Lower plasma tumor necrosis factor-α is associated with symptomatic remission in patients with schizophrenia.

We investigated the plasma tumor necrosis factor (TNF)-α levels between patients with schizophrenia remission and healthy controls, and the association between the plasma TNF-α levels and cognitive function and social function. This cross-sectional study included 48 patients with schizophrenia who fulfilled the remission criteria and 20 healthy controls. Plasma TNF-α levels were measured using the enzyme-linked immunosorbent assay, and cognitive function was assessed using the Japanese version of the Brief Assessment of Cognition in Schizophrenia (BACS-J). We measured social function using the Social Functioning Scale (SFS-J). The plasma TNF-α levels were significantly lower in the remission schizophrenia group (31.7 ± 27.4 ng/mL) compared to the heathy control group (55.1 ± 38.5 ng/mL) (P = 0.01). In contrast, no correlation was observed between the plasma levels of TNF-α and all BACS-J scores and all SFS-J scores in either group. This result suggests that plasma TNF-α levels may serve as a clinical biomarker of remission of schizophrenia and that the plasma TNF-α levels bore no association with cognitive function. Thus, TNF-α may have potential as a useful indicator of the therapeutic response in patients with schizophrenia.

Effects of neutron radiation generated in deep space-like environments on food resources.

The impact of deep space cosmic rays on food resources is as important as the risks of cosmic rays to the human body. This study demonstrates the potential for neutrons as secondary radiation in deep space spacecraft to cause meat activation and oxidative modification of proteins and lipids. We conducted a series of experiments such as the neutron irradiation experiment, the radioactivation analysis and the biochemical analysis. Neutrons with energies from 1 to 5 MeV with doses from 0.01 Gy to 4 Gy were irradiated by the RIKEN accelerated-driven neutron source (RANS). Radioactive nuclei, 24Na, 42K, and 38Cl, were detected in the neutron-irradiated meat. The modification products of the proteins by oxidative nitration, 6-nitrotryptophan (6NO2Trp), and by a lipid peroxidation, 4-hydroxy-2-nonenal (4-HNE), were detected in several proteins with neutron dose dependent. The proteome analysis showed that many oxidative modifications were detected in actin and myosin which are major proteins of myofibrils. This study is of crucial importance not only as risk factors for human space exploration, but also as fundamental effects of radiation on the components of the human body.

Prescription of Anticholinergic Drugs in Patients With Schizophrenia: Analysis of Antipsychotic Prescription Patterns and Hospital Characteristics.

In several clinical guidelines for schizophrenia, long-term use of anticholinergic drugs is not recommended. We investigated the characteristics of the use of anticholinergics in patients with schizophrenia by considering psychotropic prescription patterns and differences among hospitals. A cross-sectional, retrospective prescription survey at the time of discharge was conducted on 2027 patients with schizophrenia from 69 Japanese hospitals. We examined the relations among psychotropic drug prescriptions regarding anticholinergic prescription. We divided the hospitals into three groups-low rate group (LG), medium rate group (MG), and high rate group (HG)-according to their anticholinergic prescription rates, and analyzed the relationship between anticholinergic prescription rates and antipsychotic prescription. Anticholinergic drugs were prescribed to 618 patients (30.5%), and the prescription rates were significantly higher for high antipsychotic doses, antipsychotic polypharmacy, and first-generation antipsychotics (FGAs) use. The anticholinergic prescription rate varied considerably among hospitals, ranging from 0 to 66.7%, and it was significantly higher in patients with antipsychotic monotherapy, antipsychotic polypharmacy, and normal and high doses of antipsychotics in HG than in those LG and MG. The anticholinergics prescription rate in patients with second-generation antipsychotic monotherapy in HG was also significantly higher than in those LG and MG; however, the difference was no longer significant in patients with FGA monotherapy. Conclusively, in addition to high antipsychotic doses, antipsychotic polypharmacy, and FGA use, hospital characteristics influence the prescribing of anticholinergic drugs.

Nitration of tryptophan in ribosomal proteins is a novel post-translational modification of differentiated and naïve PC12 cells.

