Epigenetic regulation of Thy‐1 gene expression by histone modification is involved in lipopolysaccharide‐induced lung fibroblast proliferation

Lipopolysaccharide (LPS)‐induced pulmonary fibrosis is characterized by aberrant proliferation and activation of lung fibroblasts. Epigenetic regulation of thymocyte differentiation antigen 1 (Thy‐1) is associated with lung fibroblast phenotype transformation that results in aberrant cell proliferation. However, it is not clear whether the epigenetic regulation of Thy‐1 expression is required for LPS‐induced lung fibroblast proliferation. To address this issue and better understand the relative underlying mechanisms, we used mouse lung fibroblasts as model to observe the changes of Thy‐1 expression and histone deacetylation after LPS challenge. The results showed that cellular DNA synthesis, measured by BrdU incorporation, was impacted less in the early stage (24 hrs) after the challenge of LPS, but significantly increased at 48 or 72 hrs after the challenge of LPS. Meanwhile, Thy‐1 expression, which was detected by real‐time PCR and Western blot, in lung fibroblasts decreased with increased time after LPS challenge and diminished at 72 hrs. We also found that the acetylation of either histone H3 or H4 decreased in the LPS‐challenged lung fibroblasts. ChIP assay revealed that the acetylation of histone H4 (Ace‐H4) decreased in the Thy‐1 promoter region in response to LPS. In addition, all the above changes could be attenuated by depletion of TLR4 gene. Our studies indicate that epigenetic regulation of Thy‐1 gene expression by histone modification is involved in LPS‐induced lung fibroblast proliferation.     

Comparative analysis of soybean plasma membrane proteins under osmotic stress using gel‐based and LC MS/MS‐based proteomics approaches

To study the soybean plasma membrane proteome under osmotic stress, two methods were used: a gel‐based and a LC MS/MS‐based proteomics method. Two‐day‐old seedlings were subjected to 10% PEG for 2 days. Plasma membranes were purified from seedlings using a two‐phase partitioning method and their purity was verified by measuring ATPase activity. Using the gel‐based proteomics, four and eight protein spots were identified as up‐ and downregulated, respectively, whereas in the nanoLC MS/MS approach, 11 and 75 proteins were identified as up‐ and downregulated, respectively, under PEG treatment. Out of osmotic stress responsive proteins, most of the transporter proteins and all proteins with high number of transmembrane helices as well as low‐abundance proteins could be identified by the LC MS/MS‐based method. Three homologues of plasma membrane H⁺‐ATPase, which are transporter proteins involved in ion efflux, were upregulated under osmotic stress. Gene expression of this protein was increased after 12 h of stress exposure. Among the identified proteins, seven proteins were mutual in two proteomics techniques, in which calnexin was the highly upregulated protein. Accumulation of calnexin in plasma membrane was confirmed by immunoblot analysis. These results suggest that under hyperosmotic conditions, calnexin accumulates in the plasma membrane and ion efflux accelerates by upregulation of plasma membrane H⁺‐ATPase protein.     

Differential regulation of CD44 expression by lipopolysaccharide (LPS) and TNF-alpha in human monocytic cells: distinct involvement of c-Jun N-terminal kinase in LPS-induced CD44 expression

Alterations in the regulation of CD44 expression play a critical role in modulating cell adhesion, migration, and inflammation. LPS, a bacterial cell wall component, regulates CD44 expression and may modulate CD44-mediated biological effects in monocytic cells during inflammation and immune responses. In this study, we show that in normal human monocytes, LPS and LPS-induced cytokines IL-10 and TNF-alpha enhance CD44 expression. To delineate the mechanism underlying LPS-induced CD44 expression, we investigated the role of the mitogen-activated protein kinases (MAPKs), p38, p42/44 extracellular signal-regulated kinase, and c-Jun N-terminal kinase (JNK) by using their specific inhibitors. We demonstrate the involvement, at least in part, of p38 MAPK in TNF-alpha-induced CD44 expression in both monocytes and promonocytic THP-1 cells. However, neither p38 nor p42/44 MAPKs were involved in IL-10-induced CD44 expression in monocytes. To further dissect the TNF-alpha and LPS-induced signaling pathways regulating CD44 expression independent of IL-10-mediated effects, we used IL-10 refractory THP-1 cells as a model system. Herein, we show that CD44 expression induced by the LPS-mediated pathway predominantly involved JNK activation. This conclusion was based on results derived by transfection of THP-1 cells with a dominant-negative mutant of stress-activated protein/extracellular signal-regulated kinase kinase 1, and by exposure of cells to JNK inhibitors dexamethasone and SP600125. All these treatments prevented CD44 induction in LPS-stimulated, but not in TNF-alpha-stimulated, THP-1 cells. Furthermore, we show that CD44 induction may involve JNK-dependent early growth response gene activation in LPS-stimulated monocytic cells. Taken together, these results suggest a predominant role of JNK in LPS-induced CD44 expression in monocytic cells.     

