bluuebunny/crossref_metadata_2025 huggingface dataset:
Pulmonary Blastomycosis: A Case from Africa
Authors: Cheikh Rouhou Sana , Racil Hajer , Ismail Olfa , Trabelsi Sonia , Zarrouk Mourad , Chaouch Nawel , Hantous Sawssen , Khaled Samira , El Mezni Faouzi , Chabbou Abdellatif
Published: November 2008

Pulmonary blastomycosis is an uncommon pathologic condition that is quite rare in Africa compared to endemic regions of Canada and the upper Midwest of the U.S. We describe a 45-year-old patient who complained of productive cough, hemoptysis, and dorsal rachiodynia. Chest imaging revealed a necrotic tissue-density pulmonary mass involving both the upper and lower right lobes. Chest MRI showed signal abnormality of the third thoracic vertebral body and the greater trochanter, consistent with metastatic lesions. Clinical and radiological findings were strongly suggestive of lung cancer. Diagnosis of pulmonary blastomycosis was made by visualization of yeast in bronchial biopsies and further confirmed by culture of bronchoalveolar lavage specimens. The patient was treated with itraconazole and his clinical condition improved markedly. Pulmonary blastomycosis is unusual in Africa and that fact caused a considerable delay in diagnosis. We suggest that this disease may be more common in Africa than has been previously suspected.
Atomic Force Microscopy Investigation of DNA Extracted from the Vegetative Forms and the Viable but Nonculturable Forms ofMycoplasma gallisepticumS6
Authors: Chernova Olga A. , Trushin Maxim V. , Mouzykantov Alexey A. , Chernov Vladislav M.
Published: November 2008

Recent studies show that mycoplasmas have various programs of life. This means that changes in morphology and genome expression may occur once the environment of these microorganisms becomes extremely altered. In this article, we report on changes in the DNA molecule obtained from the vegetative forms and the viable but nonculturable (VBNC) forms ofMycoplasma gallisepticumS6. Atomic force microscopy studies show that the above-mentioned forms of the mycoplasma have different values of DNA parameters (height: 0.461 ± 0.141 and 0.236 ± 0.069 nm; width: 2.221 ± 0.286 and 1.291 ± 0.705 nm for the vegetative and the VBNC forms, respectively). We suppose that the observed phenomenon may be connected with the process of adaptation of these bacteria to severe environments.
BMP/SMAD1 signaling sets a threshold for the left/right pathway in lateral plate mesoderm and limits availability of SMAD4
Authors: Furtado Milena B. , Solloway Mark J. , Jones Vanessa J. , Costa Mauro W. , Biben Christine , Wolstein Orit , Preis Jost I. , Sparrow Duncan B. , Saga Yumiko , Dunwoodie Sally L. , Robertson Elizabeth J. , Tam Patrick P.L. , Harvey Richard P.
Published: November 2008

Bistability in developmental pathways refers to the generation of binary outputs from graded or noisy inputs. Signaling thresholds are critical for bistability. Specification of the left/right (LR) axis in vertebrate embryos involves bistable expression of transforming growth factor β (TGFβ) member NODAL in the left lateral plate mesoderm (LPM) controlled by feed-forward and feedback loops. Here we provide evidence that bone morphogenetic protein (BMP)/SMAD1 signaling sets a repressive threshold in the LPM essential for the integrity of LR signaling. Conditional deletion of Smad1 in the LPM led to precocious and bilateral pathway activation. NODAL expression from both the left and right sides of the node contributed to bilateral activation, indicating sensitivity of mutant LPM to noisy input from the LR system. In vitro, BMP signaling inhibited NODAL pathway activation and formation of its downstream SMAD2/4–FOXH1 transcriptional complex. Activity was restored by overexpression of SMAD4 and in embryos, elevated SMAD4 in the right LPM robustly activated LR gene expression, an effect reversed by superactivated BMP signaling. We conclude that BMP/SMAD1 signaling sets a bilateral, repressive threshold for NODAL-dependent Nodal activation in LPM, limiting availability of SMAD4. This repressive threshold is essential for bistable output of the LR system.
Parathyroid hormone signaling through low-density lipoprotein-related protein 6
Authors: Wan Mei , Yang Chaozhe , Li Jun , Wu Xiangwei , Yuan Hongling , Ma Hairong , He Xi , Nie Shuyi , Chang Chenbei , Cao Xu
Published: November 2008

