|ognizant Communication Corporation|
VOLUME 13, NUMBER 2
Gene Expression, Vol. 13, pp. 67-71
1052-2166/06 $90.00 + .00
Copyright © 2006 Cognizant Comm. Corp.
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Arginine to Cysteine Mutation (R499C) Found in a Japanese Patient With Complete Myeloperoxidase Deficiency
Amanda S. Persad,1 Yosuke Kameoka,2 Shuji Kanda,3 Yoshiyuki Niho,3 and Kazuo Suzuki1
1Department of Bioactive Molecules,
National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku,
Tokyo 162-8640, Japan
2Division of Genetic Resources, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
3Chihaya Hospital, Fukuoka, Japan
Animal models suggest that a deficiency in myeloperoxidase (MPO; EC 18.104.22.168), a lysosomal hemoprotein involved in host defense, may be associated with a decreased level of immunity. A nonsynonymous mutation, resulting in an arginine to cysteine substitution (Arg499Cys or R499C), has been identified in the exon 9 genetic coding region of a Japanese patient with complete MPO deficiency. Genetic analysis revealed that the mRNA of the patient could be correctly transcribed then further translated into a peptide sequence. However, the Western blot analysis confirmed the absence of MPO peptides. An initial screening assay of the patient's blood exhibited an abnormal hematograph, and no MPO activity was detected. To determine if this mutation might be associated with MPO deficiency, DNA samples for 387 controls were examined. Genetic analysis was performed using standard PCR techniques for amplification and sequencing. None of the control samples possessed the R499C substitution. This mutation is in close proximity to a different mutation (G501S) previously found in another Japanese MPO-deficient patient, and the amino acid, H502, which is strongly involved in heme binding, leading to the speculation that heme binding may play a role in complete MPO deficiency.
Key words: Myeloperoxidase (MPO); Myeloperoxidase deficiency; Arg499Cys; R499C
Address correspondence to Kazuo Suzuki, Chief, Department of Bioactive Molecules, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Tel: +81-3-5285-1111, ext. 2329; Fax: +81-3-5285-1160; E-mail: email@example.com
PKCd Alternatively Spliced Isoforms Modulate Cellular Apoptosis in Retinoic Acid-Induced Differentiation of Human NT2 Cells and Mouse Embryonic Stem Cells
Niketa A. Patel,1,2 Shijie S. Song,1 and Denise R. Cooper1,2
1James A. Haley Veterans Hospital,
Research Service, Tampa, FL 33612, USA
2Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33612, USA
NT2 cells are a human teratocarcinoma cell line that, upon treatment with retinoic acid (RA), begin differentiating into a neuronal phenotype. The transformation of undifferentiated NT2 cells into hNT neurons presents an opportunity to investigate the mechanisms involved in neurogenesis because a key component is cell apoptosis, which is essential for building neural networks. Protein kinase Cd (PKCd) plays an important role as a mediator of cellular apoptosis in response to various stimuli. PKCd (dI) is proteolytically cleaved at its hinge region (V3) by caspase 3 and the catalytic fragment is sufficient to induce apoptosis in various cell types. Mouse PKCdII is rendered caspase resistant due to an insertion of 78 bp within the caspase recognition site in its V3 domain. No functional role has been attributed to these alternatively spliced variants of PKCd. We sought to find a correlation between the onset of apoptosis, neurogenesis, and the expression of PKCd isoforms. Our results indicate that RA regulates the expression of PKCd alternative splicing variants in NT2 cells. Further, overexpression of PKCδI promotes apoptosis while PKCdII overexpression shields the cells from apoptosis. This is the first report to attribute physiological function to PKCdI and -dII isoforms. Next we demonstrated that mouse embryonic stem cells differentiate in vitro into dopaminergic neurons upon stimulation with RA and ciliary neurotrophic factor. These cells showed a simultaneous increase in tyrosine hydroxylase and PKCδII expression. We suggest that the molecular mechanisms regulating differentiation and apoptosis could be understood by alternative expression of PKCδ isoforms.