Quantitative proteomics analysis of BMS‐214662 effects on CD34 positive cells from chronic myeloid leukaemia patients

Chronic myeloid leukaemia (CML) arises in a haemopoietic stem cell and is driven by the Bcr‐Abl oncoprotein. Abl kinase inhibitors (protein tyrosine kinase inhibitors) represent standard treatment for CML and induce remission in the majority of patients with early disease, however these drugs do not target leukaemic stem cells (LSCs) effectively, thus preventing cure. Previously, we identified the farnesyl transferase inhibitor BMS‐214662 as a selective inducer of apoptosis in LSCs of CML patients relative to normal controls; however, the mechanism underlying LSC‐specific apoptosis remains unclear. To identify pathways involved in the favourable effects of BMS‐214662 in CML, we employed a proteomic approach (based on iTRAQ) to analyse changes in protein expression in response to drug treatment in the nuclear and cytoplasmic fractions of CD34⁺ CML cells. The study identified 88 proteins as altered after drug treatment, which included proteins known to be involved in nucleic acid metabolism, oncogenesis, developmental processes and intracellular protein trafficking. We found that expression of Ebp1, a negative regulator of proliferation, was upregulated in the nucleus of BMS‐214662‐treated cells. Furthermore, proteins showing altered levels in the cytosol, such as histones, were predominantly derived from the nucleus and BMS‐214662 affected expression levels of nuclear pore complex proteins. Validation of key facets of these observations suggests that drug‐induced alterations in protein localisation, potentially via loss of nuclear membrane integrity, contributes to the LSC specificity of BMS‐214662, possibly via Ran proteins as targets.     

Mechanisms of LPS-induced CD40 expression in human peripheral blood monocytic cells

CD40 plays important roles in cell-mediated and humoral immune responses. In this study, we explored mechanisms underlying lipopolysaccharide (LPS)-induced CD40 expression in purified human peripheral blood monocytic cells (PBMCs) from healthy volunteers. Exposure to LPS induced increases in CD40 mRNA and protein expression on PBMCs. LPS stimulation caused IκBα degradation. Inhibition of NFκB activation abrogated LPS-induced CD40 expression. LPS stimulation also resulted in phosphorylation of mitogen-activated protein kinases, however, only Jun N-terminal kinase (JNK) was partially involved in LPS-induced CD40 expression. In addition, LPS exposure resulted in elevated interferon γ (IFNγ) levels in the medium of PBMCs. Neutralization of IFNγ and IFNγ receptor using specific antibodies blocked LPS-induced CD40 expression by 44% and 37%, respectively. In summary, LPS-induced CD40 expression on human PBMCs through activation of NFκB and JNK, and partially through the induction of IFNγ production.     

Plasma Membrane Protein Profiling in Beta‐Amyloid‐Treated Microglia Cell Line

In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi‐stage aqueous two‐phase partition system. We were able to identify by 1D‐LC‐MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24‐h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity‐regulating kinase 3 (MARK3), Interferon‐induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin‐1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate.     

Regulation of lipopolysaccharide‐induced inflammatory response and endotoxemia by β‐arrestins

