bluuebunny/crossref_metadata_2025 huggingface dataset:
The PTEN-regulating microRNA miR-26a is amplified in high-grade glioma and facilitates gliomagenesis in vivo
Authors: Huse Jason T. , Brennan Cameron , Hambardzumyan Dolores , Wee Boyoung , Pena John , Rouhanifard Sara H. , Sohn-Lee Cherin , le Sage Carlos , Agami Reuven , Tuschl Thomas , Holland Eric C.
Published: June 2009

Activated oncogenic signaling is central to the development of nearly all forms of cancer, including the most common class of primary brain tumor, glioma. Research over the last two decades has revealed the particular importance of the Akt pathway, and its molecular antagonist PTEN (phosphatase and tensin homolog), in the process of gliomagenesis. Recent studies have also demonstrated that microRNAs (miRNAs) may be responsible for the modulation of cancer-implicated genes in tumors. Here we report the identification miR-26a as a direct regulator of PTEN expression. We also show that miR-26a is frequently amplified at the DNA level in human glioma, most often in association with monoallelic PTEN loss. Finally, we demonstrate that miR-26a-mediated PTEN repression in a murine glioma model both enhances de novo tumor formation and precludes loss of heterozygosity and the PTEN locus. Our results document a new epigenetic mechanism for PTEN regulation in glioma and further highlight dysregulation of Akt signaling as crucial to the development of these tumors.
Integration of microRNA miR-122 in hepatic circadian gene expression
Authors: Gatfield David , Le Martelot Gwendal , Vejnar Charles E. , Gerlach Daniel , Schaad Olivier , Fleury-Olela Fabienne , Ruskeepää Anna-Liisa , Oresic Matej , Esau Christine C. , Zdobnov Evgeny M. , Schibler Ueli
Published: June 2009

In liver, most metabolic pathways are under circadian control, and hundreds of protein-encoding genes are thus transcribed in a cyclic fashion. Here we show that rhythmic transcription extends to the locus specifying miR-122, a highly abundant, hepatocyte-specific microRNA. Genetic loss-of-function and gain-of-function experiments have identified the orphan nuclear receptor REV-ERBα as the major circadian regulator of mir-122 transcription. Although due to its long half-life mature miR-122 accumulates at nearly constant rates throughout the day, this miRNA is tightly associated with control mechanisms governing circadian gene expression. Thus, the knockdown of miR-122 expression via an antisense oligonucleotide (ASO) strategy resulted in the up- and down-regulation of hundreds of mRNAs, of which a disproportionately high fraction accumulates in a circadian fashion. miR-122 has previously been linked to the regulation of cholesterol and lipid metabolism. The transcripts associated with these pathways indeed show the strongest time point-specific changes upon miR-122 depletion. The identification of Pparβ/δ and the peroxisome proliferator-activated receptor α (PPARα) coactivator Smarcd1/Baf60a as novel miR-122 targets suggests an involvement of the circadian metabolic regulators of the PPAR family in miR-122-mediated metabolic control.
Dynamic and complex transcription factor binding during an inducible response in yeast
Authors: Ni Li , Bruce Can , Hart Christopher , Leigh-Bell Justine , Gelperin Daniel , Umansky Lara , Gerstein Mark B. , Snyder Michael
Published: June 2009

Complex biological processes are often regulated, at least in part, by the binding of transcription factors to their targets. Recently, considerable effort has been made to analyze the binding of relevant factors to the suite of targets they regulate, thereby generating a regulatory circuit map. However, for most studies the dynamics of binding have not been analyzed, and thus the temporal order of events and mechanisms by which this occurs are poorly understood. We globally analyzed in detail the temporal order of binding of several key factors involved in the salt response of yeast to their target genes. Analysis of Yap4 and Sko1 binding to their target genes revealed multiple temporal classes of binding patterns: (1) constant binding, (2) rapid induction, (3) slow induction, and (4) transient induction. These results demonstrate that individual transcription factors can have multiple binding patterns and help define the different types of temporal binding patterns used in eukaryotic gene regulation. To investigate these binding patterns further, we also analyzed the binding of seven other key transcription factors implicated in osmotic regulation, including Hot1, Msn1, Msn2, Msn4, Skn7, and Yap6, and found significant coassociation among the different factors at their gene targets. Moreover, the binding of several key factors was correlated with distinct classes of Yap4- and Sko1-binding patterns and with distinct types of genes. Gene expression studies revealed association of Yap4, Sko1, and other transcription factor-binding patterns with different gene expression patterns. The integration and analysis of binding and expression information reveals a complex dynamic and hierarchical circuit in which specific combinations of transcription factors target distinct sets of genes at discrete times to coordinate a rapid and important biological response.
The Rho-linked mental retardation protein oligophrenin-1 controls synapse maturation and plasticity by stabilizing AMPA receptors
Authors: Nadif Kasri Nael , Nakano-Kobayashi Akiko , Malinow Roberto , Li Bo , Van Aelst Linda
Published: June 2009

Oligophrenin-1 (OPHN1) encodes a Rho-GTPase-activating protein (Rho-GAP) whose loss of function has been associated with X-linked mental retardation (MR). The pathophysiological role of OPHN1, however, remains poorly understood. Here we show that OPHN1 through its Rho-GAP activity plays a critical role in the activity-dependent maturation and plasticity of excitatory synapses by controlling their structural and functional stability. Synaptic activity through NMDA receptor activation drives OPHN1 into dendritic spines, where it forms a complex with AMPA receptors, and selectively enhances AMPA-receptor-mediated synaptic transmission and spine size by stabilizing synaptic AMPA receptors. Consequently, decreased or defective OPHN1 signaling prevents glutamatergic synapse maturation and causes loss of synaptic structure, function, and plasticity. These results imply that normal activity-driven glutamatergic synapse development is impaired by perturbation of OPHN1 function. Thus, our findings link genetic deficits in OPHN1 to glutamatergic dysfunction and suggest that defects in early circuitry development are an important contributory factor to this form of MR.
Molecular orchestration of differentiation and function of regulatory T cells
Authors: Lu Li-Fan , Rudensky Alexander
Published: June 2009

