Apoptosis inhibitor of macrophage (AIM) diminishes lipid droplet-coating proteins leading to lipolysis in adipocytes.

Under fasting conditions, triacylglycerol in adipose tissue undergoes lipolysis to supply fatty acids as energy substrates. Such lipolysis is regulated by hormones, which activate lipases via stimulation of specific signalling cascades. We previously showed that macrophage-derived soluble protein, AIM induces obesity-associated lipolysis, triggering chronic inflammation in fat tissue which causes insulin resistance. However, the mechanism of how AIM mediates lipolysis remains unknown. Here we show that AIM induces lipolysis in a manner distinct from that of hormone-dependent lipolysis, without activation or augmentation of lipases. In vivo and in vitro, AIM did not enhance phosphorylation of hormone-sensitive lipase (HSL) in adipocytes, a hallmark of hormone-dependent lipolysis activation. Similarly, adipose tissue from obese AIM-deficient and wild-type mice showed comparable HSL phosphorylation. Consistent with the suppressive effect of AIM on fatty acid synthase activity, the amount of saturated and unsaturated fatty acids was reduced in adipocytes treated with AIM. This response ablated transcriptional activity of peroxisome proliferator-activated receptor (PPARγ), leading to diminished gene expression of lipid-droplet coating proteins including fat-specific protein 27 (FSP27) and Perilipin, which are indispensable for triacylglycerol storage in adipocytes. Accordingly, the lipolytic effect of AIM was overcome by a PPARγ-agonist or forced expression of FSP27, while it was synergized by a PPARγ-antagonist. Overall, distinct modes of lipolysis appear to take place in different physiological situations; one is a supportive response against nutritional deprivation achieved by enhancing lipase activity, and the other is a pathological consequence of obesity, causing subclinical inflammation and metabolic disorders, mediated by abolishing droplet-coating proteins.

N (α) Selective Acetylation of Peptides.

A chemical tag at the peptide N-terminus, in combination with MS, can be useful for quantitative analysis, N-terminal peptide identification, or peptide sequencing. Here we report on the N (α) selective acetylation of a peptide using acetic anhydride, a popular reagent for the modification of amino groups, without the need for the blocking of lysine side-chain ε-amino groups, which is usually required for N (α) selective acetylation. By controlling the amount of acetic anhydride used and running the reaction at 0°C, it is possible to preferentially acetylate the α-amino group. As a typical application of the method, a tryptic digest of an N-terminally blocked protein, cytochrome c, was directly acetylated using the present method. When deuterated acetic anhydride was used as the reagent, the N-terminal blocked peptide could be easily identified as a non-labeled ion peak while the N (α)-acetyl groups of all the other peptides were deuterated.

The application of mass spectrometry to proteomics and metabolomics in biomarker discovery and drug development.

Drugs are launched to market after the lengthy process of development. Despite careful preclinical assessment, there is still a significant risk that a drug candidate may elicit adverse effects or display a low level of efficacy during clinical trials. If a drug candidate fails in the latter stages of the clinical process, the overall loss, both in terms of time and money, is enormous. A major concern for the pharmaceutical companies is to improve the drug development process to make it faster and more cost-effective by adoption of new technologies. Biomarkers are emerging as a key tool in identifying potential drug failures at an early stage or in helping to make go/no-go decisions, which should significantly accelerate drug development. Omics technologies play an important role in biomarker discovery as well as in other stages of the drug discovery and development (e.g. target discovery, mechanism of action or predicting toxicity). In particular, recent progress in mass spectrometry techniques such as selected reaction monitoring (SRM) and novel high-resolution features have helped facilitate the realization of the inherent power of proteomics and metabolomics in biomarker discovery, validation and qualification. In this manuscript, we review the current state of proteomics and metabolomics in conjunction with recent technical advances in mass spectrometry with some examples of applications in biomarker research. In addition, we discuss the possible impact of biomarker research with these technologies in drug discovery and development.

Adipokine ganglioside GM2 activator protein stimulates insulin secretion.

Recently, we identified ganglioside GM2 activator protein (GM2AP) as a novel adipokine, and revealed that treatment of cultured cells with GM2AP impairs insulin signal transduction. The aim of this study was to examine the impact of GM2AP on glucose metabolism in vivo. Injection of recombinant GM2AP in mice significantly lowered blood glucose levels in glucose tolerance tests. Administration of GM2AP to mice for 10 days increased serum insulin levels, whereas the contents of glucose, leptin and FFA were significantly decreased. Stimulation of calcium influx and insulin secretion by GM2AP was observed in hamster insulinoma HIT-T15 cells. Blockage of GM2AP function by specific antibodies inhibited GM2AP-induced insulin secretion. These results provide novel insights into the physiological functions of GM2AP in obesity.