β‐Arrestins are scaffolding proteins implicated as negative regulators of TLR4 signaling in macrophages and fibroblasts. Unexpectedly, we found that β‐arrestin‐1 (β‐arr‐1) and ‐2 knockout (KO) mice are protected from TLR4‐mediated endotoxic shock and lethality. To identify the potential mechanisms involved, we examined the plasma levels of inflammatory cytokines/chemokines in the wild‐type (WT) and β‐arr‐1 and ‐2 KO mice after lipopolysaccharide (LPS, a TLR4 ligand) injection. Consistent with lethality, LPS‐induced inflammatory cytokine levels in the plasma were markedly decreased in both β‐arr‐1 and ‐2 KO, compared to WT mice. To further explore the cellular mechanisms, we obtained splenocytes (separated into CD11^b+ and CD11^b? populations) from WT, β‐arr‐1, and ‐2 KO mice and examined the effect of LPS on cytokine production. Similar to the in vivo observations, LPS‐induced inflammatory cytokines were significantly blocked in both splenocyte populations from the β‐arr‐2 KO compared to the WT mice. This effect in the β‐arr‐1 KO mice, however, was restricted to the CD11^b? splenocytes. Our studies further indicate that regulation of cytokine production by β‐arrestins is likely independent of MAPK and IκBα‐NFκB pathways. Our results, however, suggest that LPS‐induced chromatin modification is dependent on β‐arrestin levels and may be the underlying mechanistic basis for regulation of cytokine levels by β‐arrestins in vivo. Taken together, these results indicate that β‐arr‐1 and ‐2 mediate LPS‐induced cytokine secretion in a cell‐type specific manner and that both β‐arrestins have overlapping but non‐redundant roles in regulating inflammatory cytokine production and endotoxic shock in mice. J. Cell. Physiol. 225: 406–416, 2010. © 2010 Wiley‐Liss, Inc.     

TLR4‐dependent induction of vascular adhesion molecule‐1 in rheumatoid arthritis synovial fibroblasts: Roles of cytosolic phospholipase A2α/cyclooxygenase‐2

Lipopolysaccharide (LPS)/Toll‐like receptor 4 (TLR4)‐mediated signaling pathways have caught the attention of strategies designed for rheumatoid arthritis (RA). In this study, we identified that cPLA₂α acted as a modulator of LPS‐induced VCAM‐1 expression and THP‐1 (human acute monocytic leukemia cell line) adherence. Treatment of RA synovial fibroblasts (RASFs) with LPS, a TLR4 agonist, promoted the VCAM‐1 expression and THP‐1 adherence which were decreased by pretreatment with a selective cytosolic phospholipase A₂ (cPLA₂) inhibitor (AACOCF₃), implying the involvement of cPLA₂α in these responses. This notion was further confirmed by knockdown of cPLA₂α expression by transfection with cPLA₂α small interfering RNA (siRNA) leading to a decrease in VCAM‐1 expression and THP‐1 adherence induced by LPS. Subsequently, the LPS‐stimulated cPLA₂α phosphorylation was attenuated by pretreatment with a MEK1/2 inhibitor (U0126), suggesting that LPS‐stimulated cPLA₂α phosphorylation and activity are mediated through an ERK‐dependent mechanism. Moreover, COX‐2‐derived PGE₂ production appeared to involve in LPS‐induced VCAM‐1 expression which was attenuated by pretreatment with selective COX‐2 inhibitors (NS‐398 and celecoxib), transfection with COX‐2 siRNA, or PGE₂ receptor antagonists. In addition, pretreatment with ecosapentaenoic acid (EPA), a substrate competitor of arachidonic acid (AA), also blocked LPS‐induced VCAM‐1 mRNA and protein expression, and THP‐1 adherence. Collectively, these results suggest that LPS‐induced VCAM‐1 expression and adhesion of THP‐1 cells are mediated through the TLR4/ERK/cPLA₂α phosphorylation and COX‐2 expression/PGE₂ synthesis in RASFs. J. Cell. Physiol. 223: 480–491, 2010. © 2010 Wiley‐Liss, Inc.     

Site‐specific and endothelial‐mediated dysfunction of the alveolar‐capillary barrier in response to lipopolysaccharides

Infectious agents such as lipopolysaccharides (LPS) challenge the functional properties of the alveolar‐capillary barrier (ACB) in the lung. In this study, we analyse the site‐specific effects of LPS on the ACB and reveal the effects on the individual cell types and the ACB as a functional unit. Monocultures of H441 epithelial cells and co‐cultures of H441 with endothelial cells cultured on Transwells^® were treated with LPS from the apical or basolateral compartment. Barrier properties were analysed by the transepithelial electrical resistance (TEER), by transport assays, and immunostaining and assessment of tight junctional molecules at protein level. Furthermore, pro‐inflammatory cytokines and immune‐modulatory molecules were evaluated by ELISA and semiquantitative real‐time PCR. Liquid chromatography–mass spectrometry‐based proteomics (LS‐MS) was used to identify proteins and effector molecules secreted by endothelial cells in response to LPS. In co‐cultures treated with LPS from the basolateral compartment, we noticed a significant reduction of TEER, increased permeability and induction of pro‐inflammatory cytokines. Conversely, apical treatment did not affect the barrier. No changes were noticed in H441 monoculture upon LPS treatment. However, LPS resulted in an increased expression of pro‐inflammatory cytokines such as IL‐6 in OEC and in turn induced the reduction of TEER and an increase in SP‐A expression in H441 monoculture, and H441/OEC co‐cultures after LPS treatment from basolateral compartment. LS‐MS‐based proteomics revealed factors associated with LPS‐mediated lung injury such as ICAM‐1, VCAM‐1, Angiopoietin 2, complement factors and cathepsin S, emphasizing the role of epithelial–endothelial crosstalk in the ACB in ALI/ARDS.     