During the last decade, a unique mechanism of negative regulation of immune responses and inflammation by a dedicated population of so-called regulatory T cells (Treg) has become a focus of intensive investigation. Through the discovery of transcription factor Foxp3 as a central molecular determinant of differentiation and function of Treg cells, the complex biology of these cells, including maintenance of immunological tolerance to “self” and regulation of immune responses to pathogens, commensals, and tumors, has become amenable to mechanistic studies. In this review, we discuss the molecular aspects of Treg cell lineage commitment, maintenance, and function.
Transcription termination by nuclear RNA polymerases
Authors: Richard Patricia , Manley James L.
Published: June 2009

Gene transcription in the cell nucleus is a complex and highly regulated process. Transcription in eukaryotes requires three distinct RNA polymerases, each of which employs its own mechanisms for initiation, elongation, and termination. Termination mechanisms vary considerably, ranging from relatively simple to exceptionally complex. In this review, we describe the present state of knowledge on how each of the three RNA polymerases terminates and how mechanisms are conserved, or vary, from yeast to human.
A role for Chk1 in blocking transcriptional elongation of p21 RNA during the S-phase checkpoint
Authors: Beckerman Rachel , Donner Aaron J. , Mattia Melissa , Peart Melissa J. , Manley James L. , Espinosa Joaquin M. , Prives Carol
Published: June 2009

We reported previously that when cells are arrested in S phase, a subset of p53 target genes fails to be strongly induced despite the presence of high levels of p53. When DNA replication is inhibited, reduced p21 mRNA accumulation is correlated with a marked reduction in transcription elongation. Here we show that ablation of the protein kinase Chk1 rescues the p21 transcription elongation defect when cells are blocked in S phase, as measured by increases in both p21 mRNA levels and the presence of the elongating form of RNA polymerase II (RNAPII) toward the 3′ end of the p21 gene. Recruitment of specific elongation and 3′ processing factors (DSIF, CstF-64, and CPSF-100) is also restored. While additional components of the RNAPII transcriptional machinery, such as TFIIB and CDK7, are recruited more extensively to the p21 locus after DNA damage than after replication stress, their recruitment is not enhanced by ablation of Chk1. Significantly, ablating Chk2, a kinase closely related in substrate specificity to Chk1, does not rescue p21 mRNA levels during S-phase arrest. Thus, Chk1 has a direct and selective role in the elongation block to p21 observed during S-phase arrest. These findings demonstrate for the first time a link between the replication checkpoint mediated by ATR/Chk1 and the transcription elongation/3′ processing machinery.
L1 retrotransposition occurs mainly in embryogenesis and creates somatic mosaicism
Authors: Kano Hiroki , Godoy Irene , Courtney Christine , Vetter Melissa R. , Gerton George L. , Ostertag Eric M. , Kazazian Haig H.
Published: June 2009

Long Interspersed Element 1 (L1) is a retrotransposon that comprises ∼17% of the human genome. Despite its abundance in mammalian genomes, relatively little is understood about L1 retrotransposition in vivo. To study the timing and tissue specificity of retrotransposition, we created transgenic mouse and rat models containing human or mouse L1 elements controlled by their endogenous promoters. Here, we demonstrate abundant L1 RNA in both germ cells and embryos. However, the integration events usually occur in embryogenesis rather than in germ cells and are not heritable. We further demonstrate L1 RNA in preimplantation embryos lacking the L1 transgene and L1 somatic retrotransposition events in blastocysts and adults lacking the transgene. Together, these data indicate that L1 RNA transcribed in male or female germ cells can be carried over through fertilization and integrate during embryogenesis, an interesting example of heritability of RNA independent of its encoding DNA. Thus, L1 creates somatic mosaicism during mammalian development, suggesting a role for L1 in carcinogenesis and other disease.
Hypoxia-selective macroautophagy and cell survival signaled by autocrine PDGFR activity
Authors: Wilkinson Simon , O'Prey Jim , Fricker Michael , Ryan Kevin M.
Published: June 2009

The selective regulation of macroautophagy remains poorly defined. Here we report that PDGFR signaling is an essential selective promoter of hypoxia-induced macroautophagy. Hypoxia-induced macroautophagy in tumor cells is also HIF1α-dependent, with HIF1α integrating signals from PDGFRs and oxygen tension. Inhibition of PDGFR signaling reduces HIF1α half-life, despite buffering of steady-state protein levels by a compensatory increase in HIF1α mRNA. This markedly changes HIF1α protein pool dynamics, and consequently reduces the HIF1α transcriptome. As autocrine growth factor signaling is a hallmark of many cancers, cell-autonomous enhancement of HIF1α-mediated macroautophagy may represent a mechanism for augmenting tumor cell survival under hypoxic conditions.
The Honeybee Apis mellifera
Authors: Dearden Peter K. , Duncan Elizabeth J. , Wilson Megan J.
Published: June 2009