Intermittent administration of PTH stimulates bone formation, but the precise mechanisms responsible for PTH responses in osteoblasts are only incompletely understood. Here we show that binding of PTH to its receptor PTH1R induced association of LRP6, a coreceptor of Wnt, with PTH1R. The formation of the ternary complex containing PTH, PTH1R, and LRP6 promoted rapid phosphorylation of LRP6, which resulted in the recruitment of axin to LRP6, and stabilization of β-catenin. Activation of PKA is essential for PTH-induced β-catenin stabilization, but not for Wnt signaling. In vivo studies confirmed that PTH treatment led to phosphorylation of LRP6 and an increase in amount of β-catenin in osteoblasts with a concurrent increase in bone formation in rat. Thus, LRP6 coreceptor is a key element of the PTH signaling that regulates osteoblast activity.
PPARγ and C/EBP factors orchestrate adipocyte biology via adjacent binding on a genome-wide scale
Authors: Lefterova Martina I. , Zhang Yong , Steger David J. , Schupp Michael , Schug Jonathan , Cristancho Ana , Feng Dan , Zhuo David , Stoeckert Christian J. , Liu X. Shirley , Lazar Mitchell A.
Published: November 2008

Peroxisome proliferator-activated receptor γ(PPARγ), a nuclear receptor and the target of anti-diabetic thiazolinedione drugs, is known as the master regulator of adipocyte biology. Although it regulates hundreds of adipocyte genes, PPARγ binding to endogenous genes has rarely been demonstrated. Here, utilizing chromatin immunoprecipitation (ChIP) coupled with whole genome tiling arrays, we identified 5299 genomic regions of PPARγ binding in mouse 3T3-L1 adipocytes. The consensus PPARγ/RXRα “DR-1”-binding motif was found at most of the sites, and ChIP for RXRα showed colocalization at nearly all locations tested. Bioinformatics analysis also revealed CCAAT/enhancer-binding protein (C/EBP)-binding motifs in the vicinity of most PPARγ-binding sites, and genome-wide analysis of C/EBPα binding demonstrated that it localized to 3350 of the locations bound by PPARγ. Importantly, most genes induced in adipogenesis were bound by both PPARγ and C/EBPα, while very few were PPARγ-specific. C/EBPβ also plays a role at many of these genes, such that both C/EBPα and β are required along with PPARγ for robust adipocyte-specific gene expression. Thus, PPARγ and C/EBP factors cooperatively orchestrate adipocyte biology by adjacent binding on an unanticipated scale.
Noncoding RNA control of the making and breaking of sugars
Authors: Görke Boris , Vogel Jörg
Published: November 2008

Noncoding RNA regulators have been implicated in almost all imaginable cellular processes. Here we review how regulatory small RNAs such as Spot42, SgrS, GlmY, and GlmZ and a cis-encoded ribozyme in glmS mRNA control sugar metabolism. Besides discussing the physiological implications, we show how the study of these molecules contributed to our understanding of the mechanisms and of general principles of RNA-based regulation. These include the post-transcriptional repression or activation of gene expression within polycistronic mRNAs; novel ribonucleoprotein complexes composed of small RNA, Hfq, and/or RNase E; and the hierarchical action of regulatory RNAs.
Differentiation of trophoblast stem cells into giant cells is triggered by p57/Kip2 inhibition of CDK1 activity
Authors: Ullah Zakir , Kohn Matthew J. , Yagi Rieko , Vassilev Lyubomir T. , DePamphilis Melvin L.
Published: November 2008