MiR‐16, as a potential NF‐κB‐related miRNA,exerts anti‐inflammatory effects on LPS‐induced myocarditis via mediating CD40 expression: A preliminary study

The purpose of this study was to investigate the biological effect of miR‐16 on myocarditis and the underlying molecular mechanism. H9c2 cells were treated with 10 µg/mL lipopolysaccharide (LPS) for 12 hours to form a myocarditis injury model. We observed that LPS treatment distinctly decreased the level of miR‐16 in H9c2 cells. Upregulation of miR‐16 increased cell proliferation and reduced cell apoptosis. Then, CD40 was predicted and verified as a target gene of miR‐16 by TargetScan and luciferase reporter assay, respectively. Furthermore, the messenger RNA and protein expression of CD40 are negatively regulated by miR‐16. The relative expression of inflammatory factors was dramatically decreased by the miR‐16 mimic. Cells cotransfected with miR‐16 mimic and si‐CD40 could significantly abolish the injury of cardiomyocytes caused by myocarditis. Our study illustrated that the upregulation of miR‐16 has a protective effect on LPS‐damaged H9c2 cells, which may be achieved by regulating CD40 and the nuclear factor kappa B pathway.     

Quantitative milk proteomics – Host responses to lipopolysaccharide‐mediated inflammation of bovine mammary gland

Intramammary infusion of lipopolysaccharide (LPS) in cows induces udder inflammation that partly simulates mastitis caused by infection with Gram‐negative bacteria. We have used this animal model to characterize the quantitiative response in the milk proteome during the time course before and immediately after the LPS challenge. Milk samples from three healthy cows collected 3 h before the LPS challenge were compared with milk samples collected 4 and 7 h after the LPS challenge, making it possible to describe the inflammatory response of individual cows. Quantitative protein profiles were obtained for 80 milk proteins, of which 49 profiles changed significantly for the three cows during LPS challenge. New information obtained in this study includes the quantified increase of apolipoproteins and other anti‐inflammatory proteins in milk, which are important for the cow's ability to balance the immune response, and the upregulation of both complement C3 and C4 indicates that more than one complement pathway could be activated during LPS‐induced mastitis. In the future, this analytical approach may provide valuable information about the differences in the ability of individual cows to resist and recover from mastitis.     

New changes in the plasma‐membrane‐associated proteome of rice roots under salt stress

To gain a better understanding of salt stress responses in plants, we used a proteomic approach to investigate changes in rice (Oryza sativa) root plasma‐membrane‐associated proteins following treatment with 150 mmol/L NaCl. With or without a 48 h salt treatment, plasma membrane fractions from root tip cells of a salt‐sensitive rice cultivar, Wuyunjing 8, were purified by PEG aqueous two‐phase partitioning, and plasma‐membrane‐associated proteins were separated by IEF/SDS‐PAGE using an optimized rehydration buffer. Comparative analysis of three independent biological replicates revealed that the expressions of 18 proteins changed by more than 1.5‐fold in response to salt stress. Of these proteins, nine were up‐regulated and nine were down‐regulated. MS analysis indicated that most of these membrane‐associated proteins are involved in important physiological processes such as membrane stabilization, ion homeostasis, and signal transduction. In addition, a new leucine‐rich‐repeat type receptor‐like protein kinase, OsRPK1, was identified as a salt‐responding protein. Immuno‐blots indicated that OsRPK1 is also induced by cold, drought, and abscisic acid. Using immuno‐histochemical techniques, we determined that the expression of OsRPK1 was localized in the plasma membrane of cortex cells in roots. The results suggest that different rice cultivars might have different salt stress response mechanisms.     