INTRODUCTION

Honeybees (Apis mellifera) are distributed throughout the world and have a critical role in the pollination of crops and pasture. Honeybees have an intricate social structure and display complex behavioral traits such as sociality, language, division of labor, and exceptional feats of learning and memory. Honeybees also exhibit polyphenism, whereby the females can either develop as workers or as morphologically distinct, reproductively active queens. Although historical studies of honeybee biology have been performed, the recent advent of the genome sequence and a number of other modern tools have made dissection of the molecular details of this remarkable insect possible.
Selection and Evaluation of Tag-SNPs Using Tagger and HapMap
Authors: de Bakker Paul I.W.
Published: June 2009

INTRODUCTION

The HapMap can guide the design and analysis of genome-wide association studies by documenting not only the location of these single-nucleotide polymorphisms (SNPs) but also the correlation structure between them. This article describes how to use the Tagger web server for tag-SNP selection and evaluation using HapMap data.
Counting Chromosomes in Embryonic Stem (ES) Cells
Authors: Nagy Andras , Gertsenstein Marina , Vintersten Kristina , Behringer Richard
Published: June 2009

INTRODUCTION

Aneuploidy in embryonic stem (ES) cells is the major cause of failure in obtaining contributions to all tissues of chimeras, including the germ line. This protocol describes a simple method for counting chromosomes from metaphase spreads in ES cells. ES cell clones showing euploidy on 70%-80% of spreads are used for chimera production.
Labeling Mitochondria with Fluorescent Dyes for Imaging
Authors: Chazotte Brad
Published: June 2009

INTRODUCTION

The eukaryotic cell has evolved to compartmentalize its functions and transport various metabolites among cellular compartments. Therefore, it is important to study cellular organization and structure/function relationships. The mitochondrion is a double-membraned organelle that is important in various metabolic processes, including oxidative phosphorylation, electron transport, the Krebs cycle, β-oxidation of fatty acids, and part of the urea cycle. This protocol presents methods for labeling mitochondria in both live and fixed cells using several different fluorescent molecules. Rhodamine 123, tetramethylrhodamine ethyl ester (TMRE), and tetramethylrhodamine methyl ester (TMRM) are membrane-potential-sensitive, cationic fluorophores. Therefore, the mitochondrion must be functioning and generating a membrane potential in order to attract and maintain the dyes in the mitochondrion. These dyes are also used as sensitive indicators of the mitochondrial membrane potential. Rhodamine 123 is specific for the mitochondrion. In contrast, TMRM, TMRE, and the carbocyanine dyes can also label the endoplasmic reticulum to some degree. Mito Tracker CMTMRos has the added feature of chemical reactivity so that, once incorporated in the mitochondrion, it can chemically link to thiol groups and will not leave the mitochondrion when the membrane potential decreases as a result of fixation and/or cell death. Hence, it can be used to locate mitochondria in specimens that will subsequently be fixed. The effect of submicromolar concentrations of mitochondrial inhibitors, for example, KCN, rotenone, antimycin α, and uncouplers of oxidative phosphorylation, such as carbonylcyanide-m-chlorophenyl hydrazone (CCCP) and the related carbonylcyanide-p-trifluoromethoxyphenyl hydrazone (FCCP), can be examined on isolated mitochondria or mitochondria in intact cells.
Phenotypic Analysis of Arabidopsis Mutants: Diaminobenzidine Stain for Hydrogen Peroxide
Authors: Clarke Joseph D.
Published: June 2009

INTRODUCTION

The oxidative burst characteristic of gene-for-gene resistance responses and early events in establishment of systemic acquired resistance can be detected using 3,3′-diaminobenzidine tetrahydrochloride (DAB), a stain for hydrogen peroxide. The presence of hydrogen peroxide, which is symptomatic of the hypersensitive response, causes polymerization of DAB, yielding a brown color. This protocol describes how to stain Arabidopsis tissue with DAB.
Transcription-Based Reporters of Wnt/β-Catenin Signaling
Authors: Biechele Travis L. , Adams Allison M. , Moon Randall T.
Published: June 2009

INTRODUCTION

The Wnt/β-catenin pathway is the best characterized Wnt pathway largely as a result of powerful assays that allow measurement of in vivo and in vitro pathway activation. Early assays for Wnt/β-catenin signaling included phenotypic assays in Drosophila, dorsal axis duplication in Xenopus, and proliferation of C57MG mammary epithelial cells. More recent characterizations rely on direct measurement of pathway activation. The Wnt/β-catenin signaling pathway culminates with T-cell factor/lymphoid enhancer factor (TCF/LEF)-dependent regulation of target gene transcription; assaying such transcription with reporters containing multimerized TCF/LEF DNA-binding sites is a reliable measure of pathway activation. These reporters have helped characterize the Wnt/β-catenin signaling pathway and will be critical to further studies of pathway regulation and in the development of therapeutic mechanisms to combat diseases arising from aberrant signaling. This protocol describes a variety of methods for using an improved Wnt/β-catenin reporter system, the β-catenin-activated reporter (BAR), and its accompanying control reporter system, found-unresponsive BAR (fuBAR). Procedures are presented for transient and stable transfection of the reporter. Transient transfection is very robust and does not require lentiviral production, although it has a slightly smaller dynamic range relative to stably integrated BAR. The stable transduction system can be used to generate stable reporter cell lines, assay Wnt/β-catenin signaling in cells that might be otherwise difficult to transfect, or assay signaling in vivo. The BAR system, with its improved sensitivity, greater dynamic range, and lentiviral-based reporter system, provides a vital tool for future studies of this signaling pathway.
Fixation and Storage of Honeybee (Apis mellifera) Tissues
Authors: Dearden Peter K. , Duncan Elizabeth J. , Wilson Megan J.
Published: June 2009

