Misato Hirano

Proteomics of two cultivated mushrooms Sparassis crispa and Hericium erinaceum provides insight into their numerous functional protein components and diversity.

Mushroom can be defined as a macrofungus with a distinctive fruiting body. Mushrooms of class Basidiomycete are primarily wood degradation fungi, but serve as food and a part of traditional medicine used by humans. Although their life cycle is fairly well-established, the information on the molecular components, especially proteins are very limited. Here, we report proteomics analysis of two edible mushrooms (fruiting bodies) Sparassis crispa and Hericium erinaceum using one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) based complementary proteomics approaches. 1-DGE coupled with liquid chromatography and mass spectrometry identified 77 (60 nonredundant proteins) and 121 (88 nonredundant proteins) proteins from S. crispa and H. erinaceum, respectively. 2-DGE analysis revealed 480 and 570 protein spots stained with colloidal coomassie brilliant blue in S. crispa and H. erinaceum, respectively. Of the 71 and 115 selected protein spots from S. crispa and H. erinaceum 2D gel blots on polyvinyldifluoride (PVDF) membranes, respectively, 29 and 35 nonredundant proteins were identified by N-terminal amino acid sequencing. Identified nonredundant proteins from 1- or 2-DGE belonged to 19 functional categories. Twenty-one proteins were found common in both S. crispa and H. erinaceum proteomes, including 14-3-3 protein and septin. Together this study provides evidence for the presence of a large number of functionally diverse proteins, expressed in the fruiting body of two economically important mushrooms, S. crispa and H. erinaceum. Data obtained from 1-DGE and 2-DGE analyses is accessible through the mushroom proteomics portal http://foodfunc.agr.ibaraki.ac.jp/mushprot.html.

Systematic investigation of the hemolymph proteome of Manduca sexta at the fifth instar larvae stage using one- and two-dimensional proteomics platforms.

Manduca sexta is an excellent insect model for studying insect physiology, including hemolymph proteins. Larvae stages of this insect are highly damaging to tobacco leaves causing a drastic decrease in crop yield. Investigation on the larval biology should help in controlling its destructive potential, thus increasing crop yields. The hemolymph is the source of its immunity to disease and environmental factors, which invariably involves protein components. To better understand the physiology of M. sexta and the protein components expressed during its life cycle, two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with N-terminal amino acid sequencing and liquid chromatography-mass spectrometry, were employed to analyze the fifth instar larvae hemolymph proteins. These proteomics approaches together identified 123 proteins, which constituted a total of 58 nonredundant proteins and belonged to 10 functional categories. Defense (49%), transport and metabolism (15%), storage (9%), and metamorphosis (7%) categories were highly represented accounting for 80% of the identified proteins. Besides identification of previously reported proteins, 18 novel proteins were identified, which include the lipoprotein-releasing system transmembrane protein lolC, 50S ribosomal protein L24, inducible serine protease inhibitor 1, imaginal disk growth factor, protein disulfide-isomerase-like protein ERp57, etc. The 2-DGE data were integrated to develop a 2-D gel reference map. Data obtained from 1-DGE and 2-DGE analyses are accessible through the M. sexta proteomics portal ( http://foodfunc.agr.ibaraki.ac.jp/mansehemoprot.html). Together, this study provides evidence for the presence of a large number of functionally diverse protein families in the hemolymph of M. sexta. These proteins correlate well with the fifth instar stage, the transition from larvae to pupae.

Proteomics- and transcriptomics-based screening of differentially expressed proteins and genes in brain of Wig rat: a model for attention deficit hyperactivity disorder (ADHD) research.