Proteome of synaptosome‐associated proteins in spinal cord dorsal horn after peripheral nerve injury

Peripheral nerve injury may lead to neuroadaptive changes of cellular signals in spinal cord that are thought to contribute to central mechanisms underlying neuropathic pain. Here we used a 2‐DE‐based proteomic technique to determine the global expression changes of synaptosome‐associated proteins in spinal cord dorsal horn after unilateral fifth spinal nerve injury (SNI). The fifth lumbar dorsal horns ipsilateral to SNI or sham surgery were harvested on day 14 post‐surgery, and the total soluble and synaptosomal fractions were isolated. The proteins derived from the synaptosomal fraction were resolved by 2‐DE. We identified 27 proteins that displayed different expression levels after SNI, including proteins involved in transmission and modulation of noxious information, cellular metabolism, membrane receptor trafficking, oxidative stress, apoptosis, and degeneration. Six of the 27 proteins were chosen randomly and further validated in the synaptosomal fraction by Western blot analysis. Unexpectedly, Western blot analysis showed that only one protein in the total soluble fraction exhibited a significant expression change after SNI. The data indicate that peripheral nerve injury changes not only protein expression but also protein subcellular distribution in dorsal horn cells. These changes might participate in the central mechanism that underlies the maintenance of neuropathic pain.     

Lipopolysaccharide induces a fibrotic‐like phenotype in endothelial cells

Endothelial dysfunction is crucial in endotoxaemia‐derived sepsis syndrome pathogenesis. It is well accepted that lipopolysaccharide (LPS) induces endothelial dysfunction through immune system activation. However, LPS can also directly generate actions in endothelial cells (ECs) in the absence of participation by immune cells. Although interactions between LPS and ECs evoke endothelial death, a significant portion of ECs are resistant to LPS challenge. However, the mechanism that confers endothelial resistance to LPS is not known. LPS‐resistant ECs exhibit a fibroblast‐like morphology, suggesting that these ECs enter a fibrotic programme in response to LPS. Thus, our aim was to investigate whether LPS is able to induce endothelial fibrosis in the absence of immune cells and explore the underlying mechanism. Using primary cultures of ECs and culturing intact blood vessels, we demonstrated that LPS is a crucial factor to induce endothelial fibrosis. We demonstrated that LPS was able and sufficient to promote endothelial fibrosis, in the absence of immune cells through an activin receptor–like kinase 5 (ALK5) activity–dependent mechanism. LPS‐challenged ECs showed an up‐regulation of both fibroblast‐specific protein expression and extracellular matrix proteins secretion, as well as a down‐regulation of endothelial markers. These results demonstrate that LPS is a crucial factor in inducing endothelial fibrosis in the absence of immune cells through an ALK5‐dependent mechanism. It is noteworthy that LPS‐induced endothelial fibrosis perpetuates endothelial dysfunction as a maladaptive process rather than a survival mechanism for protection against LPS. These findings are useful in improving current treatment against endotoxaemia‐derived sepsis syndrome and other inflammatory diseases.     

Proteomic Profiling of LPS‐Induced Macrophage‐Derived Exosomes Indicates Their Involvement in Acute Liver Injury

Exosomes are typically involved in cellular communication and signaling. Macrophages play a key role in lipopolysaccharide (LPS)‐induced sepsis. However, the molecular comparison of exosomes derived from LPS‐induced macrophage has not been well analyzed. The macrophage‐exosomes are validated and the protein composition of those exosomes are investigated by isobaric tags for relative and absolute quantification (iTRAQ) mass spectrometry. A total of 5056 proteins are identified in macrophage‐exosomes. We discovered 341 increased proteins and 363 reduced proteins in LPS‐treated macrophage‐exosomes compared with control exosomes. In addition, gene ontology analysis demonstrates that macrophage‐exosomes proteins are mostly linked to cell, organelle, extracellular region, and membrane. The bioinformatics analysis also indicates that these proteins are mainly involved in cellular process, single‐organism process, metabolic process, and biological regulation. Among these 341 upregulated proteins, Kyoto Encyclopedia of Genes and Genomes analysis reveals that 22 proteins are involved in the NOD‐like receptor signaling pathway. Finally, hepatocytes can uptake macrophage‐exosomes and subsequently NLRP3 inflammasome is activated in vitro and in vivo. These data emphasize the fundamental importance of macrophage‐exosomes in sepsis‐induced liver injury. Therefore, the iTRAQ proteomic strategy brings new insights into macrophage‐derived exosomes. It may improve our understanding of macrophage‐exosomes’ functions and their possible use as therapeutic targets for sepsis.     