INTRODUCTION

This protocol describes the preparation and fixation of honeybee tissue. Honeybee embryos are very fragile and must be fixed overnight before they can be subjected to downstream applications such as in situ hybridization and immunohistochemistry.
In Situ Hybridization of Sectioned Honeybee (Apis mellifera) Tissues: Figure 1.
Authors: Dearden Peter K. , Duncan Elizabeth J. , Wilson Megan J.
Published: June 2009

INTRODUCTION

This protocol outlines how to carry out section in situ hybridization on honeybee larvae to detect expression of a gene of interest in individual cell types. It can also be modified to investigate gene expression in a sectioned honeybee brain. A digoxigenin-labeled probe is hybridized to fixed sections and the probe is detected by 4-nitro blue tetrazolium chloride (NBT)-5-bromo-4-chloro-3-indolyl-phosphate (BCIP) color development.
Immunohistochemistry on Honeybee (Apis mellifera) Embryos
Authors: Dearden Peter K. , Duncan Elizabeth J. , Wilson Megan J.
Published: June 2009

INTRODUCTION

This protocol describes the preparation and staining of honeybee embryos for immunohistochemistry. An antigen retrieval step is required in order to restore antigens that have become masked by the long fixation step required for honeybee embryos.
RNA Interference (RNAi) in Honeybee (Apis mellifera) Embryos: Figure 1.
Authors: Dearden Peter K. , Duncan Elizabeth J. , Wilson Megan J.
Published: June 2009

INTRODUCTION

This protocol describes RNA interference (RNAi)-mediated knockdown of a target gene in honeybee embryos. dsRNA is directly injected into freshly laid embryos in order to disrupt the function of a specific gene during honeybee embryogenesis.
DNA Extraction from Forensic Samples Using Chelex
Authors: Butler John M.
Published: June 2009

INTRODUCTION

This protocol describes the extraction of DNA from forensic samples using Chelex, a chelating ion exchange resin suspension.
Magnetofection: The Use of Magnetic Nanoparticles for Nucleic Acid Delivery: Figure 1.
Authors: Plank Christian , Rosenecker Joseph
Published: June 2009

INTRODUCTION

Magnetofection is defined as nucleic acid delivery guided and mediated by magnetic force acting on associations of magnetic particles and nucleic acids or nucleic acid vectors. Vectors are bound to magnetic, usually iron oxide, nanoparticles, in most cases by noncovalent bonds. Magnetic force accumulates and/or holds magnetic vectors in a target tissue against hydrodynamic forces. In cell culture, magnetic vectors are magnetically sedimented on the target cells within minutes. Thus, the diffusion barrier to nucleic acid delivery is overcome, the full vector dose comes in contact with the target cells, and introduction of genetic material is synchronized. Nucleic acid delivery is greatly accelerated and its efficiency with many, if not most, vector types is improved. Magnetofection is applicable to small and large nucleic acids. Other advantages include low-dose requirements, the possibility of confining nucleic acid introduction to a localized area (magnetic targeting), and the amenability to high-throughput automation. Due to the favorable dose-response profile and the rapid kinetics, vector-related toxicity can be kept low. This protocol describes the use of magnetic nanoparticles for delivery of nucleic acid to target cells, using either nonviral or viral vectors.
Two-Photon Calcium Imaging of Spines and Dendrites: Figure 1.
Authors: Goldberg Jesse H. , Yuste Rafael
Published: June 2009

INTRODUCTION

This protocol describes an approach to two-photon calcium imaging in dendrites and spines of living neurons. The technique can be applied to acute slices from hippocampus, cerebellum, and neocortex, as well as to slice and neuronal cultures. It uses two fluorescence detection channels to provide quantitative estimates of calcium concentration and to minimize the required concentrations of calcium indicator. A method for estimating calcium concentrations from fractional changes in fluorescent light intensity (ΔF/F) is presented, along with two methods for loading neurons with calcium indicator.
Whole-Cell Recording and Voltage-Sensitive Dye Imaging In Vivo: Figure 1.
Authors: Petersen Carl
Published: June 2009

INTRODUCTION

This protocol describes an in vivo technique for combining whole-cell (WC) recordings of membrane potentials of individual neurons with voltage-sensitive dye (VSD) imaging of the ensemble neocortical network dynamics. If careful experimental measurements and controls are performed, VSD imaging can be used to define the ensemble spatiotemporal subthreshold membrane potential dynamics within which the membrane potential changes of individual neurons are embedded. Data from these individual neurons can be recorded simultaneously with the ensemble dynamics using the WC technique. This approach has been tested on rodent barrel cortex, but it is likely to be applicable, with minor modifications, to other superficial brain areas (including other neocortical areas, cerebellum, and olfactory bulb) and other species (such as cat or monkey).
Culturing and Transfecting Zebrafish PAC2 Fibroblast Cells
Authors: Senghaas Niklas , Köster Reinhard W.
Published: June 2009

INTRODUCTION

The use of zebrafish (Danio rerio) as a model organism has seen rapid growth in recent years. Numerous new techniques have emerged for zebrafish, and many protocols used in other model organisms have been adapted to fit the special needs of this species. Zebrafish cell lines, however, have not been studied to a great extent, and their applications remain limited. The PAC2 fibroblast line, isolated from 24-h post-fertilization zebrafish embryos, is one of the few available lines. Here, we provide a basic set of methods for maintenance and handling of PAC2 cells, as well as general procedures for transfection, immunocytochemistry, and protein extraction.
Native Chromatin Preparation and Illumina/Solexa Library Construction
Authors: Cuddapah Suresh , Barski Artem , Cui Kairong , Schones Dustin E. , Wang Zhibin , Wei Gang , Zhao Keji
Published: June 2009

