|ognizant Communication Corporation|
VOLUME 11, NUMBERS 5/6
Gene Expression, Vol. 11, pp. 211-219
1052-2166/04 $20.00 + .00
Copyright © 2004 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Rubratoxin B Elicits Antioxidative and DNA Repair Responses in Mouse Brain
V. Sava,1,2 D. Mosquera,1,2 S. Song,1,2 T. Stedeford,2,4 K. Calero,1 F. Cardozo-Pelaez,3 R. Harbison,4 and J. Sanchez-Ramos1,2
1Neurology, University of South Florida, Tampa, FL
2Research Service, James Haley VA, Tampa, FL
3Department of Pharmaceutical Sciences, University of Montana, Missoula, MT
4College of Public Health, University of South Florida, Tampa, FL
Rubratoxin B (RB) is a mycotoxin with potential neurotoxic effects that have not yet been characterized. Based on existing evidence that RB interferes with mitochondrial electron transport to produce oxidative stress in peripheral tissues, we hypothesized that RB would produce oxidative damage to macromolecules in specific brain regions. Parameters of oxidative DNA damage and repair, lipid peroxidation, and superoxide dismutase (SOD) activity were measured across six mouse brain regions 24 h after administration of a single dose of RB. Lipid peroxidation and oxidative DNA damage were either unchanged or decreased in all brain regions in RB-treated mice compared with vehicle-treated mice. Concomitant with these decreased indices of oxidative macromolecular damage, SOD activity was significantly increased in all brain regions. Oxyguanosine glycosylase activity (OGG1), a key enzyme in the repair of oxidized DNA, was significantly increased in three brain regions--cerebellum (CB), caudate/putamen (CP), and cortex (CX)--but not in the hippocampus (HP), midbrain (MB), and pons/medulla (PM). The RB-enhanced OGG1 catalytic activity in these brain regions was not due to increased OGG1 protein expression, but was a result of enhanced catalytic activity of the enzyme. In conclusion, specific brain regions responded to an acute dose of RB by significantly altering SOD and OGG1 activities to maintain the degree of oxidative DNA damage equal to, or less than, that of normal steady-state levels.
Key words: Rubratoxin B; Oxidative stress; DNA damage and repair; Superoxide dismutase (SOD); Mouse brain regions
Address correspondence to Dr. Juan R. Sanchez-Ramos, The Helen E. Ellis Professor of Neurology, University of South Florida, Department of Neurology (MDC 55), 12901 Bruce B. Downs Blvd., Tampa, FL 33612. Tel: (813) 974-6022; Fax: (813) 974-7200; E-mail: firstname.lastname@example.org
Mouse Huntington's Disease Homolog mRNA Levels: Variation and Allele Effects
Karen T. Dixon,1 Jamie A. Cearley,1 Jesse M. Hunter,1 and Peter J. Detloff1,2
1Department of Biochemistry and Molecular Genetics and 2Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294
Huntington's disease homolog (Hdh) mRNA levels in mice with different Hdh alleles were measured. Brain Hdh mRNA levels varied up to threefold in genetically identical wild-type mice, indicating nongenetic factors influence Hdh expression. Striatal Hdh mRNA levels from an allele with a repeat expanded to 150 CAGs were diminished compared with wild-type and showed variation that might contribute to phenotypic variability in the Hdh(CAG)150 knock-in mouse model. To determine whether Hdh mRNA levels are tightly regulated, we assessed these levels in mice heterozygous for a deletion of the Hdh promoter. The loss of one allele reduced Hdh mRNA levels in most tissues, suggesting mechanisms to maintain Hdh mRNA levels are not in effect and should not impede therapies designed to destroy mutant huntingtin mRNA. Finally, we found a correlation between tissue mRNA levels and the susceptibility of the Hdh locus to Cre-mediated deletion. The two tissues with the highest levels of Hdh mRNA, testes and brain, were the only tissues susceptible to Cre-mediated recombination between loxP sites at Hdh locus. In contrast, the same Cre-expressing line caused recombination in every tissue for loxP sites at another genomic location. The pattern of Cre susceptibility at Hdh suggests a correlation between chromatin accessibility and high levels of Hdh expression in testes and brain.
Key words: Knock-out; Knock-in; Cre recombinase; Gene expression; Real-time PCR; Chromatin structure
Address correspondence to Peter J. Detloff, KAUL 540, 720 20th Street South, Birmingham, AL 35294. Tel: (205) 975-8157; Fax: (205) 975-2188; E-mail: email@example.com
A High Proportion of Chromosome 21 Promoter Polymorphisms Influence Transcriptional Activity
Paul R. Buckland, Sharol L. Coleman, Bastiaan Hoogendoorn, Carol Guy, S. Kaye Smith, and Michael C. O'Donovan
Department of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
We have sought to obtain an unbiased estimate of the proportion of polymorphisms in promoters of human genes that have functional effects. We carried out polymorphism discovery on a randomly selected group of 51 gene promoters mapping to human chromosome 21 and successfully analyzed the effect on transcription of 38 of the sequence variants. To achieve this, a total of 53 different haplotypes from 20 promoters were cloned into a modified pGL3 luciferase reporter gene vector and were tested for their abilities to promote transcription in HEK293t and JEG-3 cells. Up to seven (18%) of the 38 tested variants altered transcription by 1.5-fold, confirming that a surprisingly high proportion of promoter region polymorphisms are likely to be functionally important. The functional variants were distributed across the promoters of CRYAA, IFNAR1, KCNJ15, NCAM2, IGSF5, and B3GALT5. Three of the genes (NCAM2, IFNAR1, and CRYAA) have been previously associated with human phenotypes and the polymorphisms we describe here may therefore play a role in those phenotypes.