Genome endoreduplication during mammalian development is a rare event for which the mechanism is unknown. It first appears when fibroblast growth factor 4 (FGF4) deprivation induces differentiation of trophoblast stem (TS) cells into the nonproliferating trophoblast giant (TG) cells required for embryo implantation. Here we show that RO3306 inhibition of cyclin-dependent protein kinase 1 (CDK1), the enzyme required to enter mitosis, induced differentiation of TS cells into TG cells. In contrast, RO3306 induced abortive endoreduplication and apoptosis in embryonic stem cells, revealing that inactivation of CDK1 triggers endoreduplication only in cells programmed to differentiate into polyploid cells. Similarly, FGF4 deprivation resulted in CDK1 inhibition by overexpressing two CDK-specific inhibitors, p57/KIP2 and p21/CIP1. TS cell mutants revealed that p57 was required to trigger endoreduplication by inhibiting CDK1, while p21 suppressed expression of the checkpoint protein kinase CHK1, thereby preventing induction of apoptosis. Furthermore, Cdk2−/− TS cells revealed that CDK2 is required for endoreduplication when CDK1 is inhibited. Expression of p57 in TG cells was restricted to G-phase nuclei to allow CDK activation of S phase. Thus, endoreduplication in TS cells is triggered by p57 inhibition of CDK1 with concomitant suppression of the DNA damage response by p21.
New cell or new cycle?
Authors: Ganier Olivier , Mechali Marcel
Published: November 2008

In mammals, trophoblast giant (TG) cell differentiation is characterized by a physiological endoreduplication, resulting in genome size augmentation. A recent study by Ullah and colleagues (pp. 3024–3036), published in this issue of Genes & Development, now elucidates the role of the cyclin-dependent kinase inhibitors (CKIs), p21 and p57, in mammalian endocycle regulation.
DNA double-strand break processing: the beginning of the end
Authors: Raynard Steven , Niu Hengyao , Sung Patrick
Published: November 2008

Nucleolytic processing of DNA double-strand breaks (DSBs) generates 3′ ssDNA tails that are essential for the assembly of DNA damage checkpoint signaling and DNA repair protein complexes. Genetic studies have provided evidence that multiple nuclease activities are involved in DSB end resection. Three recent studies, including work by Jackson and colleagues (pp. 2767–
2772) in the October 15, 2008, issue of Genes & Development, have begun to shed some light on the intricacy of this process.
Drosophila HP1c isoform interacts with the zinc-finger proteins WOC and Relative-of-WOC to regulate gene expression
Authors: Font-Burgada Joan , Rossell David , Auer Herbert , Azorín Fernando
Published: November 2008

Heterochromatin protein 1 (HP1) proteins are conserved in eukaryotes, with most species containing several isoforms. Based on the properties of Drosophila HP1a, it was proposed that HP1s bind H3K9me2,3 and recruit factors involved in heterochromatin assembly and silencing. Yet, it is unclear whether this general picture applies to all HP1 isoforms and functional contexts. Here, we report that Drosophila HP1c regulates gene expression, as (1) it localizes to active chromatin domains, where it extensively colocalizes with the poised form of RNApolymerase II (RNApol II), Pol IIoser5, and H3K4me3, suggesting a contribution to transcriptional regulation; (2) its targeting to a reporter gene does not induce silencing but, on the contrary, increases its expression, and (3) it interacts with the zinc-finger proteins WOC (without children) and Relative-of-WOC (ROW), which are putative transcription factors. Here, we also show that, although HP1c efficiently binds H3K9me2,3 in vitro, its binding to chromatin strictly depends on both WOC and ROW. Moreover, expression profiling indicates that HP1c, WOC, and ROW regulate a common gene expression program that, in part, is executed in the context of the nervous system. From this study, which unveils the essential contribution of DNA-binding proteins to HP1c functionality and recruitment, HP1 proteins emerge as an increasingly diverse family of chromatin regulators.
Genome-wide profiling of PPARγ:RXR and RNA polymerase II occupancy reveals temporal activation of distinct metabolic pathways and changes in RXR dimer composition during adipogenesis
Authors: Nielsen Ronni , Pedersen Thomas Åskov , Hagenbeek Dik , Moulos Panagiotis , Siersbæk Rasmus , Megens Eva , Denissov Sergei , Børgesen Michael , Francoijs Kees-Jan , Mandrup Susanne , Stunnenberg Hendrik G.
Published: November 2008