Neuron growth factor (NGF) signaling in PC12 cell, which is derived from pheochromocytoma of rat adrenal medulla, induces expression of neuronal nitric oxide synthase (nNOS) and nitric oxide (NO) production. Subsequently, NO causes differentiation of PC12 cell to neuronal cell with morphological changes, such as neurite extension. In this study, we showed that 6-nitrotryptophan-containing proteins were produced in PC12 cell (naïve PC12 cell) and/or NGF-induced PC12 cell (differentiated PC12 cell). Western blot analysis of the protein extract of naïve PC12 cell and differentiated PC12 cell using anti 6-nitrotryptophan antibody showed several immunoreactive bands, which were subsequently subjected to trypsin digestion and LC-ESI-MS-MS analysis. The peptides from five ribosomal proteins, namely, 60S ribosomal protein L7 (Trp154), 60S acidic ribosomal protein P1 (Trp43), 40S ribosomal protein S2 (Trp60), 40S ribosomal protein S6 (Trp45), and 40S ribosomal protein S19 (Trp52), were identified as nitrotryptophan residue-containing proteins with significant ion score levels (p<0.05). Among these, tryptophan nitration was observed only in differentiated PC12 cell for S19 protein, and only in naïve PC12 cell for L7 protein. Tryptophan nitration of the other ribosomal proteins P1, S2, and S6 was observed in both naive and differentiated PC12 cells. The positive signal of nitrotryptophan-containing proteins in the Western blotting around 16 kDa (Band 1), which includes 40S ribosomal protein S19, was suppressed by treatment with NOS inhibitor, L-NAME. The tryptophan nitration of 40S ribosomal protein was not observed by LC-ESI-MS-MS analysis of this sample. This is the first study to identify several specific sites of nitrated tryptophan on proteins not only in viable culture cells but also in a physiological process: cell differentiation.

Mass spectrometric identification of tryptophan nitration sites on proteins in peroxynitrite-treated lysates from PC12 cells.

One of the important sites of peroxynitrite action that affects cellular function is known to be nitration of tyrosine residues. However, tryptophan residues could be another target of peroxynitrite-dependent modification of protein function, as we have shown previously using a model protein (F. Yamakura et al., J. Biochem. 138:57-69; 2005). Here, we report the identification of several proteins that allowed us to determine the position of nitrotryptophan in their amino acid sequences in a more complex system. We modified lysates from PC12 cells with and without nerve growth factor (NGF) by treatment with peroxynitrite (0.98 or 4.9 mM). Western blot analyses using anti-6-nitrotryptophan antibody showed several immunoreactive bands and spots, which were subsequently subjected to trypsin digestion and LC-ESI-MS-MS analysis. We identified several tryptic peptides including nitrotryptophan residues, which were derived from L-lactate dehydrogenase A, malate dehydrogenase 1, M2 pyruvate kinase, and heat-shock protein 90 α, in peroxynitrite-treated lysates from PC12 cells, and l-lactate dehydrogenase A, malate dehydrogenase 1, transaldorase, and lactoylglutathione lyase, in peroxynitrite-treated lysates from NGF/PC12 cells. The molar ratio of 3-nitrotyrosine to 6-nitrotryptophan in protease-digested PC12 cell lysates treated with peroxynitrite was determined to be 5.8 to 1 by using an HPLC-CoulArray system. This is the first report to identify several specific sites of nitrated tryptophan on proteins in a complex system treated with peroxynitrite and to compare the susceptibility of nitration between tryptophan and tyrosine residues of the proteins.

Glyceraldehyde-3-phosphate dehydrogenase interacts with phosphorylated Akt resulting from increased blood glucose in rat cardiac muscle.

Here we describe the interaction of phosphorylated approximately 40 kDa protein with phosphorylated Akt which is a serine/threonine kinase resulting from increased blood glucose in rat cardiac muscle. Mass spectrometry analysis revealed that this protein was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Furthermore, increase in Akt and GAPDH phosporylation and induction of their association were both observed after insulin stimulation in the H9c2 cell line derived from embryonic rat ventricle. Moreover, the activation of GAPDH was upregulated when the GAPDH phosphorylation was increased. Our data suggest that GAPDH phosphorylation and association with Akt by insulin treatment have some bearing on the enhancement of GAPDH activity.

Innovative radiographic system to improve the sharpness of radiographs: could a phase-shift effect contribute to improved image-quality for plain computed radiographs for general use?

PURPOSE: The aim of this study was to determine whether a high-resolution digital radiography system that was originally developed for mammography could be used for general radiographic purposes by means of a phantom test. MATERIALS AND METHODS: This system includes an X-ray tube with a smaller focal point than is generally used for general radiography, and a computed radiography (CR) system to provide the highest spatial resolution. The imaging object and CR plate are intentionally separated to capture an edge-enhancing effect that is the result of a phase-shift (phase contrast) of the X-rays. RESULTS: This system showed greater sharpness and spatial resolution, as well as an equal level of contrast resolution compared to a conventional X-ray system. The image sharpness in this system appears to be at least partially attributable to an edge-enhancing effect produced by the phase-contrast effect, which occurs when X-rays pass the surfaces of objects. CONCLUSION: This technique may be suitable for clinical use and may contribute to improved image-quality in general radiography.