Key words: CRYAA; Cataracts; IFNAR1; Galactosyltransferase; KCNJ15; Down syndrome; NCAM2; B3GALT5; Hepatitis C
Address correspondence to Paul R. Buckland, Department of Psychological Medicine, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK. Tel: (0)2920 744840; Fax: (0)2920 747839.
b-Catenin Associates With Human Parainfluenza Virus Type 3 Ribonucleoprotein Complex and Activates Transcription of Viral Genome RNA In Vitro
Santanu Bose and Amiya. K. Banerjee
Department of Virology, The Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, OH 44195
Several studies have indicated that human parainfluenza virus type 3 (HPIV-3) requires polymeric actin for transcription of its genome RNA in vitro and in vivo. In the current study, we have identified b-catenin, an actin-bound protein, as one of the transcriptional activators for HPIV-3 genome RNA. b-Catenin was packaged within the purified HPIV-3 virions and was associated with the HPIV-3 ribonucleoproteins (RNP) from infected cells. Moreover, purified b-catenin interacted with bacterially expressed HPIV-3 nucleocapsid protein (N) and phosphoprotein (P) fused to glutathione S-transferase (GST). Double-labeled immunofluorescent confocal microscopic analysis revealed colocalization of b-catenin with HPIV-3 RNP at cell periphery in infected cells. The HPIV-3 RNP-associated b-catenin functioned as a transactivator of HPIV-3 genome, because purified b-catenin stimulated transcription of viral RNP in an in vitro transcription assay. These results demonstrate that b-catenin, a multifunctional protein that is involved in cell-cell adhesion and embryogenesis, acts as one of the transcriptional activators of HPIV-3 genome RNA.
Key words: b-Catenin; Human parainfluenza virus type 3; In vitro transcription; Actin
Address correspondence to Dr. Santanu Bose, Ph.D., Department of Virology, The Cleveland Clinic Foundation, Lerner Research Institute, Room NN-10, 9500 Euclid Avenue, Cleveland, OH 44195. Tel: (216) 444-0626; Fax: (216) 444-2998; E-mail: firstname.lastname@example.org
Body Weight and Abdominal Fat Gene Expression Profile in Response to a Novel Hydroxycitric Acid-Based Dietary Supplement
Sashwati Roy,1 Cameron Rink,1 Savita Khanna,1 Christina Phillips,1 Debasis Bagchi,2 Manashi Bagchi,2 and Chandan K. Sen1
1Laboratory of Molecular Medicine, Department of Surgery,
The Ohio State University Medical Center, Columbus, OH 43210
2School of Pharmacy and Health Professions, Creighton University Medical Center, Omaha, NE 68178
Obesity is a global public health problem, with about 315 million people worldwide estimated to fall into the WHO-defined obesity categories. Traditional herbal medicines may have some potential in managing obesity. Botanical dietary supplements often contain complex mixtures of phytochemicals that have additive or synergistic interactions. The dried fruit rind of Garcinia cambogia, also known as Malabar tamarind, is a unique source of (-)-hydroxycitric acid (HCA), which exhibits a distinct sour taste and has been safely used for centuries in Southeastern Asia to make meals more filling. Recently it has been demonstrated that HCA-SX or Super Citrimax, a novel derivative of HCA, is safe when taken orally and that HCA-SX is bioavailable in the human plasma as studied by GC-MS. Although HCA-SX has been observed to be conditionally effective in weight management in experimental animals as well as in humans, its mechanism of action remains to be understood. We sought to determine the effects of low-dose oral HCA-SX on the body weight and abdominal fat gene expression profile of Sprague-Dawley rats. We observed that at doses relevant for human consumption dietary HCA-SX significantly contained body weight growth. This response was associated with lowered abdominal fat leptin expression while plasma leptin levels remained unaffected. Repeated high-density microarray analysis of 9960 genes and ESTs present in the fat tissue identified a small set (~1% of all genes screened) of specific genes sensitive to dietary HCA-SX. Other genes, including vital genes transcribing for mitochondrial/nuclear proteins and which are necessary for fundamental support of the tissue, were not affected by HCA-SX. Under the current experimental conditions, HCA-SX proved to be effective in restricting body weight gain in adult rats. Functional characterization of HCA-SX-sensitive genes revealed that upregulation of genes encoding serotonin receptors represent a distinct effect of dietary HCA-SX supplementation.
