First observation of N-acetyl leucine and N-acetyl isoleucine in diabetic patient hair and quantitative analysis by UPLC-ESI-MS/MS.

BACKGROUND: Type 2 diabetes patients (DP) have significantly higher plasma levels of valine, leucine, isoleucine and alanine than the controls. Specific amino acids may acutely and chronically regulate insulin secretion from the pancreatic ß-cells. We recently identified a metabolic signature of N-acetyl leucine (Ac-Leu) that strongly predicts diabetes development in mice hair. The Ac-Leu appears to be a potential biomarker candidate related to diabetes. However, the determination of Ac-Leu in human hair has not been reported. We measured the Ac-Leu, and its structure is similar to N-acetyl isoleucine (Ac-Ile) in human hair by ultra-performance liquid chromatography (UPLC) with electrospray ionization tandem mass spectrometry (ESI-MS/MS). The developed method was applied to the determination of Ac-Leu and Ac-Ile in the hair of healthy volunteers (HV) and DP. METHODS: Ac-Leu, Ac-Ile and N-acetyl norleucine (Ac-Nle, IS) were extracted from human hair samples by a micropulverized extraction procedure, then separated on a C18 column by isocratic elution of acetonitrile-0.1% formic acid in water:0.1% formic acid (14:86, vol./vol.). MRM using the fragmentation transitions of m/z 174.1→86.1 in the positive ESI mode was performed to quantify the N-acetyl leucine, N-acetyl isoleucine and IS. RESULTS: Ac-Leu, Ac-Ile and Ac-Nle in the human hair samples were completely separated by isocratic elution of a 5.0 min duration wash program using a reversed-phase column, and sensitively detected by LC-MS/MS in the ESI(+) MRM mode. The amounts of Ac-Leu and Ac-Ile in the hairs of HV and DP were determined. When comparing the concentrations between DP and those from HV, a statistically significant correlation was observed for the Ac-Leu (p<0.001) and Ac-Ile (p<0.01). CONCLUSIONS: The proposed method is useful for the determination of Ac-Leu and Ac-Ile in the hairs of DP and HV. Human hair may serve as a noninvasive biosample for the diagnosis of diabetes.

Gene network inference and visualization tools for biologists: application to new human transcriptome datasets.

Gene regulatory networks inferred from RNA abundance data have generated significant interest, but despite this, gene network approaches are used infrequently and often require input from bioinformaticians. We have assembled a suite of tools for analysing regulatory networks, and we illustrate their use with microarray datasets generated in human endothelial cells. We infer a range of regulatory networks, and based on this analysis discuss the strengths and limitations of network inference from RNA abundance data. We welcome contact from researchers interested in using our inference and visualization tools to answer biological questions.

Development of novel active acceptors possessing a positively charged structure for the transglycosylation reaction with Endo-M and their application to oligosaccharide analysis.

With Boc-Asn-GlcNAc as a basic structure, four permanently positively charged kinds of new acceptors (GP-Boc-Asn-GlcNAc, GT-Boc-Asn-GlcNAc, HMP-Boc-Asn-GlcNAc, MPDPZ-Boc-Asn-GlcNAc) and five kinds of similar structure acceptors (2-PA-Boc-Asn-GlcNAc, 3-PA-Boc-Asn-GlcNAc, 4-PA-Boc-Asn-GlcNAc, HP-Boc-Asn-GlcNAc, PDPZ-Boc-Asn-GlcNAc) were synthesized as acceptors for the resolution of oligosaccharides in glycopeptides. The synthesized acceptors enzymatically reacted with Disialo-Asn (donor) in the presence of Endo-M. The reaction yields of each transglycosylation product were not obvious, because we do not have all the authentic Disialo-Asn-Boc-acceptors. Therefore, we used the peak area of the transglycosylation product detected by mass spectrometry and evaluated the utility of each acceptor. Among the Boc-Asn-GlcNAc acceptors, the positively charged MPDPZ derivative peak area was the highest, MPDPZ-Boc-Asn-GlcNAc with a positively charged structure showed about a 2.2 times greater sensitivity of the transglycosylation product compared to the conventional fluorescence acceptor DBD-PZ-Boc-Asn-GlcNAc. As a result, the MPDPZ-Boc-Asn-GlcNAc acceptor was suitable for the transglycosylation reaction with Endo-M. The development of a qualitative determination method for the N-linked oligosaccharides in glycoproteins was attempted by combination of the transglycosylation reaction and semi-micro high-performance liquid chromatography/electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC/ESI-QTOF-MS/MS). The asparaginyl-oligosaccharides in glycoproteins, liberated by treatment with Pronase E, were separated, purified and labeled with positively charged MPDPZ. The resulting derivatives were separated by a semi-micro HPLC system. The eluted N-linked oligosaccharide derivatives were then introduced into a QTOF-MS instrument and sensitively detected in the ESI(+) mode. Various fragment ions based on the carbohydrate units appeared in the MS/MS spectra. Among the peaks, m/z 782.37 corresponding to MPDPZ-Boc-Asn-GlcNAc is the most important one for identifying the asparaginyl-oligosaccharides. Disialo-Asn-Boc-MPDPZ was easily identified by the selected-ion chromatogram at m/z 782.37 by MS/MS detection. Therefore, the identification of N-linked oligosaccharides in glycoproteins seems to be possible by the proposed semi-micro HPLC separations followed by the QTOF-MS/MS detection. Furthermore, several oligosaccharides in ovalbumin and ribonuclease B were successfully identified by the proposed procedure.