The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) is a key regulator of adipocyte differentiation in vivo and ex vivo and has been shown to control the expression of several adipocyte-specific genes. In this study, we used chromatin immunoprecipitation combined with deep sequencing to generate genome-wide maps of PPARγ and retinoid X receptor (RXR)-binding sites, and RNA polymerase II (RNAPII) occupancy at very high resolution throughout adipocyte differentiation of 3T3-L1 cells. We identify >5000 high-confidence shared PPARγ:RXR-binding sites in adipocytes and show that during early stages of differentiation, many of these are preoccupied by non-PPARγ RXR-heterodimers. Different temporal and compositional patterns of occupancy are observed. In addition, we detect co-occupancy with members of the C/EBP family. Analysis of RNAPII occupancy uncovers distinct clusters of similarly regulated genes of different biological processes. PPARγ:RXR binding is associated with the majority of induced genes, and sites are particularly abundant in the vicinity of genes involved in lipid and glucose metabolism. Our analyses represent the first genome-wide map of PPARγ:RXR target sites and changes in RNAPII occupancy throughout adipocyte differentiation and indicate that a hitherto unrecognized high number of adipocyte genes of distinctly regulated pathways are directly activated by PPARγ:RXR.
Tomato (Solanum lycopersicum): A Model Fruit-Bearing Crop
Authors: Kimura Seisuke , Sinha Neelima
Published: November 2008

INTRODUCTION

Tomato (Solanum lycopersicum) is one of the most important vegetable plants in the world. It originated in western South America, and domestication is thought to have occurred in Central America. Because of its importance as food, tomato has been bred to improve productivity, fruit quality, and resistance to biotic and abiotic stresses. Tomato has been widely used not only as food, but also as research material. The tomato plant has many interesting features such as fleshy fruit, a sympodial shoot, and compound leaves, which other model plants (e.g., rice and Arabidopsis) do not have. Most of these traits are agronomically important and cannot be studied using other model plant systems. There are 13 recognized wild tomato species that display a great variety of phenotypes and can be crossed with the cultivated tomato. These wild tomatoes are important for breeding, as sources of desirable traits, and for evolutionary studies. Current progress on the tomato genome sequencing project has generated useful information to help in the study of tomato. In addition, the tomato belongs to the extremely large family Solanaceae and is closely related to many commercially important plants such as potato, eggplant, peppers, tobacco, and petunias. Knowledge obtained from studies conducted on tomato can be easily applied to these plants, which makes tomato important research material. Because of these facts, tomato serves as a model organism for the family Solanaceae and, specifically, for fleshy-fruited plants.
Comb Jellies (Ctenophora): A Model for Basal Metazoan Evolution and Development
Authors: Pang Kevin , Martindale Mark Q.
Published: November 2008

INTRODUCTION

Ctenophores, or comb jellies, are a group of marine organisms whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. These gelatinous creatures are clearly distinct from cnidarian medusae (i.e., jellyfish). Key features present in the ctenophore body plan include biradial symmetry, an oral-aboral axis delimited by a mouth and an apical sensory organ, two tentacles, eight comb rows composed of interconnected cilia, and thick mesoglea. Other morphological features include definitive muscle cells, a nerve net, basal lamina, a sperm acrosome, and light-producing photocytes. Aspects of their development made them attractive to experimental embryologists as early as the 19th century. Recently, because of their role as an invasive species, studies on their role in ecology and fisheries-related fields have increased. Although the phylogenetic placement of ctenophores with respect to other animals has proven difficult, it is clear that, along with poriferans, placozoans, and cnidarians, ctenophores are one of the earliest diverging extant animal groups. It is important to determine if some of the complex features of ctenophores are examples of convergence or if they were lost in other animal branches. Because ctenophores are amenable to modern technical approaches, they could prove to be a highly useful emerging model.
Astyanax mexicanus, the Blind Mexican Cave Fish: A Model for Studies in Development and Morphology: Figure 1.
Authors: Borowsky Richard
Published: November 2008

