bluuebunny/crossref_metadata_2025_split huggingface dataset:
Labeling Mitochondria with Fluorescent Dyes for Imaging
Authors: Chazotte Brad 
Published: June 2009

INTRODUCTIONThe 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

INTRODUCTIONThe 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

INTRODUCTIONThe 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

INTRODUCTIONThis 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

INTRODUCTIONThis 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

INTRODUCTIONThis 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

INTRODUCTIONThis 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.
Magnetofection: The Use of Magnetic Nanoparticles for Nucleic Acid Delivery: Figure 1.
Authors: Plank Christian , Rosenecker Joseph 
Published: June 2009

INTRODUCTIONMagnetofection 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

INTRODUCTIONThis 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

INTRODUCTIONThis 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

INTRODUCTIONThe 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

INTRODUCTIONHigh-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.
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)
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)
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

AbstractThis 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

AbstractChanges 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.