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As one of the most induced genes in activated macrophages, immune-responsive gene 1 (IRG1) encodes a mitochondrial metabolic enzyme catalysing the production of itaconic acid (ITA). Although ITA has an anti-inflammatory property, the underlying mechanisms are not fully understood. Here we show that ITA is a potent inhibitor of the TET-family DNA dioxygenases. ITA binds to the same site on TET2 as the co-substrate α-ketoglutarate, inhibiting TET2 catalytic activity. Lipopolysaccharide treatment, which induces Irg1 expression and ITA accumulation, inhibits Tet activity in macrophages. Transcriptome analysis reveals that TET2 is a major target of ITA in suppressing lipopolysaccharide-induced genes, including those regulated by the NF-κB and STAT signalling pathways. In vivo, ITA decreases the levels of 5-hydroxymethylcytosine, reduces lipopolysaccharide-induced acute pulmonary oedema as well as lung and liver injury, and protects mice against lethal endotoxaemia, depending on the catalytic activity of Tet2. Our study thus identifies ITA as an immune modulatory metabolite that selectively inhibits TET enzymes to dampen the inflammatory responses.

The liver is a metabolically active organ and is also 'tolerogenic', exhibiting sophisticated mechanisms of immune regulation that prevent pathogen attacks and tumorigenesis. How metabolism impacts the tumor microenvironment (TME) in hepatocellular carcinoma (HCC) remains understudied.We investigated the role of the metabolic regulator SIRT5 in HCC development by conducting metabolomic analysis, gene expression profiling, flow cytometry and immunohistochemistry analyses in oncogene-induced HCC mouse models and human HCC samples.We show that SIRT5 is downregulated in human primary HCC samples and that Sirt5 deficiency in mice synergizes with oncogenes to increase bile acid (BA) production, via hypersuccinylation and increased BA biosynthesis in the peroxisomes of hepatocytes. BAs act as a signaling mediator to stimulate their nuclear receptor and promote M2-like macrophage polarization, creating an immunosuppressive TME that favors tumor-initiating cells (TICs). Accordingly, high serum levels of taurocholic acid correlate with low SIRT5 expression and increased M2-like tumor-associated macrophages (TAMs) in HCC patient samples. Finally, administration of cholestyramine, a BA sequestrant and FDA-approved medication for hyperlipemia, reverses the effect of Sirt5 deficiency in promoting M2-like polarized TAMs and liver tumor growth.This study uncovers a novel function of SIRT5 in orchestrating BA metabolism to prevent tumor immune evasion and suppress HCC development. Our results also suggest a potential strategy of using clinically proven BA sequestrants for the treatment of patients with HCC, especially those with decreased SIRT5 and abnormally high BAs.Hepatocellular caricinoma (HCC) development is closely linked to metabolic dysregulation and an altered tumor microenvironment. Herein, we show that loss of the metabolic regulator Sirt5 promotes hepatocarcinogenesis, which is associated with abnormally elevated bile acids and subsequently an immunosuppressive microenvironment that favors HCC development. Targeting this mechanism could be a promising clinical strategy for HCC.

There is a need to investigate the role of nuclear factor kappa B in the regulation of cyclooxygenase-2 expression in the epileptic rat brain and cultured hippocampal neurons. Immunofluorescence and polymerase chain reaction was used to detect the expression of nuclear factor kappa B and cyclooxygenase-2. In cultured hippocampal neurons and rat brain: the control group compared with the normal group, nuclear factor kappa B expression in the hippocampal dentate gyrus, cerebral cortex, the piriform cortex brain regions were significantly increased (P < 0.01). This is accompanied by a significant increase in cyclooxygenase-2 protein and mRNA expressions in the hippocampus (P < 0.01). In the experimental group compared to the control group, the nuclear factor-kappa B expression in the hippocampal dentate gyrus, cerebral cortex, piriform cortex, and other brain regions was significantly lower (P < 0.01), with the accompanying decrease in cyclooxygenase-2 protein and mRNA expression (P < 0.01) in the hippocampus. In conclusion, κB-decoy can inhibit nuclear factor kappa B activation in epileptic rat brain and cyclooxygenase-2 overexpression.