INTRODUCTION

High-throughput whole-genome analysis has become a practical and important technique to understand nuclear processes, such as transcription, replication, and genome structure. Though microarrays have been the preferred genome-scale analysis method for over a decade, new technologies, referred to as next-generation sequencing, offer distinct advantages over microarrays in both sensitivity and scale. Several next-generation sequencing platforms are currently available, including the Genome Analyzer (Solexa/Illumina), 454 (Roche), and ABI-SOLiD (Applied Biosystems). This protocol describes sample preparation for sequencing of chromatin-immunoprecipitated DNA (ChIP-Seq) to analyze histone modification patterns using native chromatin and the Genome Analyzer. One advantage of using native chromatin as compared to cross-linked chromatin is that it provides single-nucleosome-level resolution and avoids nonspecific modification signals from different nucleosomes carried over through protein-protein interactions. The protocol includes purification of human CD4+ T cells from lymphocytes and chromatin fragmentation using micrococcal nuclease (MNase) digestion, followed by chromatin immunoprecipitation (ChIP) and construction of a library for sequencing.
Molecular genetics of sarcomas: Applications to diagnoses and therapy
Authors: Toguchida Junya , Nakayama Tomitaka
Published: June 2009

Sarcomas are mesenchymal cancers consisting of tumors with various clinical and pathological features. Some of them compel affected individuals to lose important musculoskeletal functions, and some of them are highly malignant and life‐threatening. A great amount of genetic information for sarcomas has accumulated during the past two decades, contributing diagnoses and treatments. From the standpoint of molecular genetics, sarcomas are classified into two groups: those with defined genetic alterations and those with various genetic alterations. The genetic alterations in the first group include reciprocal translocations resulting in fusion oncoproteins and oncogenic mutations of defined genes such as those of the c‐kit gene in gastrointestinal stromal tumors. The function of fusion proteins includes transcription regulator, signal transducer, chromatic remodeling factor, and growth factor, some of which are suitable targets for the molecular therapy. In tumors belonging to the second group, the number of which is far larger than those of the first group, considerable genetic heterogeneity was found even among tumors with same pathological diagnosis. The disruption of the RB and p53 pathways was frequently found, resulting in the dysregulation of cell cycle and the genomic instability. The application of molecular target therapy for tumors in this group requires novel strategies to overcome cross talk between different signal pathways. Recent evidence from in vitro and in vivo experiments has indicated that the cells of origin of sarcomas are tissue stem cells such as mesenchymal stem cells, and the application of stem cell biology holds the promise of novel treatment options. (Cancer Sci 2009; 100: 1573–1580)
Midkine in plasma as a novel breast cancer marker
Authors: Ibusuki Mutsuko , Fujimori Hiromi , Yamamoto Yutaka , Ota Kazutoshi , Ueda Mitsuharu , Shinriki Satoru , Taketomi Masahiro , Sakuma Sadatoshi , Shinohara Masanori , Iwase Hirotaka , Ando Yukio
Published: June 2009

Midkine, a heparin‐binding growth factor, is up‐regulated in many types of cancer. The aim of this study was to measure plasma midkine levels in patients with breast cancer and to assess its clinical significance. We examined plasma midkine levels in 95 healthy volunteers, 11 patients with ductal carcinoma in situ (DCIS), 111 patients with primary invasive breast cancer without distant metastasis (PIBC), and 25 patients with distant metastatic breast cancer (MBC), using an automatic immunoasssay analyzer (TOSOH AIA system). In PIBC, we studied the correlation between plasma midkine levels and clinicopathological factors. Immunoreactive midkine was detectable in the plasma of healthy volunteers, and a cut‐off level of 750 pg/mL was established. In breast cancer patients, plasma midkine levels were increased above normal values. These elevated levels of midkine were seen in one (9.1%) of 11 patients with DCIS, 36 (32.4%) of 111 patients with PIBC, and 16 (64.0%) of 25 patients with MBC. Increased levels of midkine were correlated with menopausal status (P = 0.0497) and nuclear grade (P = 0.0343) in PIBC. Cancer detection rates based on midkine levels were higher than those based on three conventional markers including CA15‐3 (P < 0.0001), CEA (P = 0.0077), and NCCST‐439 (P < 0.0001). Detection rates of breast cancer using a combination of two conventional tumor markers (CA15‐3/CEA, CA15‐3/NCCST‐439, or CEA/NCCST‐439) with midkine is significantly higher than those using combination of three conventional tumor markers. Midkine may be a useful novel tumor marker for detection of breast cancer, superior to conventional tumor markers. (Cancer Sci 2009; 100: 1735–1739)
Expression of Autotaxin and Acylglycerol kinase in prostate cancer: Association with cancer development and progression
Authors: Nouh Mohammed Ahmed Abdel Muneem , Wu Xiu‐Xian , Okazoe Homare , Tsunemori Hiroyuki , Haba Reiji , Abou‐Zeid Abdel Muneem Mohammed , Saleem Mohammed Diaa , Inui Masashi , Sugimoto Mikio , Aoki Junken , Kakehi Yoshiyuki
Published: June 2009

