Requiem protein links RelB/p52 and the Brm-type SWI/SNF complex in a noncanonical NF-kappaB pathway.

The SWI/SNF chromatin remodeling complex plays pivotal roles in mammalian transcriptional regulation. In this study, we identify the human requiem protein (REQ/DPF2) as an adaptor molecule that links the NF-kappaB and SWI/SNF chromatin remodeling factor. Through in vitro binding experiments, REQ was found to bind to several SWI/SNF complex subunits and also to the p52 NF-kappaB subunit through its nuclear localization signal containing the N-terminal region. REQ, together with Brm, a catalytic subunit of the SWI/SNF complex, enhances the NF-kappaB-dependent transcriptional activation that principally involves the RelB/p52 dimer. Both REQ and Brm were further found to be required for the induction of the endogenous BLC (CXCL13) gene in response to lymphotoxin stimulation, an inducer of the noncanonical NF-kappaB pathway. Upon lymphotoxin treatment, REQ and Brm form a larger complex with RelB/p52 and are recruited to the BLC promoter in a ligand-dependent manner. Moreover, a REQ knockdown efficiently suppresses anchorage-independent growth in several cell lines in which the noncanonical NF-kappaB pathway was constitutively activated. From these results, we conclude that REQ functions as an efficient adaptor protein between the SWI/SNF complex and RelB/p52 and plays important roles in noncanonical NF-kappaB transcriptional activation and its associated oncogenic activity.

Proteomic analysis of proteins expressing in regions of rat brain by a combination of SDS-PAGE with nano-liquid chromatography-quadrupole-time of flight tandem mass spectrometry.

BACKGROUND: Most biological functions controlled by the brain and their related disorders are closely associated with activation in specific regions of the brain. Neuroproteomics has been applied to the analysis of whole brain, and the general pattern of protein expression in all regions has been elucidated. However, the comprehensive proteome of each brain region remains unclear. RESULTS: In this study, we carried out comparative proteomics of six regions of the adult rat brain: thalamus, hippocampus, frontal cortex, parietal cortex, occipital cortex, and amygdala using semi-quantitative analysis by Mascot Score of the identified proteins. In order to identify efficiently the proteins that are present in the brain, the proteins were separated by a combination of SDS-PAGE on a C18 column-equipped nano-liquid chromatograph, and analyzed by quadrupole-time of flight-tandem-mass spectrometry. The proteomic data show 2,909 peptides in the rat brain, with more than 200 identified as region-abundant proteins by semi-quantitative analysis. The regions containing the identified proteins are membrane (20.0%), cytoplasm (19.5%), mitochondrion (17.1%), cytoskeleton (8.2%), nucleus (4.7%), extracellular region (3.3%), and other (18.0%). Of the identified proteins, the expressions of glial fibrillary acidic protein, GABA transporter 3, Septin 5, heat shock protein 90, synaptotagmin, heat shock protein 70, and pyruvate kinase were confirmed by immunoblotting. We examined the distributions in rat brain of GABA transporter 3, glial fibrillary acidic protein, and heat shock protein 70 by immunohistochemistry, and found that the proteins are localized around the regions observed by proteomic analysis and immunoblotting. IPA analysis indicates that pathways closely related to the biological functions of each region may be activated in rat brain. CONCLUSIONS: These observations indicate that proteomics in each region of adult rat brain may provide a novel way to elucidate biological actions associated with the activation of regions of the brain.

Urinary trypsin inhibitor suppresses excessive generation of superoxide anion radical, systemic inflammation, oxidative stress, and endothelial injury in endotoxemic rats.

OBJECTIVE AND DESIGN: The protective effects of ulinastatin, a human urinary trypsin inhibitor (UTI), against superoxide radical (O(2)(-*)) generation, systemic inflammation, lipid peroxidation, and endothelial injury were investigated in endotoxemic rats. MATERIALS AND TREATMENT: Twenty-one Wistar rats were allocated to a control group, a UTI group, and a sham group. A bolus of lipopolysaccharide (LPS; 3 microg/g) was administered intravenously to the control group, a bolus of LPS and UTI (5 U/g) to the UTI group, and a bolus of saline to the sham group. METHODS: The O(2)(-*) generated was measured as the current in the right atrium using an electrochemical O(2)(-*) sensor. Plasma nitrite, high mobility group box 1 (HMGB1), tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, malondialdehyde, and soluble intercellular adhesion molecule-1 (sICAM-1) were measured 360 min after LPS administration. RESULTS: The O(2)(-*) current increased in the control group and was significantly attenuated in the UTI group after 55 min (P < 0.05 at 55-60 min, P < 0.01 at 65-360 min). Plasma nitrite, HMGB1, TNF-alpha, IL-6, malondialdehyde, and sICAM-1 were attenuated in the UTI group. CONCLUSIONS: UTI suppressed excessive O(2)(-*) generation, systemic inflammation, lipid peroxidation, and endothelial injury in endotoxemic rats.