Multilocus sequence typing implicates rodents as the main reservoir host of human-pathogenic Borrelia garinii in Japan.

Multilocus sequence typing of Borrelia garinii isolates from humans and comparison with rodent and tick isolates were performed. Fifty-nine isolates were divided into two phylogenetic groups, and an association was detected between clinical and rodent isolates, suggesting that, in Japan, human-pathogenic B. garinii comes from rodents via ticks.

A novel small compound that promotes nuclear translocation of YB-1 ameliorates experimental hepatic fibrosis in mice.

Transforming growth factor-ß (TGF-ß) is considered to be a major factor contributing to liver fibrosis. We have previously shown that nuclear translocation of YB-1 antagonizes the TGF-ß/Smad3 signaling in regulating collagen gene expression. More recently, we have demonstrated that the novel small compound HSc025 promotes nuclear translocation of YB-1, resulting in the improvement of skin and pulmonary fibrosis. Here, we presented evidence as to the mechanism by which HSc025 stimulates nuclear translocation of YB-1 and the pharmacological effects of HSc025 on a murine model of hepatic fibrosis. A proteomics approach and binding assays using HSc025-immobilized resin showed that HSc025 binds to the amino acid sequence within the C-tail region of YB-1. In addition, immunoprecipitation experiments and glutathione S-transferase pulldown assays identified poly(A)-binding protein (PABP) as one of the cytoplasmic anchor proteins of YB-1. HSc025 directly binds to YB-1 and interrupts its interaction with PABP, resulting in accelerated nuclear translocation of YB-1. Transfection of cells with PABP siRNA promoted nuclear translocation of YB-1 and subsequently inhibited basal and TGF-ß-stimulated collagen gene expression. Moreover, HSc025 significantly suppressed collagen gene expression in cultured activated hepatic stellate cells. Oral administration of HSc025 to mice with carbon tetrachloride-induced hepatic fibrosis improved liver injury as well as the degree of hepatic fibrosis. Altogether, the results provide a novel insight into therapy for organ fibrosis using YB-1 modulators.

A novel adipokine GM2AP impairs insulin signaling.

In an attempt to discover novel adipokines, we performed proteomics analyses using culture medium from differentiated 3T3-L1 adipocytes, and first identified GM2AP. The levels of GM2AP mRNA and protein were augmented by adipogenesis in cultured adipocytes and expression in adipose tissue and serum of obese mice or human subjects was found to be significantly higher than in lean counterparts. Exposure of 3T3-L1 adipocytes to GM2AP protein accelerated dissociation of insulin receptor-beta (IRß) from caveolin-1, and interrupted insulin signal transduction. Abrogation of GM2AP function by specific antibodies augmented glucose uptake. Furthermore, treatment of rat pheochromocytoma PC12 NS1 cells with GM2AP impaired NGF signal transduction. Taken together, these results provide novel insights into the physiological functions of GM2AP in obesity.

Hepatocyte growth factor suppresses profibrogenic signal transduction via nuclear export of Smad3 with galectin-7.

BACKGROUND & AIMS: Hepatocyte growth factor (HGF) and transforming growth factor-beta (TGF-beta) regulate diversified cellular functions and often act antagonistically against each other. For example, TGF-beta is the most potent factor accelerating liver fibrosis, whereas HGF treatment prevents its progression. Here, we propose a novel molecular mechanism by which HGF counter represses TGF-beta-stimulated profibrogenic signal transduction. METHODS: Effects of HGF on TGF-beta-responsive gene transcription of type I collagen, the major matrix component of fibrotic liver, were examined by using cultured hepatic stellate cells (HSC) and transgenic mice harboring alpha2(I) collagen gene (COL1A2) promoter. Expression and subcellular localization of Smad3 were determined by Western blot analyses and immunofluorescence staining, respectively. A mass spectrometric analysis was employed to identify immunoprecipitated proteins with antiphospho-Smad2/3 antibodies. RESULTS: Over expression of HGF inhibited COL1A2 transcription in cultured HSC and suppressed activation of COL1A2 promoter in liver tissue induced by carbon tetrachloride administration. A mass spectrometric analysis identified galectin-7 as one of the immunoprecipitated proteins with antiphospho-Smad2/3 antibodies following HGF treatment. HGF accelerated nuclear export of Smad3 by enhancing its interaction with galectin-7. Transfection of cells with galectin-7 small interfering RNA inhibited nuclear export of Smad3 and abolished suppressive effect of HGF on expression of TGF-beta-responsive genes such as COL1A2 and plasminogen activator inhibitor-1. On the other hand, over expression of galectin-7 suppressed TGF-beta-stimulated expression of those target genes. CONCLUSIONS: These results reveal a novel function of intracellular galectin-7 as a transcriptional regulator via its interaction with Smad3 and provide a molecular basis for the antifibrotic effect of HGF.