Lysophosphatidic acid (LPA) may enhance diverse biologic activities in prostate cancer. This study was conducted to analyze expression levels of LPA‐producing enzymes, autotaxin (ATX) and acylglycerol kinase (AGK), in prostate cancer with relevance to clinicopathological parameters. Real‐time RT‐PCR and western blotting were performed for ATX and AGK in non‐neoplastic prostate cells (PrECs and PrSCs) and prostate cancer cell‐lines (DU‐145, PC‐3, LNCaP, and AILNCaP). Immunohistochemical analyses were conducted in tissue specimens of 132 localized prostate cancer patients who underwent radical prostatectomy between 2001 and 2007 (median observation period, 22 months). Both enzymes were negatively expressed in PrECs and PrSCs at mRNA and protein levels. ATX expression was higher than AGK in AILNCaP, DU‐145, and PC‐3 cell‐lines, while AGK was mainly expressed in LNCaP cells. Immunohistochemically, ATX and AGK expressions were negative in non‐neoplastic epithelia, while both were weakly expressed in the majority of high‐grade intra‐epithelial neoplasia (HG‐PIN). In cancer foci, ATX and AGK expressions were strong in 49% and 62%, weak in 40% and 32%, and negative in 11% and 6%, respectively. Expressions of both enzymes were significantly correlated with primary Gleason grade of cancer foci (P < 0.0001) and capsular invasion (P = 0.03 and 0.003 respectively). ATX expression was significantly correlated with probability of prostate specific antigen (PSA)‐failure after surgery (P < 0.0001). In conclusion, LPA‐producing enzymes (ATX and AGK) were frequently expressed in prostate cancer cells and precancerous HG‐PIN. In particular, high expression levels of ATX were associated with both malignant potentials and poor outcomes. (Cancer Sci 2009; 100: 1631–1638)
Constitutive phosphorylation of a Rac GAP MgcRacGAP is implicated in v‐Src‐induced transformation of NIH3T3 cells
Authors: Doki Noriko , Kawashima Toshiyuki , Nomura Yasushi , Tsuchiya Akiho , Oneyama Chitose , Akagi Tsuyoshi , Nojima Yoshihisa , Kitamura Toshio
Published: June 2009

MgcRacGAP plays critical roles in cell division through regulating Rho family small GTPases. As we previously reported, phosphorylation of MgcRacGAP on serine 387 (S387) is induced by Aurora B kinase at the midbody during cytokinesis, which is a critical step of cytokinesis. Phosphorylation of S387‐MgcRacGAP converts it from RacGAP to RhoGAP, leading to completion of cytokinesis. Here we show that MgcRacGAP is prominently phosphorylated on S387 even in the interphase of v‐Src‐transformed NIH3T3 cells in the cytoplasm, but not in the interphase of parental NIH3T3 or H‐RasV12‐transformed NIH3T3 cells. Interestingly, levels of phosphorylation on S387 (pS387) correlated with soft agar colony‐forming abilities of v‐Src‐transformed NIH3T3 cells. Expression of a phosphorylation‐mimic mutant MgcRacGAP‐S387D enhanced colony formation of v‐Src‐transformed NIH3T3 cells. Surprisingly, a Rac1 inhibitor but not kinase inhibitors including Aurora B kinase inhibitor specifically inhibited phosphorylation of S387‐MgcRacGAP in v‐Src‐transformed NIH3T3 cells, suggesting the v‐Src‐induced pathological positive feedback mechanisms towards Rac1 activation using pS387‐MgcRacGAP. These results indicated the difference in the mechanisms between v‐Src‐ and H‐RasV12‐induced transformation, and should shed some light on pathological roles of disordered phosphorylation of MgcRacGAP at S387 in v‐Src‐induced cell transformation. (Cancer Sci 2009; 100: 1675–1679)
Effect of the alcoholic extract of Ashwagandha leaves and its components on proliferation, migration, and differentiation of glioblastoma cells: Combinational approach for enhanced differentiation
Authors: Shah Navjot , Kataria Hardeep , Kaul Sunil C. , Ishii Tetsuro , Kaur Gurcharan , Wadhwa Renu
Published: June 2009

Ashwagandha (Withania somnifera) is widely used in the Indian traditional system of medicine, Ayurveda. Although it is claimed to have a large variety of health‐promoting effects, including therapeutic effects on stress and disease, the mechanisms of action have not yet been determined. In the present study, we aimed to investigate the growth inhibition and differentiation potential of the alcoholic extract of Ashwagandha leaves (i‐Extract), its different constituents (Withaferin A, Withanone, Withanolide A) and their combinations on glioma (C6 and YKG1) cell lines. Withaferin A, Withanone, Withanolide A and i‐Extract markedly inhibited the proliferation of glioma cells in a dose‐dependent manner and changed their morphology toward the astrocytic type. Molecular analysis revealed that the i‐Extract and some of its components caused enhanced expression of glial fibrillary acidic protein, change in the immunostaining pattern of mortalin from perinuclear to pancytoplasmic, delay in cell migration, and increased expression of neuronal cell adhesion molecules. The data suggest that the i‐Extract and its components have the potential to induce senescence‐like growth arrest and differentiation in glioma cells. These assays led us to formulate a unique combination formula of i‐Extract components that caused enhanced differentiation of glial cells. (Cancer Sci 2009; 100: 1740–1747)
Phase I and pharmacokinetic study of vorinostat (suberoylanilide hydroxamic acid) in Japanese patients with solid tumors
Authors: Fujiwara Yutaka , Yamamoto Noboru , Yamada Yasuhide , Yamada Kazuhiko , Otsuki Tetsuya , Kanazu Shinichi , Iwasa Takashi , Hardwick James S. , Tamura Tomohide
Published: June 2009