G protein-coupled receptor 50 (GPR50) belongs to the G protein-coupled receptor which is highly homologous with the sequence of melatonin receptor MT1 and MT2. GPR50 expression has previously been reported in many brain regions, like cortex, midbrain, pons, amygdala. But, the distribution of GPR50 in the hippocampus and cortex and the cell types expressing GPR50 is not yet clear. In this study, we examined the distribution of GPR50 in adult male mice by immunofluorescence. Our results showed that GPR50 was localized in the CA1-3 pyramidal cells and the granule cells of the dentate gyrus. GPR50 was also expressed in excitatory and inhibitory neurons. As inhibitory neurons also contain many types, we found that GPR50 was localized in some interneurons in which it was co-expressed with the calcium-binding proteins calbindin, calretinin, and parvalbumin. Besides, similar results were seen in the cortex. The widespread expression of GPR50 in the hippocampus and cortex suggests that GPR50 may be associated with synaptic plasticity and cognitive function.

MicroRNAs, a class of small and non-encoding RNAs that transcriptionally or post-transcriptionally modulate the expression of their target genes, have been implicated as critical regulatory molecules in ischemia-/reperfusion-induced cardiac injury. In the present study, we report on the role of miR-208a in myocardial I/R injury and the underlying cardio-protective mechanism. The gain-of-function and loss-of-function were used to explore the effects of miR-208a on cardiac injury induced by H

Genipin is the major active component of Gardeniae fructus and has been shown to ameliorate diabetes and insulin resistance in rat models. In this study, we first investigated the effect of genipin on obesity and the related lipid metabolism mechanisms in diet-induced obese rats. Our results showed that genipin reduced body weight, food intake, and visceral fat mass; ameliorated dyslipidemia, glucose intolerance, insulin intolerance, adipocyte hypertrophy, and hepatic steatosis; and reduced serum tumor necrosis factor-α level in diet-induced obese rats. Quantitative real-time reverse-transcription polymerase chain reaction results further illustrated that genipin promoted lipolysis and β-oxidation of fatty acid by upregulating gene expressions of hormone-sensitive lipase and adipose triglyceride lipase in white adipose tissue (WAT) and peroxisome proliferator-activated receptor-α and carnitine palmitoyltransferase 1α in hepatic tissue. Moreover, genipin promoted browning of WAT by upregulating the mRNA and protein levels of uncoupling protein 1 and PRD1-BF1-RIZ1 homologous domain containing 16 in WAT. Additionally, genipin inhibited gene expressions of activin receptor-like kinase 7, tumor necrosis factor-α, and interlukin-6 in WAT. These results indicated that genipin had a potential therapeutic role in obesity, in which regulation of lipid mobilization and browning of WAT were involved.

Background Myocardial infarction (MI) remains the most common cause of heart failure (HF) worldwide. For almost 50 years, HF has been recognized as a determinant of adverse prognosis after MI, but efforts to promote myocardial repair have failed to be translated into clinical therapies. Aims In this study, we investigated the effects of BRD4 on cardiac function and the underlying mechanism. Material and methods The in vivo rat model of AMI and in vitro neonatal cardiomyocytes were established and cultured respectively, the BRD4 and NPPA/NPPB expression levels were detected by qPCR and Western blot, and interaction of BRD4 with acetylation RelA or NPPA/B promoters were examined by co-immunoprecipitation and chromatin immunoprecipitation assays, respectively. Results We found that BRD4 protein expression was significantly increased in cardiomyocytes of MI rat model and cardiomyocytes under hypoxia, accompanied by the expression of natriuretic peptide A (NPPA) and natriuretic peptide B (NPPB). Functionally, knockdown of BRD4 greatly downregulated the NPPA and NPPB in vivo and in vitro, improved the hemodynamic and biometric parameters in rat with heart failure, as well as decreased the apoptosis occurrence. In vitro studies further demonstrated that BRD4 bound with acetylated RelA to enhance the activation of NF-κb signaling, which resulted in activation of NPPA and NPPB transcriptions. Conclusions Taken together, our findings suggest that inhibition of BRD4 attenuated cardiomyocyte apoptosis via NF-κB pathway in myocardial infarction, and this study sheds light on developing new strategies to overcome myocardial damage.

The primary structure and posttranslational modifications of rabbit lens alpha-crystallins were examined using electrospray ionization mass spectrometry to determine the molecular weights of the intact proteins and fast atom bombardment mass spectrometry to analyze proteolytic digests of the alpha A- and alpha B-crystallins. The previously determined primary structure of alpha A-crystallin was confirmed. Posttranslational modifications detected included one phosphorylation site and the presence of a truncated form minus the five C-terminal residues. The previously undetermined amino acid sequence of rabbit alpha B-crystallin was determined to be the same as the bovine alpha B-crystallin sequence except at three residues: Thr 40, Thr 132, and Pro 153. Rabbit alpha B-crystallin showed evidence of phosphorylation at the same three sites as bovine alpha B-crystallin. The molecular weights of the intact proteins indicated that any one molecule had a maximum of two phosphorylations. Also, there was a truncated form which did not include the five C-terminal residues.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1- 14 C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH 3 . This rate of incorporation was 6–7-fold more rapid than that obtained with [[ 14 C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the cyrstallin subunits. [1- 14 ]Ascorbic acid-label led α-crystallin was separated into into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N ϵ -(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.