INTRODUCTION

The perpetual darkness of caves has two important consequences for permanent inhabitants. First, eyes and pigmentation lose their primary functions. Second, in the absence of photosynthesis, food is rare. For these reasons, cave-adapted species typically have reduced eyes and pigmentation and increased or more efficient metabolisms. Additionally, other senses are usually augmented to compensate for the loss of vision. Identifying the genetic bases underlying these phenotypic changes will enhance our understanding of the specific pathways involved in control of these phenotypes and, in general, the evolutionary process. Unfortunately, the genetics of most cave animals cannot be studied because they are not easily bred. Blind Mexican tetras, Astyanax mexicanus, are the valuable exception to this rule because fish from the various cave populations are fully interfertile with one another and with eyed sister forms still living in nearby surface streams. Hybrids between surface and cave forms permit genetic analysis of their differences, and study of the pure forms as well as of hybrids allows study of their developmental differences. Quantitative trait loci (QTL) analysis has already identified some specific genes responsible for differences between cave and surface forms as well as other likely candidates; more will be added in the future. This system is a valuable addition to the array of existing models for the study of developmental and evolutionary genetics because cave populations are repositories of numerous naturally occurring mutations affecting development of the eyes and other senses, pigmentation, bone structure, metamerism, and metabolism. These alleles have been prescreened by natural selection for high viability, which simplifies their study. In contrast, new alleles obtained through mutagenesis in other model species are typically burdened with lower viability.
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 1: Coating Glass Slides with Amino Silane
Authors: Link Andrew J. , LaBaer Joshua
Published: November 2008

INTRODUCTION

Functional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This approach, termed “Nucleic Acid Protein Programmable Array” (NAPPA), enables the simultaneous expression of proteins in microarray format without the need for individual protein purification. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes the initial preparation of slides to be used in the method.
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 2: Preparing Bacterial Cultures in a 96-Well Format: Figure 1.
Authors: Link Andrew J. , LaBaer Joshua
Published: November 2008

INTRODUCTION

Functional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This approach, termed “Nucleic Acid Protein Programmable Array” (NAPPA), enables the simultaneous expression of proteins in microarray format without the need for individual protein purification. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes preparation of the bacterial cultures in deep-well blocks.
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 3: Isolating DNA Plasmids in a 96-Well Plate Format
Authors: Link Andrew J. , LaBaer Joshua
Published: November 2008

INTRODUCTION

Functional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes a method for isolating the plasmids in a 96-well format.
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 4: DNA Biotinylation, Precipitation, and Arraying of Samples
Authors: Link Andrew J. , LaBaer Joshua
Published: November 2008

INTRODUCTION

Functional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes DNA biotinylation, precipitation, and arraying in preparation for protein expression.
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 5: Expressing Proteins on NAPPA Slides
Authors: Link Andrew J. , LaBaer Joshua
Published: November 2008

INTRODUCTION

Functional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes a method for expression of the desired proteins in situ on the printed slides.
Construction of Nucleic Acid Programmable Protein Arrays (NAPPA) 6: Detecting Proteins on NAPPA Slides: Figure 1.
Authors: Link Andrew J. , LaBaer Joshua
Published: November 2008

INTRODUCTION

Functional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains. This protocol describes antibody detection of the arrayed proteins.
Isolation, Culture, and Differentiation of Progenitor Cells from the Central Nervous System
Authors: Hutton Scott R. , Pevny Larysa H.
Published: November 2008

INTRODUCTION

The ability to prospectively identify and characterize neural progenitor cells in vivo has been difficult due to a lack of cell-surface markers specific for these cell types. A widely used in vitro culture method, known as the Neurosphere Assay (NSA), has provided a means to retrospectively identify neural progenitor cells as well as to determine both their self-renewal capacity and their ability to generate the three primary cell types of the nervous system: neurons, astrocytes, and oligodendrocytes. Today, combined with the establishment of multiple transgenic mouse strains expressing fluorescent markers and advances in cell isolation techniques such as fluorescence-activated cell sorting (FACS), the NSA provides a powerful system to prospectively elucidate neural progenitor characteristics and functions. Here we describe methods for the isolation, culture, and differentiation of neural progenitors from the developing mouse and adult cortex.
Preparing Recombinant Gonad Organ Cultures
Authors: Capel Blanche , Batchvarov Jordan
Published: November 2008