Serum glial fibrillary acidic protein is a highly specific biomarker for traumatic brain injury in humans compared with S-100B and neuron-specific enolase.

BACKGROUND: Serum glial fibrillary acidic protein (GFAP) is a specific predictor of brain damage and neurologic outcome in patients with traumatic brain injury (TBI). In this study, serum GFAP, S-100B, and neuron-specific enolase (NSE) were compared in the same samples from severe trauma patients to assess their ability to predict abnormalities detectable on head computed tomography (CT). METHODS: This study was a retrospective analysis at a single university emergency center. Thirty-four trauma patients were included. Serum samples were collected from the patients for 3 days. Serum GFAP, S-100B, and NSE concentrations were measured with enzyme-linked immunosorbent assays and compared in patients with and without TBI, as evaluated by head CT. RESULTS: Serum GFAP, S-100B, and NSE were significantly higher in the TBI patients than in the non-TBI patients (p < 0.05 for each protein). The receiver operating characteristic curves for TBI were compared for the three biomarkers for 3 days. Serum GFAP on day 1 had the largest area under the receiver operating characteristic curve (0.983), with 88.9% sensitivity and 100% specificity. CONCLUSIONS: Serum GFAP has remarkable diagnostic value for TBI, defined by abnormal head CT findings, in prehospital-triaged patients with severe trauma.

Brm transactivates the telomerase reverse transcriptase (TERT) gene and modulates the splicing patterns of its transcripts in concert with p54(nrb).

We report that a DBHS (Drosophila behaviour, human splicing) family protein, p54(nrb), binds both BRG1 (Brahma-related gene 1) and Brm (Brahma), catalytic subunits of the SWI/SNF (switch/sucrose non-fermentable) chromatin remodelling complex, and also another core subunit of this complex, BAF60a. The N-terminal region of p54(nrb) is sufficient to pull-down other core subunits of the SWI/SNF complex, suggesting that p54(nrb) binds SWI/SNF-like complexes. PSF (polypyrimidine tract-binding protein-associated splicing factor), another DBHS family protein known to directly bind p54(nrb), was also found to associate with the SWI/SNF-like complex. When sh (short hairpin) RNAs targeting Brm were retrovirally expressed in a BRG1-deficient human cell line (NCI-H1299), the resulting clones showed down-regulation of the TERT (telomerase reverse transcriptase) gene and an enhancement of ratios of exon-7-and-8-excluded TERT mRNA that encodes a beta-site-deleted inactive protein. All of these clones display growth arrest within 2 months of the Brm-knockdown. In NCI-H1299 cells, Brm, p54(nrb), PSF and RNA polymerase II phosphorylated on CTD (C-terminal domain) Ser(2) specifically co-localize at a region incorporating an alternative splicing acceptor site of TERT exon 7. These findings suggest that, at the TERT gene locus in human tumour cells containing a functional SWI/SNF complex, Brm, and possibly BRG1, in concert with p54(nrb), would initiate efficient transcription and could be involved in the subsequent splicing of TERT transcripts by accelerating exon-inclusion, which partly contributes to the maintenance of active telomerase.

MARCKS-like protein, a membrane protein identified for its expression in developing neural retina, plays a role in regulating retinal cell proliferation.

