Synthesis of (11)C-labeled retinoic acid, [(11)C]ATRA, via an alkenylboron precursor by Pd(0)-mediated rapid C-[(11)C]methylation.

Retinoids are a class of chemical compounds which include both natural dietary vitamin A (retinol) metabolites and active synthetic analogs. Both experimental and clinical studies have revealed that retinoids regulate a wide variety of essential biological processes. In this study, we synthesized (11)C-labeled all-trans-retinoic acid (ATRA), the most potent biologically active metabolite of retinol and used in the treatment of acute promyelocytic leukemia. The synthesis of (11)C-labeled ATRA was accomplished by a combination of rapid Pd(0)-mediated C-[(11)C]methylation of the corresponding pinacol borate precursor prepared by 8 steps and hydrolysis. [(11)C]ATRA will prove useful as a PET imaging agent, particularly for elucidating the improved therapeutic activity of ATRA (natural retinoid) for acute promyelocytic leukemia by comparing with the corresponding PET probe [(11)C]Tamibarotene (artificial retinoid).

Endovascular Treatment for Primary Tumor Hemorrhage in Oropharyngeal Carcinoma: A Report of Two Cases.

Acute arterial hemorrhage is a damaging and sometimes lethal complication that occurs in patients with head and neck cancer. However, achieving hemostasis can be challenging because of the difficulty in applying pressure in the throat and oral cavity. In this context, endovascular treatment (ET) has been performed in recent years. This report aims to describe the benefits of ET for acute bleeding. Additionally, our findings emphasize the importance of early diagnosis and treatment of tumor-related bleeding, not only for immediate life-saving benefits but also for the potential resumption of irradiation and chemotherapy, which can lead to favorable long-term prognoses in some instances. We describe two cases of primary tumor bleeding where treatment was successful with ET. Neurosurgeons performed these treatments, and effective hemostasis was achieved in both cases. No complications or rebleeding were observed. ET is a better option for hemorrhage from oropharyngeal tumors than for hemorrhage from the main trunk of the carotid artery. The efficacy of ET is dependent on the vessels involved, and early identification of the culprit artery can predict the prognosis. ET should be considered an option for acute arterial hemorrhage in head and neck cancer.

Efficient sequential synthesis of PET Probes of the COX-2 inhibitor [11C]celecoxib and its major metabolite [11C]SC-62807 and in vivo PET evaluation.

Synthesis of [(11)C]celecoxib, a selective COX-2 inhibitor, and [(11)C]SC-62807, a major metabolite of celecoxib, were achieved and the potential of these PET probes for assessing the function of drug transporter in biliary excretion was evaluated. The synthesis of [(11)C]celecoxib was achieved in one-pot by reacting [(11)C]methyl iodide with an excess of the corresponding pinacol borate precursor using Pd(2)(dba)(3), P(o-tolyl)(3), and K(2)CO(3) (1:4:9) in DMF. The radiochemical yield of [(11)C]celecoxib was 63±23% (decay-corrected, based on [(11)C]CH(3)I) (n=7) with a specific radioactivity of 83±23GBq/µmol (n=7). The average time of synthesis from end of bombardment including formulation was 30min with >99% radiochemical purity. [(11)C]SC-62807 was synthesized from [(11)C]celecoxib by further rapid oxidation in the presence of excess KMnO(4) with microwave irradiation. The radiochemical yield of [(11)C]SC-62807 was 55±9% (n=3) (decay-corrected, based on [(11)C]celecoxib) with a specific radioactivity of 39±4GBq/µmol (n=3). The average time of synthesis from [(11)C]celecoxib including formulation was 20min and the radiochemical purity was >99%. PET studies in rats and the metabolite analyzes of [(11)C]celecoxib and [(11)C]SC-62807 showed largely different excretion processes, and consequently, [(11)C]SC-62807 was rapidly excreted via hepatobiliary excretion without further metabolism. [(11)C]SC-62807 was shown to have a high potential as a PET probe for evaluating drug transporter function in biliary excretion.

Efficient synthesis of [¹¹C]ramelteon as a positron emission tomography probe for imaging melatonin receptors involved in circadian rhythms.