INTRODUCTION

It can be useful to assay migration between any two adjacent tissues during development. This protocol assays cell migration between the gonad and mesonephros using tissue recombination between genetically marked and unmarked tissue, combined with an organ culture technique. First, agar blocks are prepared in a custom-built mold. The size and shape of the wells are important to maintain the authentic three-dimensional morphology of the organ; the molds here are designed specifically to accommodate the gonad/mesonephros complex. Freshly dissected organs are then transferred to grooves within the agar blocks, where they are allowed to grow over 24-48 h. Using this protocol, organs develop with good morphology, and show only an ~12-h delay relative to in vivo development.
How to Grow Tomatoes
Authors: Kimura Seisuke , Sinha Neelima
Published: November 2008

INTRODUCTION

Tomatoes can be easily grown in a field, in a greenhouse, or in a growth cabinet. They need acidic soil (pH 6.0-6.8), a lot of light, and water. The optimum temperature for growing tomato plants and fruit is 18°C-24°C. This protocol describes how to germinate tomato seeds, cultivate adult plants, and harvest seeds from fruit.
Crossing Tomato Plants
Authors: Kimura Seisuke , Sinha Neelima
Published: November 2008

INTRODUCTION

This protocol describes how to cross tomato plants. Crossing is important for the genetic analysis and breeding of tomatoes. Tomatoes are self-pollinating plants; thus, emasculation (removal of the anthers from the female parent) is essential. All wild tomato species can be crossed with cultivated tomatoes (although it may be difficult); this is useful because wild tomatoes are a great source of desirable traits. Most commercial tomatoes are F1 hybrids, and the seeds for them were produced by crossing two parent tomatoes.
Mnemiopsis leidyi Spawning and Embryo Collection
Authors: Pang Kevin , Martindale Mark Q.
Published: November 2008

INTRODUCTION

Ctenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Because of its large size, fecundity, abundance in coastal areas, and recent introduction to European waters, Mnemiopsis leidyi (commonly called “sea walnuts”) is the most highly studied ctenophore. Under optimal conditions, these self-fertile hermaphrodites are capable of reproduction at 2 wk of age and can release up to 10,000 eggs per day. Adults can be maintained in large aquaria with gentle aeration as long as they are well fed and can be spawned daily; multiple generations can be raised in the laboratory. This protocol describes how to collect embryos from M. leidyi. Under natural conditions, spawning normally occurs ~8 h after sunset, such that eggs are released under the cover of darkness. Because spawning is triggered by the onset of darkness, keeping animals under an artificial light regimen in the laboratory can alter the time of spawning. This protocol is designed to induce animal spawning at approximately 11:00 a.m.; however, it can be adjusted for other times. The duration from spawning to hatching of larvae is 18-24 h.
Ctenophore Whole-Mount Antibody Staining
Authors: Pang Kevin , Martindale Mark Q.
Published: November 2008

INTRODUCTION

Ctenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. Because of the ease of embryo collection, their size (up to 1 mm in some species), and their rapid development, ctenophores have been attractive animals for experimental embryologists. This protocol describes how to fix ctenophore embryos and their cydippid larvae for antibody staining. Once the samples have been fixed, tissues are incubated with an antibody to the epitope of interest. A secondary antibody conjugated to a fluorescent molecule then reveals the expression pattern of the epitope. Fluorescent microscopy is used to visualize and document the signal. The protocol also includes methods for staining or counterstaining with a fluorescent derivative of phalloidin, which reveals F-actin in muscles and cell borders. Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.
Ctenophore Whole-Mount In Situ Hybridization
Authors: Pang Kevin , Martindale Mark Q.
Published: November 2008