Association analysis of adenosine A1 receptor gene (ADORA1) polymorphisms with schizophrenia in a Japanese population.

OBJECTIVE: The human adenosine A1 receptor gene (ADORA1) localizes to chromosome 1q32 is 76.8 kbp in length and contains six exons. ADORA1 is ubiquitously expressed in the central nervous system and clinical and pharmacological evidence suggest the involvement of adenosine neurotransmission in the pathogenesis of schizophrenia. Therefore, we investigated the contribution of genetic variations of ADORA1 to the pathophysiological mechanisms of Japanese schizophrenia patients. METHODS: We performed genetic analysis of 29 polymorphic markers in 200 schizophrenic patients and 210 healthy controls from the Kyushu region of Japan. In statistical analysis, we performed the univariate analysis with genotypes and allele frequencies, linkage disequilibrium (LD) analyses, multivariate analysis, haplotype analysis, and sliding window haplotype analysis. RESULTS: In univariate analysis, no statistical difference was shown, after Bonferroni correction. By LD analysis, however, we could not find any LD blocks. In haplotype analysis, a total of 359 haplotypes were estimated. In multivariate analysis, we found three statistically different markers. In sliding window haplotype analysis, there were four statistically different haplotypes. CONCLUSION: This is the first study describing the involvement of ADORA1 polymorphisms in the pathophysiological mechanisms of schizophrenia in a Japanese population. These results corroborate our previous pharmacological and neurochemical studies in the rat that have suggested an association between ADORA1 neurotransmission and the schizophrenic effects of the N-methyl-D-aspartate receptor antagonist phencyclidine. Thus, ADORA1 polymorphisms may represent good candidate markers for schizophrenia research and ADORA1 may be involved in the pathophysiological mechanisms of schizophrenia in Japanese populations.

Lysophosphatidic acid acyltransferase-beta is a prognostic marker and therapeutic target in gynecologic malignancies.

Lysophosphatidic acid, the substrate for lysophosphatidic acid acyltransferase beta (LPAAT-beta), is a well-studied autocrine/paracrine signaling molecule that is secreted by ovarian cancer cells and is found at elevated levels in the blood and ascites fluid of women with ovarian cancer. LPAAT-beta converts lysophosphatidic acid to phosphatidic acid, which functions as a cofactor in Akt/mTOR and Ras/Raf/Erk pathways. We report that elevated expression of LPAAT-beta was associated with reduced survival in ovarian cancer and earlier progression of disease in ovarian and endometrial cancer. Inhibition of LPAAT-beta using small interfering RNA or selective inhibitors, CT32521 and CT32228, two small-molecule noncompetitive antagonists representing two different classes of chemical structures, induces apoptosis in human ovarian and endometrial cancer cell lines in vitro at pharmacologically tenable nanomolar concentrations. Inhibition of LPAAT-beta also enhanced the survival of mice bearing ovarian tumor xenografts. Cytotoxicity was modulated by diacylglycerol effectors including protein kinase C and CalDAG-GEF1. LPAAT-beta was localized to the endoplasmic reticulum and overexpression was associated with redistribution of protein kinase C-alpha. These findings identify LPAAT-beta as a potential prognostic and therapeutic target in ovarian and endometrial cancer.

Chemistry-based RNA technologies: demonstration of usefulness of libraries of ribozymes and short hairpin RNAs (shRNAs).

Mechanism of action of hammerhead ribozymes has been investigated and their intracellular activities have been improved. Based on the improved ribozymes and more recently discovered natural RNAi, we have created libraries of both ribozymes and short hairpin RNAs (shRNAs). The introduction of a library of active ribozymes or shRNAs into cells, and the subsequent screening for phenotypic changes, allows the rapid identification of gene function.

Exploration of human miRNA target genes in neuronal differentiation.