Selective isolation of N-blocked peptides by isocyanate-coupled resin.

We developed a method for selective isolation of N-blocked peptides from a complex mixture such as an enzymatic digest of a protein. The approach is based on a newly designed isocyanate-resin (resin-NCO), which specifically reacts with alpha-amino or imino groups. This method, then, permits the isolation of N-blocked peptides, even those containing Lys, from a peptide mixture as intact forms by trapping N-free peptides via covalent bonding to the resin-NCO. The present study demonstrates the performance of this method for the selective isolation of N-blocked peptides by applying it to several peptide mixtures, including proteolytic digests.

An outbreak of gastroenteritis during school trip caused by serotype G2 group A rotavirus.

Between May14 and 18, 2001, there was an outbreak of acute gastroenteritis involving 45 school children out of a total of 107 (aged 11-12 years) attending a 3-day school trip. The epidemic curve characterized by a rapid onset and decline with a single peak incidence over a 5-day period resembled the pattern typical of a food-borne gastroenteritis outbreak. Epidemiological and virological investigations concluded, however, that this outbreak was caused by a single strain of serotype G2 group A rotavirus spreading to schoolmates from the primary case-pupil who had already been ill at the start of the trip. Efficient person-to-person transmission was likely to have occurred due to prolonged and close contacts under the conditions typical of such school trips. This study emphasizes the importance of including group A rotavirus infection as a possible cause of gastroenteritis outbreaks even in older children and adults.

Ubiquitination-mediated regulation of biosynthesis of the adhesion receptor SHPS-1 in response to endoplasmic reticulum stress.

Misfolding of proteins during endoplasmic reticulum (ER) stress results in the formation of cytotoxic aggregates. The ER-associated degradation pathway counteracts such aggregation through the elimination of misfolded proteins by the ubiquitin-proteasome system. We now show that SHP substrate-1 (SHPS-1), a transmembrane glycoprotein that regulates cytoskeletal reorganization and cell-cell communication, is a physiological substrate for the Skp1-Cullin1-NFB42-Rbx1 (SCF(NFB42)) E3 ubiquitin ligase, a proposed mediator of ER-associated degradation. SCF(NFB42) mediated the polyubiquitination of immature SHPS-1 and its degradation by the proteasome. Ectopic expression of NFB42 both suppressed the formation of aggresome-like structures and the phosphorylation of the translational regulator eIF2alpha induced by overproduction of SHPS-1 as well as increased the amount of mature SHPS-1 at the cell surface. An NFB42 mutant lacking the F box domain had no such effects. Our results suggest that SCF(NFB42) regulates SHPS-1 biosynthesis in response to ER stress.

Detection, quantitation, and phylogenetic analysis of noroviruses in Japanese oysters.

Noroviruses (NVs) cause many cases of oyster- or clam-associated gastroenteritis in various countries. We collected 191 samples from Japanese oysters intended for raw consumption that had been harvested from the sea in two different areas between December 2001 and February 2002. To detect, quantitate, and phylogenetically analyze the NV genome in purified concentrates from the stomachs and digestive diverticula of these oysters, we amplified the NV capsid gene by reverse transcription-PCR. Phylogenetic analysis was performed by using the neighbor-joining method. We detected the NV genome in 17 of 191 oysters (9%). Phylogenetic analysis indicated genogroup I (Norwalk virus type) in 3 of the 17 oysters and genogroup II (Snow Mountain virus type) in the other 14. Both genogroups showed wide genetic diversity. To quantify the NV capsid gene in these oysters, we performed real-time PCR using genogroup-specific probes. More than 10(2) copies of the NV genome were detected in 11 of 17 oysters. The results suggested that about 10% of Japanese oysters intended for raw consumption harbored NVs, and more than 50% of those oysters in which NVs were detected had a large amount.