INTRODUCTION

Ctenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. In some cases, gene expression patterns appear to be conserved. Of particular interest is the finding that some genes are expressed in regions of the ctenophore body that are not morphologically distinct from the adjacent areas. However, it has proven difficult to determine the orthology of some genes, possibly because of the extreme divergence of ctenophore representatives. This protocol describes how to fix, prepare, and hybridize antisense RNA probes in ctenophore embryos and cydippid larvae, as well as how to detect the probes using an alkaline phosphatase-conjugated antibody and colorimetric substrates. Using these techniques, it is possible to determine which cells or tissues express the gene of interest. Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.
Ctenophore Tissue Preparation and Extraction of DNA
Authors: Pang Kevin , Martindale Mark Q.
Published: November 2008

INTRODUCTION

Ctenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. In some cases, gene expression patterns appear to be conserved. Of particular interest is the finding that some genes are expressed in regions of the ctenophore body that are not morphologically distinct from the adjacent areas. However, it has proven difficult to determine the orthology of some genes, possibly because of the extreme divergence of ctenophore representatives. This protocol describes how to isolate genomic DNA from ctenophores. The procedure can be applied to adult tissues, but it is best to use embryos and larvae. After washing and concentrating the embryos or larvae, DNA is extracted using DNAzol reagent, a guanidine-detergent lysing solution. The resulting DNA can be used for polymerase chain reaction (PCR) or other applications.
Ctenophore Tissue Preparation and Extraction of RNA
Authors: Pang Kevin , Martindale Mark Q.
Published: November 2008

INTRODUCTION

Ctenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. In some cases, gene expression patterns appear to be conserved. Of particular interest is the finding that some genes are expressed in regions of the ctenophore body that are not morphologically distinct from the adjacent areas. However, it has proven difficult to determine the orthology of some genes, possibly because of the extreme divergence of ctenophore representatives. This protocol describes how to isolate total RNA from ctenophore embryos and larvae. After the specimens are sorted, cleaned, and concentrated, they are placed into TRI Reagent, a solution containing phenol and guanidine thiocyanate that allows for the effective isolation of total RNA. The resulting RNA can be used for various applications (e.g., to generate cDNA for reverse transcriptase-polymerase chain reactions). Although the protocol focuses on embryonic and larval samples, the technique can also be applied to adult tissues.
Determining the Sex of Adult Astyanax mexicanus
Authors: Borowsky Richard
Published: November 2008

INTRODUCTION

This protocol describes methods for determining the sex of an individual Astyanax mexicanus. Adult males and females differ most obviously in body shape and in form of the anal fin. However, if the sex of an adult cannot be determined by these differences, it can be assessed by testing the anal fin for the presence (or absence) of denticle or hook-like bony elements on the anterior fin rays of the anal fin. These features are observed as an opacity toward the anterior half of the male’s anal fin, detected as described in the method presented here.
Breeding Astyanax mexicanus through Natural Spawning
Authors: Borowsky Richard
Published: November 2008

INTRODUCTION

Male and female Astyanax mexicanus can be bred successfully in tanks under appropriate conditions. Females should be maintained on a diet high in fats for 10-14 d before breeding. The transfer of a male and female into clean water in a fresh tank and a change (increase) in water temperature are cues for breeding. Newly fertilized eggs may also be obtained through in vitro fertilization. Note that blind fish should never be paired with eyed fish in illuminated aquaria, because the eyed fish are aggressive and will kill even much larger blind fish. Such matings must be carried out in the dark or by using in vitro fertilization.
Handling Astyanax mexicanus Eggs and Fry
Authors: Borowsky Richard
Published: November 2008

INTRODUCTION

In this protocol, fertilized Astyanax mexicanus eggs are transferred into larger dishes to reduce crowding and are observed until they begin to hatch into fry.
Conditioned media from mouse osteosarcoma cells promote MC3T3‐E1 cell proliferation using JAKs and PI3‐K/Akt signal crosstalk
Authors: Mori Kanji , Blanchard Frederic , Charrier Celine , Battaglia Severine , Ando Kosei , Duplomb Laurence , Shultz Leonard D. , Redini Francoise , Heymann Dominique
Published: November 2008