Comparative Proteomic Analyses of the Liver in D‐Galactosamine‐Sensitized Mice Treated with Different Toll‐Like Receptor Agonists

Acute liver failure (ALF) is a severe consequence of abrupt hepatocyte injury and has lethal outcomes. Three toll‐like receptor agonists, including polyinosinic‐polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS), and cytosine‐phosphate‐guanine (CpG) DNA, cause acute and severe hepatitis, respectively, in D‐galactosamine (D‐GalN)‐sensitized mice. However, the molecular differences among three ALF models (LPS/D‐GalN, poly(I:C)/D‐GalN, and CpG DNA/D‐GalN), are unclear. Here, tandem mass tag based quantitative proteomic analyses of three ALF mouse models are performed. 52 common differentially expressed proteins (DEPs) are identified, in three ALF groups, compared to the control. Gene ontology analyses show that among the common DEPs, ten proteins are involved in immune system process, and 39 proteins in metabolic process. Among 80,195, and 23 specifically‐expressed proteins in poly(I:C)/D‐GalN, LPS/D‐GalN, and CpG DNA/D‐GalN groups, LPS/D‐GalN‐specific proteins are mostly distributed in the endoplasmic reticulum and more enriched in metabolic pathways, whereas poly (I:C)/D‐GalN‐specific proteins are mainly in the membrane and CpG DNA/D‐GalN‐specific proteins are related to the ribosome structural composition. In conclusion, the common and specific DEPs in three ALF mouse models at molecular level are identified; and determined a close‐to‐complete reference map of mouse liver proteins which will be useful for clinical diagnosis and treatment of liver failure in humans.     

Expression of complement regulatory proteins [membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59)] in endometrial stressed cells

In the female reproductive tract, the complement system represents a defense mechanism that can act directly against pathogens and cells, and mediates inflammatory response. Endometrial cells are protected from autologous complement attack by membrane-bound complement regulatory proteins (CRPs) that prevent complement activation: membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59). In this work we show that all CRPs were overexpressed after LPS exposure. Maximal stimulatory effect was detected after 6h, and was declining after 12h, reaching control levels in 24h. CD59 was the protein showing the more prominent effect. There seems to be a slight increase of CRP expression in the endometrium of sterile patients that have anti-endometrial antibodies (AEA) in their serum. Our results suggest that under stress, the high expression of CRPs (CD46, CD55, and CD59) could protect endometrial injured cells against complement mediated lysis. The survival of these cells with some biochemical modifications would enable autoimmune response.     

iTRAQ proteomic analysis of N‐acetylmuramic acid mediated anti‐inflammatory capacity in LPS‐induced RAW 264.7 cells

Lactobacillus acidophilus probiotic bacteria have lasting beneficial health effects in the gastrointestinal tract, including protecting against pathogens, improving immunomodulation, and producing beneficial bacteria‐derived molecules. In lipopolysaccharide (LPS) induced RAW 264.7 cells treated with peptidoglycan or N‐acetylmuramic acid (NAM) from L. acidophilus, 390 differentially expressed proteins (8.76%) were identified by iTRAQ analysis, 257 (5.77%) of which were upregulated and 133 (2.99%) were downregulated under LPS‐induced conditions. Most of these proteins were grouped into the following inflammation‐related cellular signaling: lysosome pathway, calcium signaling pathway, and Toll‐like receptor (TLR) signaling pathway. Among them, clathrin, SERCA, and interleukin 1 receptor antagonist were differentially expressed to a significant degree in peptidoglycan or NAM pretreated RAW 264.7 cells. Bioinformatics analysis indicated that NAM may mediate an anti‐inflammatory process via a Ca²⁺‐dependent NF‐κB pathway. These observations reveal new insights into the molecular mechanisms involved in the suppression of LPS‐induced macrophage inflammation by L. acidophilus.     

Analysis of the rat hypothalamus proteome by data‐independent label‐free LC‐MS/MS

Studies of neuronal, endocrine, and metabolic disorders would be facilitated by characterization of the hypothalamus proteome. Protein extracts prepared from 16 whole rat hypothalami were measured by data‐independent label‐free nano LC‐MS/MS. Peptide features were detected, aligned, and searched against a rat Swiss‐Prot database using ProteinLynx Global Server v.2.5. The final combined dataset comprised 21 455 peptides, corresponding to 622 unique proteins, each identified by a minimum of two distinct peptides. The majority of the proteins (69%) were cytosolic, and 16% were membrane proteins. Important proteins involved in neurological and synaptic function were identified including several members of the Ras‐related protein family and proteins involved in glutamate biosynthesis.