Key words: Alternative splicing; Apoptosis; Mouse embryonic stem cells; NT2 cells; Neurons; PKCd Retinoic acid
Address correspondence to Niketa A. Patel, Ph.D., J. A. Haley Veterans Hospital (VAR 151), 13000 Bruce B. Downs Blvd., Tampa, FL 33612, USA. Tel: (813) 972-2000, ext. 7121; Fax: (813) 972-7623; E-mail: firstname.lastname@example.org
Transcriptional Profiling of the Cell Cycle Checkpoint Gene Krüppel-Like Factor 4 Reveals a Global Inhibitory Function in Macromolecular Biosynthesis
Erika M. Whitney,1 Amr M. Ghaleb,1 Xinming Chen,1 and Vincent W. Yang1,2
1Division of Digestive Diseases, Department
of Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA
2Department of Hematology and Oncology, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322, USA
Krüppel-like factor 4 (KLF4; also known as gut-enriched Krüppel-like factor or GKLF) is known to exhibit checkpoint function during the G1/S and G2/M transitions of the cell cycle. The mechanism by which KLF4 exerts these effects is not fully established. Here we investigated the expression profile of KLF4 in an inducible system over a time course of 24 h. Using oligonucleotide microarrays, we determined that the fold changes relative to control in expression levels of KLF4 exhibited a time-dependent increase from 3- to 20-fold between 4 and 24 h following KLF4 induction. During this period and among a group of 473 cell cycle regulatory genes examined, 96 were positively correlated and 86 were negatively correlated to KLF4's expression profile. Examples of upregulated cell cycle genes include those encoding tumor suppressors such as MCC and FHIT, and cell cycle inhibitors such as CHES1 and CHEK1. Examples of downregulated genes include those that promote the cell cycle including several cyclins and those required for DNA replication. Unexpectedly, several groups of genes involved in macromolecular synthesis, including protein biosynthesis, transcription, and cholesterol biosynthesis, were also significantly inhibited by KLF4. Thus, KLF4 exerts a global inhibitory effect on macromolecular biosynthesis that is beyond its established role as a cell cycle inhibitor.
Key words: Cell cycle; Checkpoint; Microarray; Cholesterol; Ribosomal proteins; Transcription
Address correspondence to Vincent W. Yang, Division of Digestive Diseases, Department of Medicine, Emory University School of Medicine, 201 Whitehead Biomedical Research Building, 615 Michael Street, Atlanta, GA 30322, USA. Tel: (404) 727-5638; Fax: (404) 727-5767; E-mail: email@example.com
Inhibition of Translation by Consecutive Rare Leucine Codons in E. coli: Absence of Effect of Varying mRNA Stability
Ping Shu,* Huacheng Dai,** Wenwu Gao,*** and Emanuel Goldman
Department of Microbiology & Molecular Genetics, New Jersey Medical School, University of Medicine & Dentistry of New Jersey, Newark, NJ 07101-1709, USA
Consecutive homologous codons that are rarely used in E. coli are known to inhibit translation to varying degrees. As few as two consecutive rare arginine codons exhibit a profound inhibition of translation when they are located in the 5? portion of a gene in E. coli. We have previously shown that nine consecutive rare CUA leucine codons cause almost complete inhibition of translation when they are placed after the 13th codon of a test message (although they do not inhibit translation when they are placed in the middle of the message). In the present work, we report that five consecutive rare CUA leucine codons exhibit approximately a threefold inhibition of translation when they are similarly placed after the 13th codon of a test message, compared to five consecutive common CUG leucine codons, in a T7 RNA polymerase-driven system. Further, by removing RNase III processing sites at the 3´ ends of the mRNAs, we have manipulated the stability of the mRNAs encoding the test and control messages to see if decreasing mRNA stability might have an effect on the extent of translation inhibition by the rare leucine codons. However, the inhibition with the less stable mRNAs was similar to that with the stable mRNAs, approximately 3.4-fold, indicating that mRNA stability per se does not have a major influence on the effects of rare codons in this system.
Key words: CUA leucine codon; Codon bias; Inhibition by rare codons; T7 expression systems; RNase III and mRNA stability
Address correspondence to Emanuel Goldman, Department of Microbiology & Molecular Genetics, New Jersey Medical School, University of Medicine & Dentistry of New Jersey, 225 Warren Street, P.O.B. 1709, Newark, NJ 07101-1709, USA. Tel: 973-972-4483, ext. 24367; Fax: 972-972-3644; E-mail: firstname.lastname@example.org
*Present address: Department of Surgery, New Jersey
Medical School, University of Medicine & Dentistry of New Jersey, 185
South Orange Avenue, Newark, NJ 07103, USA.
**Present address: Department of Pathology, Robert Wood Johnson Medical School, University of Medicine & Dentistry of New Jersey, 675 Hoes Lane, Piscataway, NJ 08854, USA.
***Present address: Department of Veterans Affairs, VA Medical Center, 200 Springs Road, Building 17, Bedford, MA 01730, USA.