Two global omics approaches were applied to develop an inventory of differentially expressed proteins and genes in Wig rat, a promising animal model of attention-deficit hyperactivity disorder (ADHD). The frontal cortex, striatum, and midbrain of Wig rat at 4 weeks of age were dissected for proteomics and transcriptomics analyses. Two-dimensional gel electrophoresis detected 13, 1, and 16 differentially expressed silver nitrate-stained spots in the frontal cortex, striatum, and midbrain, respectively. Peptide mass fingerprinting/tandem mass spectrometry identified 19 nonredundant proteins, belonging to 7 functional categories, namely, signal transduction, energy metabolism, cellular transport, protein with binding function, protein synthesis, cytoskeleton, and cell rescue. Interestingly, 10 proteins that were indentified in the present study were also previously reported in studies involving neurodegenerative diseases and psychiatric disorders, such as Alzheimers disease (AD), Parkinsons disease, and Schizophrenia. Moreover, some of the proteins identified in the midbrain were involved in synaptic vesicular transport, suggesting abnormality in neurotransmitter release in this region. On the other hand, transcriptomics analysis of combined frontal cortex, striatum, and midbrain by rat whole genome 44K DNA oligo microarray revealed highly up-regulated (28) and down-regulated (33) genes. Functional categorization of these genes showed cellular transport, metabolism, protein fate, signal transduction, and transcription as the major categories, with 26% genes of unknown function. Some of the identified genes were related to AD, fragile X syndrome, and ADHD. This is a first comprehensive study providing insight into molecular components in Wig rat brain, and will help to elucidate the roles of identified proteins and genes in Wig rat brain, hopefully leading to uncovering the pathogenesis of ADHD.

Omic analyses unravels global molecular changes in the brain and liver of a rat model for chronic sake (Japanese alcoholic beverage) intake

The effects of chronic administration of Sake (Japanese alcoholic beverage, Nihonshu) on brain and liver of female F334 (Fisher) rats were surveyed via global omic analyses using DNA microarray, 2‐DE, and proton nuclear magnetic resonance. Rats weaned at 4 wk of age were given free access to Sake (15% alcohol), instead of water. At 13 months of age, and 24 h after withdrawal of Sake supply, rats were sacrificed, and the whole brain and liver tissues dissected for analyses. In general, molecular changes in brain were found to be less than those in liver. Transcriptomics data revealed 36 and 9, and 80 and 62 up‐ and down‐regulated genes, in the brain and liver, respectively, with binding and catalytic activity gene categories the most prominently changed. Results suggested Sake‐induced fragility of brain and liver toxicity/damage, though no significant abnormalities in growth were seen. At protein level, a striking decrease was found in the expression of NADH dehydrogenase (ubiquinone) Fe‐S protein 1 in brain, suggesting attenuation of mitochondrial metabolism. In liver, results again suggested an attenuation of mitochondrial function and, in addition, glycoproteins with unknown function were induced at protein and gene levels, suggesting possible changes in glycoprotein binding in that organ. Metabolomic analysis of brain revealed significant increases in valine, arginine/ornithine, alanine, glutamine, and choline with decreases in isoleucine, N‐acetyl aspartate, taurine, glutamate, and gamma aminobutyric acid. Our results provide a detailed inventory of molecular components of both brain and liver after Sake intake, and may help to better understand effects of chronic Sake drinking.

Effects of Coffee Bean Aroma on the Rat Brain Stressed by Sleep Deprivation: A Selected Transcript- and 2D Gel-Based Proteome Analysis