Key words: Herbal; Obesity; Leptin; Rat; Serotonin
Address correspondence to Dr. Chandan K. Sen, 512 Davis Heart & Lung Research Institute, The Ohio State University Medical Center, 473 W. 12th Avenue, Columbus, OH 43210. Tel: (614) 247-7658; Fax: (614) 247-7818; E-mail: email@example.com
Septin 3 Gene Polymorphism in Alzheimer's Disease
Masanori Takehashi,1 Tyler Alioto,2 Todd Stedeford,3 Amanda S. Persad,4 Marek Banasik,3 Eliezer Masliah,5 Seigo Tanaka,1 and Kunihiro Ueda1
1Laboratory of Molecular Clinical Chemistry, Institute for
Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
2Department of Molecular and Cell Biology, University of California Berkeley,
3Laboratory of Toxicology and Risk Assessment, Institute of Coal Chemistry, Polish Academy of Sciences, 44-121 Gliwice, Poland
4Infection Control Department, Florida Hospital, Orlando, FL 32803-1248
5Department of Neurosciences, School of Medicine, University of California San Diego, La Jolla, CA 92093-0624
Septin 3 is a novel member of the septin subfamily of GTPase domain proteins that was recently identified in human neuronal cells. These proteins are involved in vesicle trafficking, neurite outgrowth, and neurofibrillary tangle formation; however, the expression and functional role of septin 3 in normal neuronal tissues and as an etiological agent in neurological disorders is currently unclear. To further characterize these parameters, the present study analyzed the expression of three isoforms of septin 3 (A, B, and C) in fetal and adult human brains and polymorphism of the septin 3 exon 11 microsatellite in control, pure Alzheimer's disease (AD), Lewy body variant (LBV) of AD, and Parkinson's disease. Septin 3 mRNAs for isoforms A and B, but not C, were detected in the frontal cortex of fetus and adult human samples, as measured by reverse transcription-coupled polymerase chain reaction. Genotype analyses indicated that polymorphic septin 3 alleles were distributed in two peaks of frequency in both control and disease groups. Categorization of the alleles into short (S) and long (L) types revealed a significant difference between AD patients and controls (p = 0.034 by chi-square test). Furthermore, the S-allele homozygosity was significantly underrepresented in AD compared with control (p = 0.015 by chi-square test). These results suggest that polymorphism in exon 11 of septin 3 may have a determinative role in the pathogenesis of AD.
Key words: Septin 3; Isoforms; Polymorphism; Alzheimer's disease; Lewy body variant of Alzheimer's disease; Parkinson's disease; CYP2D6
Address correspondence to Seigo Tanaka, M.D., Ph.D., Laboratory of Molecular Clinical Chemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. Tel.: +81-774-38-3225; Fax: +81-774-38-3226; E-mail: firstname.lastname@example.org
Expression of Septin 3 Isoforms in Human Brain
Masanori Takehashi,1 Seigo Tanaka,1 Todd Stedeford,2 Marek Banasik,2 Hiroe Tsukagoshi-Nagai,3 Noriaki Kinoshita,3 Toshio Kawamata,4 and Kunihiro Ueda1
1Laboratory of Molecular Clinical Chemistry, Institute for
Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan
2Laboratory of Toxicology and Risk Assessment, Institute of Coal Chemistry, Polish Academy of Sciences, 44-121 Gliwice, Poland
3Department of Research and Development, IBL Co., Ltd., Fujioka, Gunma 375-0005, Japan
4Faculty of Health Science, Kobe University School of Medicine, Kobe 654-0142, Japan
Septin 3 is a novel member of the septin subfamily of GTPase domain proteins. Human septin 3 was originally cloned during a screening of genes expressed in human teratocarcinoma cells induced to differentiate with retinoic acid. Alternative splicing of the septin 3 gene transcript produces two isoforms, A and B, in the human brain, though their regional expression and physiological function remain to be determined. The purpose of the present study was to identify the expression patterns of human septin 3 isoforms in normal human brain and a human neuroblastoma cell line, SH-SY5Y, after retinoic acid-induced differentiation. The expression and distribution patterns of septin 3 isoforms A and B were similar and resembled that of another septin, CDCrel-1. Septin 3A and 3B were expressed in normal human brain in a region-specific manner, with the highest level in the temporal cortex and hippocampus and the lowest level in the brainstem regions. Prominent immunoreactivity was observed diffusely in the neocortices in association with neuropils and punctate structures suggestive of synaptic junctions. Immunoprecipitation studies revealed that septin 3A, 3B, and CDCrel-1 form a complex in the frontal cortex of human brain. These findings, taken together, suggest that septin 3A and 3B, along with CDCrel-1, play some fundamental role(s) in synaptogenesis and neuronal development.
Key words: CNS; Neurons; Septins; Differentiation
Address correspondence to Seigo Tanaka, M.D., Ph.D., Laboratory of Molecular Clinical Chemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan. Tel: +81-774-38-3225; Fax: +81-774-38-3226; E-mail: email@example.com