Analysis of PPARalpha-dependent and PPARalpha-independent transcript regulation following fenofibrate treatment of human endothelial cells.

Fenofibrate is a synthetic ligand for the nuclear receptor peroxisome proliferator-activated receptor (PPAR) alpha and has been widely used in the treatment of metabolic disorders, especially hyperlipemia, due to its lipid-lowering effect. The molecular mechanism of lipid-lowering is relatively well defined: an activated PPARalpha forms a PPAR-RXR heterodimer and this regulates the transcription of genes involved in energy metabolism by binding to PPAR response elements in their promoter regions, so-called "trans-activation". In addition, fenofibrate also has anti-inflammatory and anti-athrogenic effects in vascular endothelial and smooth muscle cells. We have limited information about the anti-inflammatory mechanism of fenofibrate; however, "trans-repression" which suppresses production of inflammatory cytokines and adhesion molecules probably contributes to this mechanism. Furthermore, there are reports that fenofibrate affects endothelial cells in a PPARalpha-independent manner. In order to identify PPARalpha-dependently and PPARalpha-independently regulated transcripts, we generated microarray data from human endothelial cells treated with fenofibrate, and with and without siRNA-mediated knock-down of PPARalpha. We also constructed dynamic Bayesian transcriptome networks to reveal PPARalpha-dependent and -independent pathways. Our transcriptome network analysis identified growth differentiation factor 15 (GDF15) as a hub gene having PPARalpha-independently regulated transcripts as its direct downstream children. This result suggests that GDF15 may be PPARalpha-independent master-regulator of fenofibrate action in human endothelial cells.

Unraveling dynamic activities of autocrine pathways that control drug-response transcriptome networks.

Some drugs affect secretion of secreted proteins (e.g. cytokines) released from target cells, but it remains unclear whether these proteins act in an autocrine manner and directly effect the cells on which the drugs act. In this study, we propose a computational method for testing a biological hypothesis: there exist autocrine signaling pathways that are dynamically regulated by drug response transcriptome networks and control them simultaneously. If such pathways are identified, they could be useful for revealing drug mode-of-action and identifying novel drug targets. By the node-set separation method proposed, dynamic structural changes can be embedded in transcriptome networks that enable us to find master-regulator genes or critical paths at each observed time. We then combine the protein-protein interaction network with the estimated dynamic transcriptome network to discover drug-affected autocrine pathways if they exist. The statistical significance (p-values) of the pathways are evaluated by the meta-analysis technique. The dynamics of the interactions between the transcriptome networks and the signaling pathways will be shown in this framework. We illustrate our strategy by an application using anti-hyperlipidemia drug, Fenofibrate. From over one million protein-protein interaction pathways, we extracted significant 23 autocrine-like pathways with the Bonferroni correction, including VEGF-NRP1-GIPC1-PRKCA-PPARalpha, that is one of the most significant ones and contains PPARalpha, a target of Fenofibrate.

Effects of nonylphenol and phytoestrogen-enriched diet on plasma vitellogenin, steroid hormone, hepatic cytochrome P450 1A, and glutathione-S-transferase values in goldfish (Carassius auratus).

The effects of nonylphenol (NP) on plasma vitellogenin (VTG) and steroid hormone values, as well as hepatic cytochrome P450 1A (CYP1A) and glutathione-S-transferase (GST) activities, were measured in goldfish (Carassius auratus) fed a diet with a low (formulated diet, FD) or high (commercial diet, CD) content of phytoestrogens, including genistein and daidzein. Male goldfish with secondary sexual characteristics were exposed to nominal NP concentrations of 0.1, 1.0, 10, and 100 microg/L in the water for 28 days while being fed either the FD or CD diet at 1.0% of body weight daily. Plasma VTG concentration in male goldfish exposed to 100 microg of NP/L and fed FD was significantly higher than that in the FD-fed control fish at seven, 21, and 28 days. However, fish of the CD-fed group exposed to 100 microg of NP/ L had significantly higher plasma VTG concentration than did fish of the CD-fed control group at 28 days only. Moreover, plasma VTG concentration in fish of the CD-fed control group was about 100-fold higher than that in fish of the FD-fed control group. Although the estrogenic effects of a phytoestrogen-enriched diet caused a decrease in testosterone and/or 11-ketotestosterone values in the CD-fed fish, there was no dose-response relationship between androgen and amount of NP to which the FD-fed fish were exposed. Nonylphenol does not have appreciable effects on hepatic CYP1A and GST activities in male goldfish at concentrations as low as 100 microg/L. These results suggest that NP has estrogenic activity in male goldfish at the nominal concentration of 100 microg/L, and that phytoestrogens, such as genistein and daidzein, in the CD inhibit an aspect(s) of steroid release and/or synthesis common to testosterone and 11-ketotestosterone. However, results of in vivo screening assays for endocrine-disrupting chemicals may be seriously affected by phytoestrogens in the diet, depending on content or potency of estrogenic activity; therefore, we recommend use in research of a standardized, open-formula diet in which estrogenic substances have been reduced to amounts that do not alter the results of studies that are influenced by exogenous estrogens.