Membrane proteins are expressed in a specific manner in developing tissues, and characterization of these proteins is valuable because it allows them to be used as cell surface markers. Furthermore, they are potentially important for the regulation of organogenesis because some may participate in signal transduction. In the present study, we used proteomics to examine the comprehensive protein expression profile of the membrane fraction in the embryonic and adult mouse retina. We purified the retinal membrane fraction by sucrose-density-gradient centrifugation and analysed total proteins using shotgun analysis on a nanoflow LC-MS/MS (liquid chromatography tandem MS) system. Approximately half of the 326 proteins from the adult retina and a quarter of the 310 proteins from the embryonic retina (day 17) appeared to be membrane-associated proteins. Among these, MLP [MARCKS (myristoylated alanine-rich C-kinase substrate)-like protein], which shares approx. 50% amino acid identity with MARCKS, was selected for further characterization. The mRNA and surface protein expression of MLP decreased as retinal development progressed. Overexpression of MLP by retrovirus-mediated gene transfer enhanced the proliferation of retinal progenitor cells without affecting differentiation or cell migration in a retinal explant culture system. In contrast, MLP overexpression did not promote proliferation in fibroblasts (NIH 3T3 cells). Mutation analysis of MLP demonstrated that myristoylation was necessary to promote proliferation and that phosphorylation inhibited proliferation, indicating the functional importance of membrane localization.

Glycosylation engineering of therapeutic IgG antibodies: challenges for the safety, functionality and efficacy.

Glycosylation of the Fc region of IgG has a profound impact on the safety and clinical efficacy of therapeutic antibodies. While the biantennary complex-type oligosaccharide attached to Asn297 of the Fc is essential for antibody effector functions, fucose and outer-arm sugars attached to the core heptasaccharide that generate structural heterogeneity (glycoforms) exhibit unique biological activities. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for the development and quality control of therapeutic antibodies, and glycan profiles of the Fc are recognized as critical quality attributes. In the past decade our understanding of the influence of glycosylation on the structure/function of IgG-Fc has grown rapidly through X-ray crystallographic and nuclear magnetic resonance studies, which provides possibilities for the design of novel antibody therapeutics. Furthermore, the chemoenzymatic glycoengineering approach using endoglycosidase-based glycosynthases may facilitate the development of homogeneous IgG glycoforms with desirable functionality as next-generation therapeutic antibodies. Thus, the Fc glycans are fertile ground for the improvement of the safety, functionality, and efficacy of therapeutic IgG antibodies in the era of precision medicine.

Prothrombin and beta 2-glycoprotein I frequently contribute to antiphospholipid antibody interactions with phospholipids and the generation of abnormal waveform profiles in coagulation assays.

Transmittance waveforms are generated during clot formation on photo-optical coagulation analyzers. We previously showed that 61.5% of patients with antiphospholipid antibodies (APLA) exhibited a negative deflection in the pre-coagulation phase of the prothrombin time (PT slope 1). The current studies investigated the 'molecular basis' of this abnormal parameter. We found that the negative PT slope 1 is IgG-mediated and is not dependent on the presence of fibrinogen or thrombin activity. We also found that IgG from most of the patients required a specific thromboplastin and the presence of prothrombin or beta(2)-glycoprotein I beta(2) GPI) to produce an abnormal IgG wave-form assay. In addition, the abnormal IgG waveform required cofactor binding to phospholipids when beta(2) GPI was the cofactor, and annexin V could partially block this interaction. In conclusion, these results showed that the interactions of IgG with phospholipids via beta(2) GPI or prothrombin constitute the core mechanisms of the abnormal waveforms.

Anti-beta(2)-glycoprotein I antibody-mediated inhibition of activated protein C requires binding of beta(2)-glycoprotein I to phospholipids.

To clarify the role(s) of anti-beta(2) GPI antibodies on thrombosis in anti-phospholipid antibody syndromes (APS), the effect of IgG from three patients on activated protein C (APC) was investigated using phospholipid vesicles and purified proteins. Two of the total IgG inhibited APC activity in the presence of beta(2)GPI, whereas the third IgG did not. In addition, one IgG inhibited APC activity without beta(2)GPI. Anti-beta(2)GPI IgG from the two inhibitory IgG preparations inhibited APC activity only in the presence of beta(2)GPI. Inhibition was suppressed partially by excess APC and almost completely by excess phospholipid vesicles. Cleaved beta(2)GPI, a non-phospholipid-binding form, did not support inhibitory activity, even though the anti-beta(2)GPI IgG bound to the cleaved molecule. This study confirms that anti-beta(2)GPI antibodies from APS patients inhibit APC activity, and demonstrates the requirement of phospholipid binding of beta(2)GPI for expression of the inhibitory activity of these antibodies.

Prediction of the neurological outcome with intrathecal high mobility group box 1 and S100B in cardiac arrest victims: a pilot study.