Although the precise mechanisms of transient hypotension after intravenous infusion of hypertonic saline (HTS) are not yet clarified, a rapid infusion of HTS is widely used as the initial therapy of hypovolemia. We investigated the effect of the intravenous infusion of a small dose of 0.97-9.7% NaCl solutions in anesthetized rats. Intravenous infusion of HTS at a rate of 0.3 ml/kg/min for 1 min produced the transient hypotension lasting for several minutes. The depressor response to HTS was not abolished by bilateral cervical vagotomy. The HTS infusion into the femoral vein evoked the depressor response with a larger magnitude and a shorter latency than that into the aortic arch. While the arterial baroreceptor pressure was kept constant at the baseline level of systemic arterial pressure, HTS-induced hypotension was significantly augmented. The gain factor of the arterial baroreflex was reduced by intravenous HTS. Pretreatment with bretylium tosylate completely abolished the depressor response without affecting the baseline level of arterial pressure. These results suggest that the depressor response to the very small dose of intravenous HTS is the sympathosympathetic neural reflex with cardiopulmonary afferents and vasomotor efferents.

Fungi in the genus Malassezia are ubiquitous skin residents of humans and other warm-blooded animals. Malassezia are involved in disorders including dandruff and seborrheic dermatitis, which together affect >50% of humans. Despite the importance of Malassezia in common skin diseases, remarkably little is known at the molecular level. We describe the genome, secretory proteome, and expression of selected genes of Malassezia globosa . Further, we report a comparative survey of the genome and secretory proteome of Malassezia restricta , a close relative implicated in similar skin disorders. Adaptation to the skin environment and associated pathogenicity may be due to unique metabolic limitations and capabilities. For example, the lipid dependence of M. globosa can be explained by the apparent absence of a fatty acid synthase gene. The inability to synthesize fatty acids may be complemented by the presence of multiple secreted lipases to aid in harvesting host lipids. In addition, an abundance of genes encoding secreted hydrolases (e.g., lipases, phospholipases, aspartyl proteases, and acid sphingomyelinases) was found in the M. globosa genome. In contrast, the phylogenetically closely related plant pathogen Ustilago maydis encodes a different arsenal of extracellular hydrolases with more copies of glycosyl hydrolase genes. M. globosa shares a similar arsenal of extracellular hydrolases with the phylogenetically distant human pathogen, Candida albicans , which occupies a similar niche, indicating the importance of host-specific adaptation. The M. globosa genome sequence also revealed the presence of mating-type genes, providing an indication that Malassezia may be capable of sex.

Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.

Cytotoxic T-lymphocytes are critical in the clearance of chronic viral infections such as HTLV-1. Peptide-based vaccines may have potential application in invoking antiviral CTL responses. In the development of vaccination strategies, it is becoming increasingly important to elicit a broad immune response against several epitopes simultaneously that may provide large population coverage. In the present study, we addressed this issue, namely the processing and presentation of multiple CTL epitopes simultaneously for the generation of multispecific CTL responses. We designed a novel multivalent peptide consisting of three HLA-A(*)0201 restricted CTL epitopes, with intervening double arginine residues in tandem. These epitopes were derived from the HTLV-1 regulatory protein Tax, which is an attractive target for vaccine development against HTLV-1. Arginine residues were included to provide cleavage sites for proteasomes, to generate the intended MHC Class I ligands. Proteasomal digestion studies and mass spectrometry analysis showed cleavage of the multivalent construct to generate the individual epitopes. Immunization of HLA-A(*)0201 transgenic mice with this construct efficiently elicited cellular responses to each intended epitope in vivo, further validating the applicability of this approach. These data may have potential in the development of immunotherapeutic strategies for the treatment of HTLV-1 disease and in the future design of multivalent subunit peptide vaccines.