Ramelteon (TAK-375) is a novel melatonin receptor agonist that is used for clinical treatment of insomnia. The present report describes radiolabeling of ramelteon with the short-lived positron-emitter ¹¹C (T(1/2)=20.4 min) by 2 methods. One method was [¹¹C]methylation of an acetoamide precursor and the other was [¹¹C]acylation of the corresponding amine precursor. First, [¹¹C]methylation method showed the low reproducibility together with the production of many kinds of side products from which the [¹¹C-methyl]Ramelteon was separated with chemical purity of <28% and radiochemical purity of >98%. Whereas, the [¹¹C]acylation method showed high efficiency and reproducibility with a good radiochemical yield (22-43%, decay corrected), high chemical and radiochemical purities (>99% each), and high specific activity (43-162 GBq/µmol) (n=5) after HPLC purification. [¹¹C]Ramelteon is a potential positron emission tomography (PET) probe for imaging the melatonin receptor.

General method for the (11)C-labeling of 2-arylpropionic acids and their esters: construction of a PET tracer library for a study of biological events involved in COXs expression.

Cyclooxygenase (COX) is a critical enzyme in prostaglandin biosynthesis that modulates a wide range of biological functions, such as pain, fever, and so on. To perform in vivo COX imaging by positron emission tomography (PET), we developed a method to incorporate (11)C radionuclide into various 2-arylpropionic acids that have a common methylated structure, particularly among nonsteroidal anti-inflammatory drugs (NSAIDs). Thus, we developed a novel (11)C-radiolabeling methodology based on rapid C-[(11)C]methylation by the reaction of [(11)C]CH(3)I with enolate intermediates generated from the corresponding esters under basic conditions. One-pot hydrolysis of the above [(11)C]methylation products also allows the synthesis of desired (11)C-incorporated acids. We demonstrated the utility of this method in the syntheses of six PET tracers, [(11)C]Ibuprofen, [(11)C]Naproxen, [(11)C]Flurbiprofen, [(11)C]Fenoprofen, [(11)C]Ketoprofen, and [(11)C]Loxoprofen. Notably, we found that their methyl esters were particularly useful as proradiotracers for a study of neuroinflammation. The microPET studies of rats with lipopolysaccharide (LPS)-induced brain inflammation clearly showed that the radioactivity of PET tracers accumulated in the inflamed region. Among these PET tracers, the specificity of [(11)C]Ketoprofen methyl ester was demonstrated by a blocking study. Metabolite analysis in the rat brain revealed that the methyl esters were initially taken up in the brain and then underwent hydrolysis to form pharmacologically active forms of the corresponding acids. Thus, we succeeded in general (11)C-labeling of 2-arylpropionic acids and their methyl esters as PET tracers of NSAIDs to construct a potentially useful PET tracer library for in vivo imaging of inflammation involved in COXs expression.

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.

Pd(0)-Mediated rapid coupling between methyl iodide and heteroarylstannanes: an efficient and general method for the incorporation of a positron-emitting (11)C radionuclide into heteroaromatic frameworks.

The Pd(0)-mediated rapid trapping of methyl iodide with an excess amount of a heteroaryl-substituted tributylstannane has been investigated with the aim of incorporating a short-lived (11)C-labelled methyl group into the heteroaromatic carbon frameworks of important organic compounds, such as drugs with various heteroaromatic structures, in order to execute a positron emission tomography (PET) study of vital systems. The reaction was first performed by using our previously developed CH(3)I/stannane/[Pd(2)(dba)(3)]/P(o-CH(3)C(6)H(4))(3)/CuCl/K(2)CO(3) (1:40:0.5:2:2:2) system in DMF at 60 degrees C for 5 min (conditions A), however, the reaction gave low yields for various heteroaromatic compounds. Increasing the amount of phosphine ligand (conditions B) led to a significant improvement in the yield, but the conditions were still not suitable for a range of basic heteroaromatic structures. Use of the CuBr/CsF system (conditions C) also provided a result similar to that obtained under conditions B with an increased amount of the phosphine. Thus, pyridine and related heteroaromatic compounds remained less reactive substrates. The problem was overcome by replacing the DMF solvent with N-methyl-2-pyrolidinone (NMP). The reaction in NMP at 60-100 degrees C for 5 min using a CH(3)I/stannane/[Pd(2)(dba)(3)]/P(o-CH(3)C(6)H(4))(3)/CuBr/CsF (1:40:0.5:16:2:5) combination (conditions D) gave the methylated products in yields of more than 80 % (based on the reaction of CH(3)I) for all of the heteroaromatic compounds listed in this study. Thus, the combined use of NMP and an increased amount of phosphine is important for promoting the reaction efficiently. The use of this general approach to rapid methylation has been well demonstrated by the synthesis of the PET tracers 2- and 3-[(11)C]methylpyridines by using [Pd(2)(dba)(3)]/P(o-CH(3)C(6)H(4))(3)/CuBr/CsF (1:16:2:5) in NMP at 60 degrees C for 5 min, which gives the desired products in HPLC analytical yields of 88 and 91 %, respectively.