Global Gene Expression Profiling of Dimethylnitrosamine-Induced Liver Fibrosis: From Pathological and Biochemical Data to Microarray Analysis
Li-Jen Su,1,2 Shih-Lan Hsu,3 Jyh-Shyue Yang,3 Huei-Hun Tseng,4 Shiu-Feng Huang,4 and Chi-Ying F. Huang1,2,4,5,6
1Graduate Institute of Life Sciences,
National Defense Medical Center, Taipei 114, Taiwan
2National Institute of Cancer Research, National Health Research Institutes, Taipei 114, Taiwan
3Department of Education and Research, Taichung Veterans General Hospital, Taichung 407, Taiwan
4Division of Molecular and Genomic Medicine, National Health Research Institutes, Miaoli County 350, Taiwan
5Institute of Biotechnology in Medicine, National Yang-Ming University, Taipei 112, Taiwan
6Department of Computer Science and Information Engineering, National Taiwan University, Taipei 106, Taiwan
The development of hepatocellular carcinoma (HCC) is generally preceded by cirrhosis, which occurs at the end stage of fibrosis. This is a common and potentially lethal problem of chronic liver disease in Asia. The development of microarrays permits us to monitor transcriptomes on a genome-wide scale; this has dramatically speeded up a comprehensive understanding of the disease process. Here we used dimethylnitrosamine (DMN), a nongenotoxic hepatotoxin, to induce rat necroinflammatory and hepatic fibrosis. During the 6-week time course, histopathological, biochemical, and quantitative RT-PCR analyses confirmed the incidence of necroinflammatory and hepatic fibrosis in this established rat model system. Using the Affymetrix microarray chip, 256 differentially expressed genes were identified from the liver injury samples. Hierarchical clustering of gene expression using a gene ontology database allowed the identification of several stage-specific characters and functionally related clusters that encode proteins related to metabolism, cell growth/maintenance, and response to external challenge. Among these genes, we classified 44 potential necroinflammatory-related genes and 62 potential fibrosis-related markers or drug targets based on histopathological scores. We also compared the results with other data on well-known markers and various other microarray datasets that are available. In conclusion, we believe that the molecular picture of necroinflammatory and hepatic fibrosis from this study may provide novel biological insights into the development of early liver damage molecular classifiers than can be used for basic research and in clinical applications. A public accessible website is available at http://LiverFibrosis.nchc.org.tw:8080/LF.
Key words: Dimethylnitrosamine; Histopathology; Necroinflammatory; Fibrosis; Biochemical data; Microarray; Quantitative RT-PCR; Tgfb1; Timp1; Spp1
Address correspondence to Chi-Ying F. Huang, National Institute of Cancer Research, National Health Research Institutes, 9F Room 9320, No. 161, Sec. 6, Min-Chuan East Road, Taipei 114, Taiwan. Tel: (886)-2-26534401, ext. 25180 or 25181; Fax: (886)-2-2792-9654; E-mail: email@example.com
Cholinergic Differentiation Occurs Early in Mouse Sympathetic Neurons and Requires Phox2b
K. Huber and U. Ernsberger
Institut für Anatomie und Zellbiologie III, Interdisziplinäres Zentrum für Neurowissenschaften, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany
The generation of neurotransmitter identity in the autonomic nervous system is a classical model system to study the development of neuronal diversity. Analysis of the expression of genes coding for enzymes of noradrenaline biosynthesis in the sympathoadrenal system allowed the characterization of factors involved in the differentiation of the noradrenergic transmitter phenotype. The development of cholinergic properties in the autonomic system is less well understood. Here we show that expression of mRNAs for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT), both encoded by the cholinergic gene locus, is induced in mouse sympathetic ganglia at embryonic day 11 (E11). Positive cells amount to more than 50% of Phox2b-positive sympathetic cells at cervical levels. The proportion declines caudally, decreasing to ~20% of Phox2b-positive cells at lower thoracic levels. In the adrenal anlage, ChAT and VAChT mRNA are largely undetectable at E11 and E13. In mice homozygous for a mutational inactivation of the transcription factor Phox2b, ChAT and VAChT mRNA expression is absent from sympathetic ganglia. The data show that expression from the cholinergic gene locus is regulated differently in sympathetic neurons and adrenal chromaffin cells. Phox2b is required for development of cholinergic neurons but does not suffice to support cholinergic properties in chromaffin cells.
Key words: Sympathetic neuron; Adrenal chromaffin cell; Choline acetyltransferase; Vesicular acetylcholine transporter; Phox2b mutant; Development
Address correspondence to Uwe Ernsberger, Institut für Anatomie und Zellbiologie III, Interdisziplinäres Zentrum für Neurowissenschaften, Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany. Tel: 06221-548344; Fax: 06221-545604; E-mail: firstname.lastname@example.org