A proteomic analysis was performed comparing normal slow twitch type fiber rat soleus muscle and normal fast twitch type fiber tibialis anterior muscle to immobilized soleus and tibialis anterior muscles at 0.5, 1, 2, 4, 6, 8 and 10 days post immobilization. Muscle mass measurements demonstrate mass changes throughout the period of immobilization. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 17 proteins. Proteomic analysis of normal and atrophied tibialis anterior muscle demonstrated statistically significant changes in the relative levels of 45 proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both soleus and tibialis anterior muscles. Four differentially regulated soleus proteins and six differentially regulated tibialis anterior proteins were identified. The identified proteins can be grouped according to function as metabolic proteins, chaperone proteins, and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the proteome occur during immobilization-induced atrophy in both slow twitch and fast twitch fiber type skeletal muscle.

A proteomic analysis was performed comparing normal rat soleus muscle to soleus muscle that had undergone either 0.5, 1, 2, 4, 7, 10 and 14 days of hindlimb suspension-induced atrophy or hindlimb suspension-induced atrophied soleus muscle that had undergone 1 hour, 8 hour, 1 day, 2 day, 4 day and 7 days of reweighting-induced hypertrophy. Muscle mass measurements demonstrated continual loss of soleus mass occurred throughout the 21 days of hindlimb suspension; following reweighting, atrophied soleus muscle mass increased dramatically between 8 hours and 1 day post reweighting. Proteomic analysis of normal and atrophied soleus muscle demonstrated statistically significant changes in the relative levels of 29 soleus proteins. Reweighting following atrophy demonstrated statistically significant changes in the relative levels of 15 soleus proteins. Protein identification using mass spectrometry was attempted for all differentially regulated proteins from both atrophied and hypertrophied soleus muscle. Five differentially regulated proteins from the hindlimb suspended atrophied soleus muscle were identified while five proteins were identified in the reweighting-induced hypertrophied soleus muscles. The identified proteins could be generally grouped together as metabolic proteins, chaperone proteins and contractile apparatus proteins. Together these data demonstrate that coordinated temporally regulated changes in the skeletal muscle proteome occur during disuse-induced soleus muscle atrophy and reweighting hypertrophy.

A combination of mass spectrometric techniques has been used to investigate the amino acid sequence and post-translational modifications of alpha B-crystallin isolated from bovine lenses by gel filtration chromatography and reversed-phase high performance liquid chromatography. Chromatographic fractions were analyzed by electrospray ionization mass spectrometry to determine the homogeneity and molecular weights of proteins in the fractions. The alpha B-crystallin primary gene product, its mono- and diphosphorylated forms, its N- and C-terminal truncated forms, as well as other lens proteins unrelated to the alpha B-crystallins were identified by their molecular weights. Detailed information about the sites of phosphorylation, as well as evidence supporting reassignment of Asn to Asp at position 80, was obtained by analyzing proteolytic digests of these proteins by fast atom bombardment mass spectrometry. Results of this investigation indicate that alpha B-crystallin is phosphorylated in vivo at Ser 45, Ser 59, and either Ser 19 or 21. From the specificity of phosphorylation of alpha-crystallins, it appears that there may be two different kinases responsible for their phosphorylation.

A new HPLC electrochemical detector that can be used to detect selectively and simultaneously both thiol- and disulfide-containing peptides is described. One electrode responds only to thiol-containing peptides, while a second electrode located downstream from a third electrode responds to thiol- as well as disulfide-containing peptides. All three electrodes are located in a single block and sample the HPLC effluent simultaneously. Since the electrochemical detector responds only to peptides that contain thiol or disulfide functionalities, it is ideal for detecting these types of peptides when they are present in complex mixtures. Peptides can be identified by their retention times if reference peptides are available, or by their molecular weights if the appropriate chromatographic fractions are collected and analyzed by mass spectrometry. The utility of the three-electrode electrochemical detector for detecting thiol- and disulfide-containing peptides in a single chromatographic analysis is illustrated with proteolytic digests of bovine alpha A-crystallin and partially reduced bovine insulin.

A new procedure to selectively identify disulfide-containing peptides in extracts of biological tissues is described. Disulfide-containing peptides are detected by their UV absorbance and electrochemical (EC) activity after chromatographic separation, and subsequently identified by fast atom bombardment mass spectrometry (FABMS). This combination of fractionation by HPLC and selective detection is attractive because it is rapid, highly specific for disulfide-containing peptides, and applicable to all disulfide-containing peptides that may be present in complex biological mixtures. Useful procedures for applying the method are demonstrated with tissue extracts from bovine pituitary and catfish pancreas. In addition to finding the expected disulfide-containing peptides, evidence for two forms of catfish insulin are presented. The merits of this and other methods used to detect peptides in similar tissue extracts are discussed.