ti ions in the SBF was influenced by its pH and by crevice corrosion. The results of this study elucidate the conditions that lead to the elution of Ti ions in humans, which results in implant corrosion and Ti allergy.     

Mechanism of neuronal coordination of the rhythmic activities of developing flight muscles and a neurosecretory cell population in the silkmoth, Bombyx mori

Ultradian rhythmic firing activity (period of 40-90 min) of a population of neurosecretory cells (NSCs) producing FXPRLamide peptides in the subesophageal ganglion (SG) of the silkmoth, Bombyx mori, is closely coordinated with periodically occurring electrical activity of developing flight muscles (FMs) during metamorphosis. To examine neuronal mechanisms and pathways that mediate the coordination of the NSC and flight motor systems, the ventral nerve cord (VNC) or circumesophageal connectives were transected. Transection of the VNC between the SG and thoracic ganglia greatly shortened the period of activity rhythm of the FMs (5-15 min) with no effect on the rhythmicity of NSCs. Bilateral transsection of the circumesophageal connectives between the brain and SG abolished the rhythmic activity of NSCs, thereby suggesting that the coordination of the two systems may be mediated by a common oscillatory mechanism in the brain. Further, bisection of the brain in the midline failed to abolish the ultradian rhythmicity of FMs. Thus, each brain hemisphere may have an ultradian oscillator that activates the NSC system in the SG, and modulates the short-period oscillation of the flight motor system located in the thoracic ganglia.     

Characterization of two cysteine residues in cytochrome P-450scc: chemical identification of the heme-binding cysteine residue

It was found that there were only two cysteine residues in highly purified cytochrome P-450scc molecule from bovine adrenocortical mitochondria by titration with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) in denatured conditions. Only one cysteine residue at position 303 of cytochrome P-450scc could be specifically modified with DTNB in the native state. The resulting cytochrome P-450scc-5-thio-2-nitrobenzoic acid complex (cytochrome P-450scc-TNB) showed no distinct differences in absorption spectra, cholesterol binding, or electron transferring from adrenodoxin, compared to those of untreated cytochrome P-450scc. These observations indicated that the 303rd cysteine residue does not play a role in heme binding, cholesterol (substrate) binding or adrenodoxin binding. The other cysteine residue at 461 could be modified with DTNB only in a denatured condition. These assignments of cysteine residues were made by the subsequent S-cyanylation with KCN followed by incubation in 6 M guanidine hydrochloride at alkaline pH, which causes enhanced cleavage of peptide bonds adjacent to the cyanylated cysteine residues. Analyses of fragmented polypeptides by SDS-polyacrylamide gel electrophoresis confirmed that there were only two cysteine residues in the molecule and indicated that the cleavage rate of the peptide bond between 460 and 461 becomes high only when both cysteine residues (303 and 461) are cyanylated. These results clearly established that the 461st cysteine residue in cytochrome P-450scc plays a role as the heme fifth ligand on the basis of the general agreement that a thiolated cysteine residue coordinates to the heme iron.     

Electrophoretic Studies on Plant Protoplasts I. pH Dependence of Zeta Potentials of Protoplasts from Various Sources

Electrophoretic mobilities of plant protoplasts from varioussources were measured, as a function of the pH of the medium,by a micro-electrophoresis technique to characterize the protoplastsin terms of curves of zeta potential vs. protoplast surfacepH (pH_s). The shape of the curves of zeta potential vs. pH_scurves differed among preparations of protoplasts isolated fromvarious species and strains. The isoelectric points (pI) ofthe protoplasts measured in this study were between 3.0 and4.0. These differences among the protoplasts suggest that itmay be possible to develop an electrophoretic method for theselection of protoplasts. The shape of the curves of zeta potentialvs. pH_s also indicated that carboxyl groups, as well as phosphategroups, may contribute to the negative charges on the surfaceof protoplasts. (Received October 14, 1988; Accepted February 22, 1989)