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 gamma-irradiation caused cellular damage, including oxidative stress, in the striata of both hemispheres of the brains of treated animals.

New protein extraction/solubilization protocol for gel-based proteomics of rat (female) whole brain and brain regions.

The rat is an accepted model for studying human psychiatric/neurological disorders. We provide a protocol for total soluble protein extraction using trichloroacetic acid/acetone (TCA/A) from rat (female) whole brain, 10 brain regions and the pituitary gland, and show that two-dimensional gel electrophoresis (2-DGE) using pre-cast immobilized pH (4-7) gradient (IPG) strip gels (13 cm) in the first dimension yields clean silver nitrate stained protein profiles. Though TCA/A precipitation may not be "ideal", the important choice here is the selection of an appropriate lysis buffer (LB) for solubilizing precipitated proteins. Our results reveal enrichment of protein spots by use of individual brain regions rather than whole brain, as well as the presence of differentially expressed spots in their proteomes. Thus individual brain regions provide improved protein coverage and are better suited for differential protein detection. Moreover, using a phosphoprotein-specific dye, in-gel detection of phosphoproteins was demonstrated. Representative high-resolution silver nitrate stained proteome profiles of rat whole brain total soluble protein are presented. Shortcomings apart (failure to separate membrane proteins), gel-based proteomics remains a viable option, and 2-DGE is the method of choice for generating high-resolution proteome maps of rat brain and brain regions.

Fasted/fed states regulate postsynaptic hub protein DYNLL2 and glutamatergic transmission in oxytocin neurons in the hypothalamic paraventricular nucleus.

The neurons in the hypothalamus regulate food intake and energy metabolism on reception of systemic energy states. Accumulating evidences have indicated that synaptic transmission on the hypothalamic neurons is modulated by the metabolic condition related to fasted/fed states, and that this modulation of synaptic plasticity plays a role in regulation of feeding. It has been shown that oxytocin (Oxt) neurons in the paraventricular nucleus (PVN) of the hypothalamus sense and integrate various peripheral and central signals and thereby induce satiety. However, whether metabolic conditions regulate the synaptic transmission on Oxt neurons in PVN remains unclear. The present study examined whether the fasted/fed states regulate synaptic transmission on Oxt neurons in PVN. The miniature excitatory postsynaptic currents (mEPSCs) onto Oxt neurons in PVN were increased under ad lib fed condition compared to 24h fasted condition. Furthermore, the NMDA receptor-mediated EPSC on Oxt neurons was increased under fed, compared to fasted, condition. In Oxt neurons, dynein light chain 2 (DYNLL2), a protein suggested to be implicated in the NMDA receptor trafficking to the postsynaptic site, was increased under fed, compared to fasted, condition. The present results suggest that feeding increases excitatory synaptic input on PVN Oxt neurons via mechanisms involving DYNLL2 upregulation and NMDA receptor-mediated synaptic reorganization.

Evaluation of breast cancer resistance protein function in hepatobiliary and renal excretion using PET with 11C-SC-62807.

UNLABELLED: A quantitative PET imaging method was used to assess the in vivo kinetics of hepatobiliary and renal excretion of the breast cancer resistance protein (Bcrp) substrate (11)C-SC-62807 in mice. METHODS: Serial abdominal PET scans were collected in wild-type and Bcrp knockout (Bcrp(-/-)) mice after intravenous injection of (11)C-SC-62807. Venous blood samples and PET images were obtained at frequent intervals up to 30 min after radiotracer administration. Dynamic PET data were analyzed to determine the canalicular and brush-border efflux clearances in the liver and kidney (CL(int,bile,liver) and CL(int,urine,kidney), respectively). RESULTS: SC-62807 is an in vitro substrate of mouse Bcrp and human BCRP. Radioactivity associated with (11)C-SC-62807 was predominantly found in the blood, liver, bile, and urine 30 min after administration. Both biliary and urinary excretion of radioactivity was markedly lower in Bcrp(-/-) mice than in wild-type mice, suggesting greater systemic exposure in Bcrp(-/-) mice. Both the CL(int,bile,liver) and the CL(int,urine,kidney) were significantly lower in Bcrp(-/-) mice (74% ± 10% and 99% ± 1% lower than controls, respectively). We also found that (11)C-SC-62807 is a substrate of the organic anion-transporting polypeptides OATP1B1 and OATP1B3 in vitro. CONCLUSION: The present study demonstrated that Bcrp plays a significant role in the efflux of (11)C-SC-62807 in mouse liver and kidney. We also demonstrated the feasibility of PET using (11)C-SC-62807 to study the activity of BCRP in humans.