OBJECTIVES: To investigate whether high mobility group box 1 (HMGB1) and S100B in cerebrospinal fluid (CSF) and the serum predict the neurological outcome in patients resuscitated from out-of-hospital cardiac arrest (OHCA). MATERIALS AND METHODS: This study was designed as a prospective observational study. Twenty-five patients, who received standard cardiopulmonary resuscitation and post-resuscitation intensive care, were enrolled in this study. The patients were divided into two groups according to Glasgow-Pittsburgh Cerebral Performance categories (CPCs) at 6 months after return of spontaneous circulation (ROSC), Group G (n = 7, CPC 1 or 2) and Group P (n = 18, CPC ≥ 3). Their blood samples were taken at 6, 24, and 48h after ROSC. The patients, whose CSF was sampled at 48h, were also divided into either sub-Group G (n = 6) or sub-Group P (n = 8) at 6 months after ROSC. RESULTS: HMGB1 and S100B in CSF in sub-Group P were significantly higher than those in sub-Group G (HMGB1, <1.0 vs. 12.4 ng/ml, P = 0.009; S100B, 2.68 vs. 84.2 ng/ml, P = 0.007, respectively). HMGB1 in CSF was strongly correlated with S100B (σ = 0.81, P = 0.001). HMGB1 was elevated in serum at 6h and normalized within 48 h after ROSC without any significant differences between the two groups. Serum S100B in Group P was significantly higher than that in Group G at each time point. CONCLUSIONS: The significant elevations of HMGB1 and S100B in CSF, and S100B in serum are associated with the neurologically poor outcome in OHCA patients.

Effects of a constant light environment on hippocampal neurogenesis and memory in mice.

Because the environmental light-dark cycle is a key factor involved in modulating circadian rhythm in mammals, disruption of cyclic light conditions has a variety of effects on physiology and behavior. In the hippocampus, neurogenesis, which continues to occur throughout life, has been reported to exhibit circadian variation under cyclic light-dark conditions. In the present study, we examined whether a constant light environment affected hippocampal neurogenesis in mice. Half of the animals were exposed to continuous light conditions (L/L group), while the other half remained under normal cyclic light-dark conditions (L/D group). In the L/L group, the number of BrdU-labeled cells (proliferating cells) and that of BrdU and class III ß-tubulin double-labeled cells (newborn neurons) in the granule cell layer were significantly decreased compared with the L/D group. Because hippocampal neurogenesis is involved in memory and learning, we also investigated the effects on performance in water maze tasks to assess spatial learning. Exposure to L/L treatment for 3 weeks impaired spatial learning task performance, although there was no difference in the open field behaviors between the groups. These findings demonstrate that the constant light conditions impaired hippocampal neurogenesis as well as cognitive performance, and suggest an important role for the cyclic light-dark environment in appropriate maintenance of the hippocampal system.

Prognostic value of biochemical markers of brain damage and oxidative stress in post-surgical aneurysmal subarachnoid hemorrhage patients.

The aim of this study is to determine effective biochemical markers and optimal sampling timing for prediction of neurological prognosis in post-surgical aneurysmal subarachnoid hemorrhage (SAH) patients. Subjects were a sequential group of SAH patients admitted to our centre who underwent aneurysm clipping before Day 3 and who received a cerebrospinal fluid (CSF) drain. CSF samples from 32 patients were collected on Days 3, 7, and 14. Neurological outcome was assessed by neurosurgeons using the Glasgow outcome scale (GOS) at 6 months after onset. CSF levels of neuron-specific enolase (NSE), S100B, and glial fibrillary acidic protein (GFAP) were determined using enzyme-linked immunosorbent assay, and the CSF concentrations of malondialdehyde (MDA) were determined using spectrophotometric assay. In univariate analysis, S100B on Days 3 and 14, GFAP on Days 3 and 7, and MDA on Day 14 were significantly higher in the poor outcome group (GOS 1-4) than in the good outcome group (GOS 5). In multivariate analysis, only MDA on Day 14 was identified as a significant predictor of poor neurological outcome at 6 months after onset. The area under the receiver-operating characteristic (ROC) curve for MDA on Day 14 was 0.841. For a threshold of 0.3 microM, sensitivity and specificity were 0.875 and 0.750, respectively. Our findings suggest that these biochemical markers, especially MDA, show significant promise as predictors of neurological outcome in clinical practice.

Cholinergic agonist physostigmine suppresses excessive superoxide anion radical generation in blood, oxidative stress, early inflammation, and endothelial injury in rats with forebrain ischemia/reperfusion.