MicroRNAs (miRNAs) are endogenous non-coding RNA molecules that inhibit protein translation in a sequence specific manner. We carried out microarray analyses of 180 human pre-miRNAs in neuroblastoma cell, SH-SY5Y, following stimulation by TPA. Twelve of the pre-miRNAs were up-regulated by the TPA stimulation. We also explored miRNA target genes associated with neuronal differentiation. Some miRNAs have complementarity with 3'UTR of the Notch1 gene, a regulator of neuronal differentiation, and luciferase assay showed that overexpression of these miRNAs reduced the luciferase activity of reporter genes containing the Notch1-3'UTR sequence. Our results suggest that miRNAs can be associated with TPA induced signalling pathways and expression of Notch1 gene.

Cell migration and metastasis as targets of small RNA-based molecular genetic analyses.

Metastatic tumor cells can migrate from one place to another in the body. This involves their adherence to host cell layers and subsequent transcellular movements by a complex process, molecular basis of which are yet to be clarified. Elucidation of genes functionally involved in metastasis may lead to deeper understanding of the mechanism of cell migration, and identification and designing of metastasis-modulating strategies for cancer therapeutics. We review here cell migration in tumor metastasis and the use of small RNA-based approaches to identify functional genes. We then describe our promising novel approach that uses randomized ribozyme libraries for identification of genes involved in cell migration, a consistent feature of metastatic cells.

Induction of DNA methylation and gene silencing by short interfering RNAs in human cells.

Double-stranded RNAs (dsRNAs) induce post-transcriptional gene silencing in several species of animal and plant. In plants, dsRNAs targeted to CpG islands within a promoter can also induce RNA-directed DNA methylation; however, it remains unclear whether gene silencing mediated by DNA methylation can be induced by dsRNAs in mammalian cells. Here, we demonstrate that short interfering RNAs (siRNAs; 21-25-nucleotide RNA molecules) induce DNA methylation and histone H3 methylation in human cells. Synthetic siRNAs targeted to CpG islands of an E-cadherin promoter induced significant DNA methylation and histone H3 lysine 9 methylation in both MCF-7 and normal mammary epithelial cells. As a result, these siRNAs repressed expression of the E-cadherin gene at the transcriptional level. In addition, disrupting the expression of either one of two DNA methyltransferases (DNMT1 or DNMT3B) by specific siRNAs abolished the siRNA-mediated methylation of DNA. Moreover, vector-based siRNAs targeted to the erbB2 (also known as HER2) promoter also induced DNA methylation in MCF-7 cells. Thus, siRNAs targeted to CpG islands within the promoter of a specific gene can induce transcriptional gene silencing by means of DNA-methyltransferase-dependent methylation of DNA in human cells, and might have potential as a new type of gene therapeutic agent.

Identification of metastasis-related genes in a mouse model using a library of randomized ribozymes.

Libraries of randomized ribozymes have considerable potential as tools for the identification of functional genes critically involved in a biological phenotype of interest in vitro. We have used a ribozyme library in an in vivo mouse model to identify genes related to metastasis. We injected weakly metastatic melanoma cells that had been treated with the library intravenously into mice. We then isolated ribozymes that accelerated metastasis from pulmonary tumors that had developed from metastasizing cells. As candidates for metastasis-related genes that were targets of the isolated ribozymes, we identified five unknown and three known genes: stromal interaction molecule 1 (STIM1), polymerase gamma2 accessory subunit (Polg2), and cytochrome P450, family 2, subfamily d, polypeptide 22 (Cyp2d22). Repression of four of these by small interfering RNAs indeed resulted in the accelerated mobility of cells in in vitro scratch-wound assay. The further characterization of these candidate genes would provide clues to the complex mechanism(s) of metastasis.

Non-kinase second-messenger signaling: new pathways with new promise.

Intercellular signaling by growth factors, hormones and neurotransmitters produces second messenger molecules such as cyclic adenosine monophosphate (cAMP) and diacylglycerol (DAG). Protein Kinase A and Protein Kinase C are the principal effector proteins of these prototypical second messengers in certain cell types. Recently, novel receptors for cAMP and DAG have been identified. These proteins, designated EPAC (Exchange Protein directly Activated by cAMP) or cAMP-GEF (cAMP regulated Guanine nucleotide Exchange Factor) and CalDAG-GEF (Calcium and Diacylglycerol regulated Guanine nucleotide Exchange Factor) or RasGRP (Ras Guanine nucleotide Releasing Protein) are able to mediate some of the physiologic effects of the second messengers in a protein-kinase-independent fashion. These proteins are exchange factors for Ras family GTPases that operate in pathways that run parallel to the classic kinase-dependent pathways. The rapidly emerging recognition of the functions of these "non-kinase" effectors in diverse processes such as insulin secretion, thymocyte development, asthma and malignant transformation creates new opportunities for discovery and identifies potential new therapeutic targets.