Synthesis of [(11)C]Am80 via Novel Pd(0)-Mediated Rapid [(11)C]Carbonylation Using Arylboronate and [(11)C]Carbon Monoxide.

(11)C-labeled methylbenzoates [(11)C]4a-d were synthesized using Pd(0)-mediated rapid cross-coupling reactions employing [(11)C]carbon monoxide and arylboronic acid neopentyl glycol esters 3a-d under atmospheric pressure in methanol-dimethylformamide (MeOH-DMF), in radiochemical yields of 12 ± 5-26 ± 13% (decay-corrected based on [(11)C]O). The reaction conditions were highly favorable for the synthesis of [(11)C]Am80 ([(11)C]2) and [(11)C]methyl 4-((5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)carbamoyl)benzoate ([(11)C]2-Me) using 4-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)-N-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)benzamide (5), both of which produced a decay-corrected radiochemical yield (RCY) of 26 ± 13%, with >99% radiochemical purity and an average specific radioactivity of 44 GBq/µmol. The yields of [(11)C]4a, [(11)C]2-Me, and [(11)C]2 were improved by the use of a 2-fold excess of the solvents and reagents under the same conditions to give respective yields of 66 ± 8, 65 ± 7, and 48 ± 2%.

In vivo expression of cyclooxygenase-1 in activated microglia and macrophages during neuroinflammation visualized by PET with 11C-ketoprofen methyl ester.

UNLABELLED: Cyclooxygenase (COX)-1 and -2 are prostanoid-synthesizing enzymes that play important roles in the regulation of neuroinflammation and in the development of neurodegenerative disorders. However, the specific functions of these isoforms are still unclear. We recently developed (11)C-labeled ketoprofen methyl ester as a PET probe that targets the COXs for imaging neuroinflammation, though its responsible isoform is yet to be determined. In the present study, we performed ex vivo and in vivo imaging studies with (11)C-ketoprofen methyl ester and determined the contributions of the COX isoforms during the neuroinflammatory process. METHODS: To identify the COX isoform responsible for (11)C-ketoprofen methyl ester in the brain, we examined the ex vivo autoradiography of (11)C-ketoprofen methyl ester using COX-deficient mice. Time-dependent changes in accumulation of (11)C-ketoprofen methyl ester during the neuroinflammatory process were evaluated by PET in rats with hemispheric neuroinflammation induced by intrastriatal injection of lipopolysaccharide or quinolinic acid. In both rat models, cell-type specificity of COX isoform expression during neuroinflammation was identified immunohistochemically. RESULTS: Ex vivo autoradiographic analysis of COX-deficient mice revealed a significant reduction of (11)C-ketoprofen methyl ester accumulation only in COX-1-deficient mice, not COX-2-deficient mice. PET of rats after intrastriatal injection of lipopolysaccharide showed a significant increase in accumulation of (11)C-ketoprofen methyl ester in the inflamed area. This increase was evident at the early phase of 6 h, peaked at day 1, and then returned to basal levels by day 7. In addition, immunohistochemical analysis revealed that the population of activated microglia and macrophages was elevated at the early phase with COX-1 expression but not COX-2. A significant increase in (11)C-ketoprofen methyl ester accumulation was also observed at day 1 after intrastriatal injection of quinolinic acid, with increased COX-1-expressing activated microglia and macrophages. CONCLUSION: We have identified (11)C-ketoprofen methyl ester as a COX-1-selective PET probe, and using this, we have also demonstrated a time-dependent expression of COX-1 in activated microglia and macrophages during the neuroinflammatory process in the living brain. Thus, COX-1 may play a crucial role in the pathology of neuroinflammation and might be a critical target for the diagnosis and therapy of neurodegenerative disorders.

11C-PK11195 PET for the in vivo evaluation of neuroinflammation in the rat brain after cortical spreading depression.