The aim of this study was 2-fold: (i) to demonstrate influences of roasted coffee bean aroma on rat brain functions by using the transcriptomics and proteomics approaches and (ii) to evaluate the impact of roasted coffee bean aroma on stress induced by sleep deprivation. The aroma of the roasted coffee beans was administered to four groups of adult male Wistar rats: 1, control group; 2, 24 h sleep deprivation-induced stress group (the stress group); 3, coffee aroma-exposed group without stress (the coffee group); and 4, the stress with coffee aroma group (the stress with coffee group). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of some known genes responsive to aroma or stress was performed using total RNA from these four groups. A total of 17 selected genes of the coffee were differently expressed over the control. Additionally, the expression levels of 13 genes were different between the stress group and the stress with coffee group: Up-regulation was found for 11 genes, and down-regulation was seen for two genes in the stress with coffee group. We also looked to changes in protein profiles in these four samples using two-dimensional (2D) gel electrophoresis; 25 differently expressed gel spots were detected on 2D gels stained by silver nitrate. Out of these, a total of nine proteins were identified by mass spectrometry. Identified proteins belonged to five functional categories: antioxidant; protein fate; cell rescue, defense, and virulence; cellular communication/signal transduction mechanism; and energy metabolism. Among the differentially expressed genes and proteins between the stress and the stress with coffee group, NGFR, trkC, GIR, thiol-specific antioxidant protein, and heat shock 70 kDa protein 5 are known to have antioxidant or antistress functions. In conclusion, the roasted coffee bean aroma changes the mRNA and protein expression levels of the rat brain, providing for the first time clues to the potential antioxidant or stress relaxation activities of the coffee bean aroma.

Transcriptomic analysis of rat brain tissue following gamma knife surgery: Early and distinct bilateral effects in the un-irradiated striatum

Gamma knife surgery (GKS) is used for the treatment of various human brain disorders. However, the biological effects of gamma ray irradiation on both the target area, and the surrounding tissues are not well studied. The effects of gamma ray exposure to both targeted and untargeted regions were therefore evaluated by monitoring gene expression changes in the unilateral irradiated (60 Gy) and contralateral un-irradiated striata in the rat. Striata of irradiated and control brains were dissected 16 hours post-irradiation for analysis using a whole genome 44K DNA oligo microarray approach. The results revealed 230 induced and 144 repressed genes in the irradiated striatum and 432 induced and 239 repressed genes in the un-irradiated striatum. Out of these altered genes 39 of the induced and 16 of the reduced genes were common to both irradiated and un-irradiated tissue. Results of semiquantitative, confirmatory RT-PCR and western blot analyses suggested that γ-irradiation caused cellular damage, including oxidative stress, in the striata of both hemispheres of the brains of treated animals.

Alterations in the expression of proteins and genes in the brain of 6-hydroxydopamine-treated rat, a model for attention-deficit hyperactivity disorder

We previously reported that brain thromboxane A2 is positively involved in the central regulation of plasma catecholamine levels in uretrhane-anesthetized rats. In the present study, we pharmacologically analyzed the role of inflammatory mediators in the restraint stress-induced elevation of plasma catecholamines (noradrenaline and adrenaline). The Restraint stress (8 h) elevated plasma catecholamines levels (adrenaline > noradrenaline). The elevations of both catecholamines reached the peak about 30 min after the restraint stress. Intraperitoneal administration of indomethacin (10 mg/kg, a non-selective cyclooxygenase inhibitor), NS-398 (5 mg/kg, a selective cyclooxygenase 2 inhibitor), daltroban (20 mg /kg, a selective thromboxane A2 receptor antagonist), CP-154,526 (20 mg/kg, a selective CRF1 receptor antagonist), PDTC (200 mg/kg, a NF B inhibitor) or MK 801 (2 mg/kg, a NMDA receptor antagonist) reduced the stress-induced elevation of plasma catecholamines. These results suggest that brain inflammatory mediators are important component of the restraint stress-induced elevation of plasma catecholamines.

Transcriptomics of unilateral irradiated and contralateral rat striata after gamma knife surgery

O1P-KØ8 Transcriptomics of unilateral irradiated and contralateral rat striata after gamma knife surgery Randeep Rakwal1, Junko Shibato1, Misato Hirano1, Nobuo Kouyama2, Yoko Katayama2, Motohiro Hayashi3, Hitoshi Iwahashi4, Yoshinori Masuo1 1 Human Stress Signal Research Center (HSS), AIST, Tsukuba, Japan; 2 Department of Physiology, School of Medicine, Tokyo Women’s Medical University, Tokyo, Japan; 3 Department of Neurosurgery, School of Medicine, Tokyo Women’s Medical University, Tokyo, Japan; 4 HSS, AIST, Ikeda, Japan