Vorinostat (suberoylanilide hydroxamic acid), a potent, oral histone deacetylase inhibitor, has demonstrated clinical activity in non‐Japanese patients with various hematological and solid tumors. We sought to determine the maximum tolerated dose and a recommended phase II dose for 18 Japanese patients with solid tumors (median age, 58 years; range, 25–72 years) who failed standard therapy. Patients received vorinostat for 14 days followed by a 7‐day rest. The initial dose was 100 mg twice daily escalating by 100 mg twice daily. Once‐daily dosing was tested at 400 and 500 mg. A maximum tolerated dose could not be identified. Dose‐limiting toxicities (thrombocytopenia, anorexia, and fatigue) were observed in two of six patients receiving 200 mg twice daily and in one of six patients receiving 500 mg once daily. In the 100–500 mg dose range, vorinostat area under the concentration–time curve increased in proportion to dose with a pharmacokinetic profile similar to that established in non‐Japanese patients. Vorinostat doses of 200 mg twice daily or 500 mg once daily for 14 days followed by a 7‐day rest were well tolerated and are candidate doses for phase II trials, although a maximum tolerated dose for vorinostat was not reached. (Cancer Sci 2009; 100: 1728–1734)
Mutations in the mitochondrial genome confer resistance of cancer cells to anticancer drugs
Authors: Mizutani Satoshi , Miyato Yasuyuki , Shidara Yujiro , Asoh Sadamitsu , Tokunaga Akira , Tajiri Takashi , Ohta Shigeo
Published: June 2009

The majority of cancer cells harbor homoplasmic somatic mutations in the mitochondrial genome. We show here that mutations in mitochondrial DNA (mtDNA) are responsible for anticancer drug tolerance. We constructed several trans‐mitochondrial hybrids (cybrids) with mtDNA derived from human pancreas cancer cell lines CFPAC‐1 and CAPAN‐2 as well as from healthy individuals. These cybrids contained the different mitochondrial genomes with the common nuclear background. We compared the mutant and wild‐type cybrids for resistance against an apoptosis‐inducing reagent and anticancer drugs by exposing the cybrids to staurosporine, 5‐fluorouracil, and cisplatin in vitro, and found that all mutant cybrids were more resistant to the apoptosis‐inducing and anticancer drugs than wild‐type cybrids. Next, we transplanted mutant and wild‐type cybrids into nude mice to generate tumors. Tumors derived from mutant cybrids were more resistant than those from wild‐type cybrids in suppressing tumor growth and inducing massive apoptosis when 5‐fluorouracil and cisplatin were administered. To confirm the tolerance of mutant cybrids to anticancer drugs, we transplanted a mixture of mutant and wild‐type cybrids at a 1:1 ratio into nude mice and examined the effect by the drugs on the drift of the ratio of mutant and wild‐type mtDNA. The mutant mtDNA showed better survival, indicating that mutant cybrids were more resistant to the anticancer drugs. Thus, we propose that mutations in the mitochondrial genome are potential targets for prognosis in the administration of anticancer drugs to cancer patients. (Cancer Sci 2009; 100: 1680–1687)
Genetic variants of C1orf10 and risk of esophageal squamous cell carcinoma in a Chinese population
Authors: Zhang Wei , Chen Xiabin , Luo Aiping , Lin Dongxin , Tan Wen , Liu Zhihua
Published: June 2009

Chromosome 1 open reading frame 10 (C1orf10) is either down‐regulated or absent in esophageal squamous cell carcinoma (ESCC) tissues compared to its corresponding normal counterparts, and it is involved in heat shock and ethanol response and is expected to protect esophageal epithelium from damage. In the present study, we sequenced DNA samples from 32 individuals to search for genetic variants in the promoter region, coding region, and the untranslated region of C1orf10. Genotypes were analyzed in 991 patients and 984 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated by logistic regression. Luciferase assays were carried out to find the functional SNPs. Six strongly linked single nucleotide polymorphisms (SNPs) spanning a region of 7 kb, –1747G/T, –1139G/C, –1079G/A, –900G/T, Gly480Ser, and 4666G/A were identified (D′= 1, r2 = 1). Only one SNP –1139G/C was selected to analyze the association between C1orf10 genotypes and risk of ESCC. Subjects with the –1139CC genotype had a greater risk of developing ESCC compared with those with the –1139GG genotype (adjusted OR = 1.34; 95% CI, 1.02–1.76). There appears to be an interaction between the –1139G/C polymorphism and tobacco smoking that contributes to the risk for ESCC. However, we did not detect any obvious difference in reporter gene assay driven by each allele of C1orf10 promoter or 3′ UTR. These data showed that C1orf10 haplotypes containing –1747G/T, –1139G/C, –1079G/A, –900G/T, Gly480Ser, and 4666G/A are significantly associated with susceptibility to ESCC. (Cancer Sci 2009; 100: 1695–1700)
Sacoglottis gabonensis– a keystone fruit for forest elephants in the Réserve de Faune du Petit Loango, Gabon
Authors: Morgan Bethan J.
Published: June 2009

This study suggests that the fruits ofSacoglottis gabonensis(Baill.) Urb. (Humiriaceae) are a keystone resource for forest elephants (Loxodonta cyclotisMatschie) in a coastal rain forest, the Réserve de Faune du Petit Loango, Gabon (now part of Loango National Park). Faecal counts demonstrated that forest elephants usedSacoglottis‐dominated forest more whenSacoglottiswas abundant and electivity indices suggest thatSacoglottisis a preferred food. The flora of Petit Loango is characterized by the absence of herbaceous vegetation such as Marantaceae and Zingiberaceae, and during the prolonged dry season few fleshy fruits are present other thanSacoglottisfruits, which are produced in a glut during this time. While inter‐annual fruiting reliability remains to be confirmed, fruit production in 1998 and high stem density relative to other study sites provide indirect evidence thatSacoglottisfruits are a reliable inter‐annual resource at Petit Loango. It is thus proposed thatSacoglottis gabonensisfruits fulfil an important role as a keystone ‘fallback’ resource for forest elephants during the dry season at Petit Loango.
Changes in the structure and composition of miombo woodlands mediated by elephants (Loxodonta africana) and fire over a 26‐year period in north‐western Zimbabwe
Authors: Mapaure Isaac , Moe Stein R.
Published: June 2009