World of small RNAs: from ribozymes to siRNA and miRNA.

RNAs, besides bridging genetic information to proteins, the major determinants of bio-structures and functions, serve as active regulators of gene expression. Initiated nearly 20 years ago with ribozymes (the small RNAs with catalytic activity providing fine tuning of gene expression and function, used as molecular scissors and tools for gene discovery), an era of more complex and coordinated gene regulation by small RNAs, siRNA, and miRNA has recently started. Simple nucleotide complementarity results in highly ordered and regulated events, such as assembly of RNA and proteins, resulting in gene silencing either by mRNA degradation or suppression of translation. This article reviews our contributions to the understanding of structure, the function of small RNAs, their use in biotechnology, and the understanding of phenotypes such as apoptosis, metastasis, and differentiation.

Maxizyme technology.

Ribozymes are small and versatile nucleic acids that can cleave RNAs at specific sites. These molecules have great potential to be used as effective gene-therapeutic agents. However, because of the limitation for cleavable sequences within the target mRNA, in some cases conventional ribozymes have failed to exhibit precise cleavage specificity. A maxizyme is the dimer of minimized ribozymes (minizymes), which can specifically cleave two distinct target sites. The maxizyme also has an allosteric function in that it can form an active conformation and cleave the two target sites only when it recognizes two distinct target sites. We demonstrated previously that an allosterically controllable maxizyme was a powerful tool in the disruption of an abnormal chimeric RNA (bcr-abl) in cells and in mice. Furthermore, more than five custom-designed maxizymes have clearly demonstrated these allosteric functions in vitro and in vivo. Thus, maxizyme technology is not limited to one specific case, but may have broad general applicability in molecular biology and in molecular gene therapy.

LIM kinase-2 targeting as a possible anti-metastasis therapy.

BACKGROUND: Metastatic properties of tumors involve movement of cancerous cells from one place to another and tissue invasion. Metastatic cells have altered cell adhesion and movement that can be examined by in vitro chemotaxis assays. The Rho/ROCK/LIM kinase pathway is one of the major signaling pathways involved in tumor metastasis. It is involved in the regulation of the actin cytoskeleton. Using the randomized ribozyme library, we initially found that metastatic human fibrosarcoma cells harboring ribozyme specific for ROCK lose their metastatic properties. In this study, we have determined the effect of ribozymes specific for LIM kinase-2 on metastatic and proliferative phenotypes of human fibrosarcoma cells. METHODS: We attempted to target LIM kinase-2 (LIMK-2) expression by hammerhead ribozymes (Rz) in human metastatic fibrosarcoma cells. An effective ribozyme was selected based on the expression analysis. Cells were stably transfected with Rz specifically effective for LIMK-2 and were examined for metastatic and proliferative properties. RESULTS: Analyses of cellular phenotypes such as cell proliferation, cell migration and colony-forming efficiency revealed that the suppression of LIMK-2 expression in human fibrosarcoma cells limits their migration and dense colony-forming efficiency without affecting cell proliferation rate or viability. CONCLUSIONS: Specific targeting of metastatic and malignant properties of tumor cells by LIMK-2 ribozyme may serve as an effective therapy for invasive tumors with minimum effect on the surrounding normal cells.

An RNA-dependent protein kinase is involved in tunicamycin-induced apoptosis and Alzheimer's disease.

Various types of stress, such as disruption of calcium homeostasis, inhibition of protein glycosylation and reduction of disulfide bonds, result in accumulation of misfolded proteins in the endoplasmic reticulum (ER). The initial cellular response involves removal of such proteins by the ER, but excessive and/or long-term stress results in apoptosis. In this study, we used a randomized ribozyme library and ER stress-mediated apoptosis (tunicamycin-induced apoptosis) in SK-N-SH human neuroblastoma cells as a selective phenotype to identify factors involved in this process. We identified a double-stranded RNA-dependent protein kinase (PKR) as one of the participants in this process. The level of nuclear PKR was elevated, but the level of cytoplasmic PKR barely changed in tunicamycin-treated SK-N-SH cells. Furthermore, tunicamycin also raised levels of phosphorylated PKR in the nucleus. We also detected the accumulation of phosphorylated PKR in the nuclei of autopsied brain tissues in Alzheimer's disease. Thus, PKR might play a role in ER stress-induced apoptosis and in Alzheimer's disease.