The cholinergic anti-inflammatory pathway is reportedly important in modulating the inflammatory response in local and systemic diseases, including ischemia/reperfusion pathophysiology. In this study, we investigated the effects of the cholinergic agonist, physostigmine, on jugular venous superoxide radical (O(2)(-)) generation, oxidative stress, early inflammation, and endothelial activation during forebrain ischemia/reperfusion (FBI/R) in rats. Fourteen male Wistar rat were allocated to the control group (n=7) or physostigmine group (n=7). The physostigmine group received 80 ng/g physostigmine intraperitoneally 24 h and 1 h before forebrain ischemia was established. The jugular venous O(2)(-) current was measured for 10 min during forebrain ischemia and for 120 min after reperfusion. The O(2)(-) current increased gradually during forebrain ischemia in both groups. The current increased markedly immediately after reperfusion in the control group but was significantly attenuated in the physostigmine group after reperfusion. Brain and plasma malondialdehyde, high-mobility group box 1 (HMGB1) protein, and intercellular adhesion molecule 1 (ICAM1) were significantly attenuated in the physostigmine group compared with the control group, except for brain HMGB1. The amount of O(2)(-) generated during FBI/R correlated with malondialdehyde, HMGB1, and ICAM1 in both the brain and plasma. In conclusion, the cholinergic agonist physostigmine suppressed jugular venous O(2)(-) generation, oxidative stress, early inflammation, and endothelial activation in the brain and plasma in the acute phase of cerebral ischemia/reperfusion. Therefore, the suppression of O(2)(-) is a key mechanism of the cholinergic anti-inflammatory pathway in the pathophysiology of cerebral ischemia/reperfusion.

Moderate hypothermia suppresses jugular venous superoxide anion radical, oxidative stress, early inflammation, and endothelial injury in forebrain ischemia/reperfusion rats.

The aim of this study was to assess the effect of moderate hypothermia (MH) on generation of jugular venous superoxide radical (O2-.), oxidative stress, early inflammation, and endothelial injury in forebrain ischemia/reperfusion (FBI/R) rats. Twenty-one Wistar rats were allocated to a control group (n=7, 37 degrees C), a pre-MH group (n=7, 32 degrees C before ischemia), and a post-MH group (n=7, 32 degrees C after reperfusion). MH was induced before induction of ischemia in the pre-MH group and just after reperfusion in the post-MH group. Forebrain ischemia was induced by occlusion of bilateral common carotid arteries with hemorrhagic hypotension for 10 min, followed by reperfusion. O(2)(-)(.) in the jugular vein was measured from the produced current using a novel O2-. sensor. The O2-. current showed a gradual increase during forebrain ischemia in the control and post-MH groups but was attenuated in the pre-MH group. Following reperfusion, the current showed a marked increase in the control group but was strongly attenuated in the pre- and post-MH groups. Concentrations of malondialdehyde, high-mobility group box 1 (HMGB1) protein, and intercellular adhesion molecule-1 (ICAM-1) in the brain and plasma 120 min after reperfusion in the pre- and post-MH groups were significantly lower than those in the control group, except for plasma HMGB1 in the post-MH group. In conclusion, MH suppressed O2-. measured in the jugular vein, oxidative stress, early inflammation, and endothelial injury in FBI/R rats.

Urinary trypsin inhibitor suppresses excessive superoxide anion radical generation in blood, oxidative stress, early inflammation, and endothelial injury in forebrain ischemia/reperfusion rats.

OBJECTIVES: To investigate the effects of ulinastatin, a urinary trypsin inhibitor (UTI), on jugular venous superoxide radical (O2⁻·) generation, oxidative stress, early inflammation, and endothelial activation in forebrain ischemia/reperfusion (FBI/R) rats. METHODS: Fourteen Wistar rats were allocated to a control group (n = 7) and a UTI group (n = 7). Throughout the experiments, O2⁻· in the jugular vein was measured by the produced current using a novel electrochemical O2⁻· sensor. Forebrain ischemia was induced by occlusion of the bilateral common caroti darteries with hemorrhagic hypotension for 20 min, followed by reperfusion. In the UTI group, UTI (5 U/g) was administered intravenously immediately after reperfusion. At 60 min after reperfusion, plasma and brain were harvested, and malondialdehyde, high-mobility group box 1 (HMGB1) protein, and intercellular adhesion molecule-1 (ICAM-1) were measured. RESULTS: O2⁻· current increased gradually during forebrain ischemia in both groups. The current increased markedly in the control group immediately after reperfusion but was significantly attenuated in the UTI group after reperfusion. Brain and plasma malondialdehyde, HMGB1, and ICAM-1 were significantly attenuated in the UTI group compared with those in the control group, except for brain HMGB1, which was associated with the amount of O2⁻· generated during FBI/R. DISCUSSION: UTI suppressed jugular venous O2⁻· generation, oxidative stress, early inflammation, and endothelial activation in FBI/R rats. Therefore, UTI might be a useful agent for the therapy of the cerebral ischemia/reperfusion pathophysiology.