UNLABELLED: Neurogenic inflammation triggered by extravasation of plasma protein has been hypothesized as a key factor in the generation of the pain sensation associated with migraine. The principal immune cell that responds to this inflammation is the parenchymal microglia of the central nervous system. METHODS: Using a PET technique with (11)C-(R)-[1-(2-chlorophenyl)-N-methyl-N-(1-methyl-propyl)-3-isoquinolinecarboxamide] ((11)C-PK11195), a PET ligand for peripheral type-benzodiazepine receptor, we evaluated the microglial activation in the rat brain after generation of unilateral cortical spreading depression, a stimulation used to bring up an experimental animal model of migraine. RESULTS: We found a significant increase in the brain uptake of (11)C-PK11195, which was completely displaceable by the excess amounts of unlabeled ligands, in the ipsilateral hemisphere of the spreading depression-generated rats. Moreover, the binding potential of (11)C-PK11195 in the spreading depression-generated rats was significantly higher than that in the sham-operated control rats. CONCLUSION: These results suggest that as an inflammatory reaction, microglial cells are activated in response to the nociceptive stimuli induced by cortical spreading depression in the rat brain. Therefore, the (11)C-PK11195 PET technique could have a potential for diagnostic and therapeutic monitoring of neurologic disorders related to neuroinflammation such as migraine.

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.

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.

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 Alzheimer's disease (AD), Parkinson's 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.

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.

Gel-based proteomics of unilateral irradiated striatum after gamma knife surgery.

Gamma knife surgery (GKS) is used for the treatment of various brain disorders. The biological effects of focal gamma ray irradiation on targeted or surrounding areas in the brain are not well-known. In the present study, we evaluated protein expression changes in the unilateral irradiated (60 Gy) striatum in rat. Striata of irradiated and control brains were dissected 16 h post-irradiation for analysis by large-format two-dimensional gel electrophoresis (2-DGE). In parallel, we also examined the un-targeted contralateral striatum over the control for potential changes in proteins patterns that may have occurred due to the effects of irradiation to the unilateral striatum. A total of 17 reproducible and differentially expressed silver nitrate-stained protein spots in the irradiated striatum was detected on 2-D gel. Their subsequent analysis by tandem mass spectrometry (nESI-LC-MS/MS) resulted in the identification of 13 nonredundant proteins. Interestingly, out of these 13 changed proteins, 2 proteins were also detected in the contralateral striatum. Some of the significantly changed proteins identified were creatine kinase, protein disulfide isomerase A3 precursor (PDA3), and peroxiredoxin 2 (Prx2). Western analysis with anti-PDA3 and anti-Prx2 antibodies revealed 4 and 2 cross-reacting protein spots on 2-D gel blots. Interestingly, after GKS, in the irradiated and un-irradiated striata, these spots showed a shift toward the acidic side, suggesting post-translational modifications. Taken together, these results indicate that unilateral irradiation during GKS triggers molecular changes in the bilateral striata.

Gel-based proteomics reveals potential novel protein markers of ozone stress in leaves of cultivated bean and maize species of Panama.

We examined responses of cultivated bean (Phaseolus vulgaris L. cv. IDIAP R-3) and maize (Zea mays L. cv. Guarare 8128) plants exposed to ozone (O(3)) using a leaf injury assessment and proteomics approach. Plants grown for 16 days in greenhouse were transferred to an O(3) chamber and exposed continuously to 0.2 ppm O(3) or filtered pollutant-free air for up to 72 h. CBB-stained gels revealed changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) protein. By Western analysis changes in marker proteins for O(3) damage in leaves by 1-DE were checked. In bean leaves, two superoxide dismutase (SOD) protein (19 and 20 kDa) were dramatically decreased, while ascorbate peroxidase (APX, 25 kDa), small heat shock protein (HSP, 33 kDa), and a naringenin-7-O-methyltransferase (NOMT, 42 kDa) were increased by O(3). In maize leaves, expression levels of catalase (increased), SOD (decreased), and APX (increased) were drastically changed by O(3) depending on the leaf stage, whereas crossreacting HSPs (24 and 30 kDa) and NOMT (41 kDa) proteins were strongly increased in O(3)-stressed younger leaves. These results indicated a clear modulation of oxidative stress-, heat shock-, and secondary metabolism-related proteins by O(3). Finally, 2-DE at 72 h after O(3) exposure revealed changes (induction/suppression) in expression levels of 25 and 12 protein spots in bean and maize leaves, respectively. Out of these, ten and nine nonredundant proteins in bean and maize, respectively, were identified by MS. A novel pathogenesis-related protein 2 may serve as a potential marker for O(3) stress in bean.