Changes in structure and composition of miombo woodlands mediated by elephants and fire were studied in 26‐year‐old permanent transects established in 1972 in north‐western Zimbabwe. Elephants caused 48% decline in proportions of large trees (>11 cm diameter), significant reductions (30.9–90.9%) in tree heights, reductions in stem areas (43.5%) and densities (2.5%) of all trees. There were increases in proportions of small trees (64.8%), shrub canopy volumes (271%) and shrub densities (172%). These increases are attributed to natural recruitment because of longer fire‐free periods and reduction of tree suppression effects on lower strata as a result of elephant‐induced tree declines. Frequencies of occurrence of most species dropped by 28–89.6%. Brachystegia boehmii was replaced by Pseudolachnostylis maprouneifolia as the most dominant tree, largely because of high elephant preference for Brachystegia boehmii. A new suite of species, dominated by Combretaceae, increased in dominance resulting in local floristic changes. Reductions in old elephant (33.4%), old unknown (89.9%) and new elephant (13.7%) damage suggest that elephant occupancy of miombo woodlands has declined, possibly because of limited availability of preferred browse species. This study clearly shows that elephants and fire have contributed significantly to the changes in miombo woodlands in the area.
Early age reproduction in female savanna elephants (Loxodonta africana) after severe poaching
Authors: Owens M. J. , Owens D.
Published: June 2009

A 10‐year study revealed that after severe poaching (>93% killed) of elephants (Loxodonta africana) in Zambia’s North Luangwa National Park (NLNP) during the 1970s and 1980s, the age of reproduction in females was greatly reduced. Fifty‐eight per cent of births were delivered by females aged 8.5–14 years, an age at which elephants were reported to be sexually immature in nearby South Luangwa National Park (SLNP) before poaching. The mean age of females at first birth (AFB) (1993, 1994) was 11.3 years. Prior to poaching, the mean age AFB in SLNP was 16 years. The NLNP age structure and sex ratio were skewed, mean family unit size was reduced, and 37% of family units contained no females older than 15 years. Twenty‐eight per cent of family units were comprised entirely of a single mother and her calf, and 8% of units consisted only of orphans who would have been considered sexually immature prior to poaching. Only 6% of survivors were older than 20 years, the age at which females in little‐poached populations generally become most reproductively active. After a community‐based conservation programme and the UN‐CITES ban on the ivory trade were introduced, no elephants were recorded killed. In spite of a high reproductive rate, 6 years after poaching decreased, the density of the NLNP population had not increased, supporting predictions that the removal of older matriarchs from family units will have serious consequences on the recovery of this species.
Elephants caught in the middle: impacts of war, fences and people on elephant distribution and abundance in the Caprivi Strip, Namibia
Authors: Chase Michael J. , Griffin Curtice R.
Published: June 2009

We conducted wet [26 March–4 April 2003 (Apr03)] and dry [1–8 November 2005 (Nov05)] season aerial surveys of African elephants (Loxodonta africanaBlumenbach) in the Caprivi Strip, Namibia to provide an updated status report on elephant numbers and distribution and assist with a historical analysis of elephant distribution and abundance in the Caprivi Strip. During the wet season when water was available in seasonal pans, elephants were widely distributed throughout the survey area. In contrast, during the dry season, a majority of elephant herds occurred within 30 km of the perennial Kwando, Linyanti and Okavango rivers and few herds occurred within the West Caprivi Game Reserve where water in the seasonal pans was limited. We estimated 5318 elephants for the 7731‐km2survey area (0.71 elephants km−2) for the Apr03 wet season survey and 6474 elephants for the 8597‐km2survey area (0.75 elephants km−2) for the Nov05 dry season survey. Based on our aerial surveys and reports of elephant numbers and distribution from historical aerial surveys and telemetry studies, civil war, veterinary fences and human activities appear to have effected changes in African elephant abundance, distribution and movements in the Caprivi Strip, Namibia since 1988 when the first comprehensive aerial surveys were conducted.
Elephant distribution around a volcanic shield dominated by a mosaic of forest and savanna (Marsabit, Kenya)
Authors: Ngene Shadrack M. , Skidmore Andrew K. , Van Gils Hein , Douglas‐Hamilton Iain , Omondi Patrick
Published: June 2009

We investigated the factors that influenced the distribution of the African elephant around a volcanic shield dominated by a mosaic of forest and savanna in northern Kenya. Data on elephant distribution were acquired from four female and five bull elephants, collared with satellite‐linked geographical positioning system collars. Based on the eigenvalues (variances) of the correlation matrix, the six factors that contributed significantly to high total variances were distance from drinking water (24%), elevation (15%), shrubland (10%), forest (9%), distance from settlements (8%) and distance from minor roads (7%), contributing to 73% in the observed variation of the elephant distribution. The elephants were found at high forested elevations during the dry season but they moved to the lowlands characterized by shrubland during the wet season. Elevation acts as a proxy for the vegetation structure. The presence of elephants near permanent water points (13%) and seasonal rivers (11%) during the dry and wet seasons, respectively, demonstrates that water is the most important determinant of their distribution throughout the year. We conclude that the distribution of elephants in Marsabit Protected Area and its adjacent areas is influenced mainly by drinking water and vegetation structure.