Hyperoxia suppresses excessive superoxide anion radical generation in blood, oxidative stress, early inflammation, and endothelial injury in forebrain ischemia/reperfusion rats: laboratory study.

This study used an electrochemical O2. sensor to investigate the effects of hyperoxia on generation of the superoxide radical (O2.) in the jugular vein during forebrain I/R in rats. Twenty-eight male Wistar rats were allocated to a sham group (n = 7; sham-treated rats with inspired oxygen fraction [FiO2] of 0.4), a hemorrhagic shock and reperfusion (HS/R) group (n = 7; HS without carotid artery occlusion and reperfusion with FiO2 of 0.4), a normoxia group (n = 7; forebrain ischemia produced by bilateral carotid arteries occlusion with HS and reperfusion with FiO2 of 0.4), and a hyperoxia group (n = 7; forebrain ischemia with FiO2 of 0.4 and reperfusion with FiO2 of 1.0). The jugular venous O2. current was measured for 10 min during forebrain ischemia and for 120 min after reperfusion. The O2. current increased gradually during forebrain ischemia in the three groups other than the sham group. Immediately after reperfusion, the current showed a marked increase in the normoxia group and a pronounced decrease in the hyperoxia group. Levels of brain and plasma malondialdehyde, high-mobility group box 1 protein, and intercellular adhesion molecule 1 were significantly attenuated in the hyperoxia group relative to those in the normoxia group. In conclusion, hyperoxia suppressed jugular venous O2. generation and malondialdehyde, high-mobility group box 1, and intercellular adhesion molecule 1 in the brain and plasma in the acute phase of cerebral I/R. Thus, the administration of 100% oxygen immediately after reperfusion suppresses oxidative stress and early inflammation in cerebral I/R.

Activation of synoviolin promoter in rheumatoid synovial cells by a novel transcription complex of interleukin enhancer binding factor 3 and GA binding protein alpha.

OBJECTIVE: Synoviolin is an E3 ubiquitin ligase, and its overexpression is implicated in the pathogenesis of rheumatoid arthritis (RA). We reported previously that Ets binding site 1 (EBS-1) within the synoviolin promoter is crucial for the expression of synoviolin, and GA binding protein (GABP) binds to this site. This study was undertaken to elucidate the precise mechanisms of transcriptional regulation via EBS-1. METHODS: We performed purification and identification of complex components that bind to EBS-1 and inspected their contributions to the transcriptional regulation of synoviolin in rheumatoid synovial cells. We biochemically purified proteins that had EBS-1 binding activity and identified the proteins using liquid chromatography tandem mass spectrometry analysis. The identified proteins were verified to recruit and form the complex on EBS-1 using electrophoretic mobility shift assay and coimmunoprecipitation assay. Furthermore, their transcription activities were tested by reporter assays and RNA interference experiments. RESULTS: We identified interleukin enhancer binding factor 3 (ILF-3) as a novel factor in the complex. ILF-3 was demonstrated to activate the synoviolin promoter via association with GABPalpha in rheumatoid synovial cells. In addition, further activation was observed with ILF-2 and GABPbeta, previously reported interactants of ILF-3 and GABPalpha, respectively. Moreover, ILF-3-knockdown experiments showed reduced expression of the synoviolin gene. CONCLUSION: Our findings indicate that ILF-3, which has been known to regulate IL-2 expression in T cells, up-regulates synoviolin expression with GABPalpha in rheumatoid synovial cells. ILF-3 might be a target for RA treatment through its effect on IL-2 in T cells and synoviolin in rheumatoid synovial cells.

Early induction of moderate hypothermia suppresses systemic inflammatory cytokines and intracellular adhesion molecule-1 in rats with caerulein-induced pancreatitis and endotoxemia.

OBJECTIVES: To evaluate the systemic effects of moderate hypothermia (MH) and the timing of induction on acute pancreatitis (AP) and endotoxemia in rats. METHODS: The effects of MH were compared in 4 groups, that is, sham group (38 degrees C), control group (38 degrees C), early MH group (32 degrees C on administration of lipopolysaccharide [LPS]), and delayed MH group (32 degrees C 1 hour after LPS). AP and endotoxemia were induced by intramuscular injection of caerulein and intraperitoneal injection of LPS. RESULTS: Serum interleukin 6 (IL-6) in both MH groups was significantly lower than that in the control group at 3 hours. Serum interleukin 10 (IL-10) in the early MH group was significantly higher than those in the other 3 groups at 1 hour. IL-10/IL-6 ratios in both MH groups were significantly higher than that in the control group at 3 hours. Serum soluble intercellular adhesion molecule (sICAM-1) in both MH groups was significantly lower than that in the control group at 3 hours. Serum sICAM-1 in the early MH group was significantly lower than that in the delayed MH group. The tendency of pancreatic ICAM-1 was similar to that of serum sICAM-1. CONCLUSIONS: Early induction of MH might be protective against pancreatic injury and systemic inflammation in AP and endotoxemia.

Large-scale identification of proteins expressed in mouse embryonic stem cells.

A protein subset expressed in the mouse embryonic stem (ES) cell line, E14-1, was characterized by mass spectrometry-based protein identification technology and data analysis. In total, 1790 proteins including 365 potential nuclear and 260 membrane proteins were identified from tryptic digests of total cell lysates. The subset contained a variety of proteins in terms of physicochemical characteristics, subcellular localization, and biological function as defined by Gene Ontology annotation groups. In addition to many housekeeping proteins found in common with other cell types, the subset contained a group of regulatory proteins that may determine unique ES cell functions. We identified 39 transcription factors including Oct-3/4, Sox-2, and undifferentiated embryonic cell transcription factor I, which are characteristic of ES cells, 88 plasma membrane proteins including cell surface markers such as CD9 and CD81, 44 potential proteinaceous ligands for cell surface receptors including growth factors, cytokines, and hormones, and 100 cell signaling molecules. The subset also contained the products of 60 ES-specific and 41 stemness genes defined previously by the DNA microarray analysis of Ramalho-Santos et al. (Ramalho-Santos et al., Science 2002, 298, 597-600), as well as a number of components characteristic of differentiated cell types such as hematopoietic and neural cells. We also identified potential post-translational modifications in a number of ES cell proteins including five Lys acetylation sites and a single phosphorylation site. To our knowledge, this study provides the largest proteomic dataset characterized to date for a single mammalian cell species, and serves as a basic catalogue of a major proteomic subset that is expressed in mouse ES cells.

Cell surface labeling and mass spectrometry reveal diversity of cell surface markers and signaling molecules expressed in undifferentiated mouse embryonic stem cells.

Although interactions between cell surface proteins and extracellular ligands are key to initiating embryonic stem cell differentiation to specific cell lineages, the plasma membrane protein components of these cells are largely unknown. We describe here a group of proteins expressed on the surface of the undifferentiated mouse embryonic stem cell line D3. These proteins were identified using a combination of cell surface labeling with biotin, subcellular fractionation of plasma membranes, and mass spectrometry-based protein identification technology. From 965 unique peptides carrying biotin labels, we assigned 324 proteins including 235 proteins that have putative signal sequences and/or transmembrane segments. Receptors, transporters, and cell adhesion molecules were the major classes of proteins identified. Besides known cell surface markers of embryonic stem cells, such as alkaline phosphatase, the analysis identified 59 clusters of differentiation-related molecules and more than 80 components of multiple cell signaling pathways that are characteristic of a number of different cell lineages. We identified receptors for leukemia-inhibitory factor, interleukin 6, and bone morphogenetic protein, which play critical roles in the maintenance of undifferentiated mouse embryonic stem cells. We also identified receptors for growth factors/cytokines, such as fibroblast growth factor, platelet-derived growth factor, ephrin, Hedgehog, and Wnt, which transduce signals for cell differentiation and embryonic development. Finally we identified a variety of integrins, cell adhesion molecules, and matrix metalloproteases. These results suggest that D3 cells express diverse cell surface proteins that function to maintain pluripotency, enabling cells to respond to various external signals that initiate differentiation into a variety of cell types.