The maintenance of bone mass requires a strict balance between bone formation by osteoblasts and bone resorption by osteoclasts. In tumoral bone environment, tumor cells frequently disturb this balance by interaction with bone cells to create a favorable site for tumor growth, and promote pathological bone changes. Thus, elucidation of the mechanisms underlying interaction between tumor cells and bone cells might eventually lead to a more rational strategy for therapeutic intervention for bone tumors and better understanding of bone biology. In the present study, the effects of mouse osteosarcoma cells on mouse preosteoblastic cells were determined by assessment of cell viability, osteoblastic differentiation and signal transduction pathways. MOS‐J/POS‐1 conditioned media (CM) significantly induced MC3T3‐E1 cell proliferation in a dose‐dependent manner and reduced both alkaline phosphatase activity and mineralized nodule formation. Piceatannol, AG490, LY294002 and rapamycin significantly abrogated this up‐regulated cell proliferation; however, UO126 and STAT3 inhibitor peptide did not affect this up‐regulated cell proliferation. MOS‐J/POS‐1 CM activated ERK 1/2, STAT3 and Akt signal transduction pathways; however, pro‐proliferating signal induced by MOS‐J/POS‐1 CM was transmitted via Akt not ERK 1/2 and STAT3 pathways. Furthermore, Western blot analyses clearly revealed novel signal crosstalk between JAKs and PI3‐K/Akt in osteoblastic cells. The specific factor(s) involved in MOS‐J/POS‐1 CM‐induced MC3T3‐E1 cell proliferation that use JAKs/PI3‐K/Akt/mTOR pathway remain(s) to be determined. Determination of the specific factor(s) responsible for JAKs and PI3‐K/Akt signal crosstalk that results in up‐regulated preosteoblast proliferation will offer new insight into the pathology of osteosarcoma as well as other bone‐related diseases. (Cancer Sci 2008; 99: 2170–2176)
Hospital procedure volume and prognosis with respect to testicular cancer patients: a population‐based study in Osaka, Japan
Authors: Suzumura Shigeo , Ioka Akiko , Nakayama Tomio , Tsukuma Hideaki , Oshima Akira , Ishikawa Osamu
Published: November 2008

Establishment of gold‐standard chemotherapy and the subsequent development of salvage therapy have much improved the prognosis of patients with testicular germ cell cancer. However, not all patients with this disease get the best possible outcome in Osaka, Japan. A population‐based study of testicular cancer cases diagnosed during 1993–1999 was performed using the Osaka Cancer Registry database to verify changes in prognosis and to examine their relationship with hospital procedure volume. Patients’ referrals to hospitals during 1990–2001 were also examined to evaluate the degree of centralization. The 5‐year relative survival rate of testicular cancer as well as testicular germ cell cancer in Osaka during 1993–1999 was markedly improved compared with that of previous periods (1975–1992); but remained lower than those reported in the EUROCARE‐4 and Surveillance Epidemiology and End Results program. There appeared to be a significant association between survival and hospital procedure volume, even with adjustment made for clinical stage, age and histology. Centralization of referrals had not progressed during the past 12 years. In conclusion, the prognosis of testicular cancer in Osaka has improved remarkably, but nevertheless has remained lower than in the EU and/or the USA. Little progress has been made in centralization of hospital referrals. (Cancer Sci 2008; 99: 2260–2263)
Prognostic value of kallikrein‐related peptidase 6 protein expression levels in advanced ovarian cancer evaluated by automated quantitative analysis (AQUA)
Authors: Kountourakis Panteleimon , Psyrri Amanda , Scorilas Andreas , Camp Robert , Markakis Sonia , Kowalski Diane , Diamandis Eleftherios P. , Dimopoulos Meletios A.
Published: November 2008

Kallikrein‐related peptidases, a subgroup of the serine protease enzyme family, are considered important prognostic biomarkers in cancer. In the present study, we sought to determine the prognostic value of kallikrein‐related peptidase 6 (KLK6) in ovarian cancer using a novel method of compartmentalized in situ protein analysis. A tissue array composed of 150 advanced stage ovarian cancers, uniformly treated with surgical debulking followed by platinum‐paclitaxel combination chemotherapy, was constructed. For evaluation of KLK6 protein expression, we used an immunofluorescence‐based method of automated in situ quantitative measurement of protein analysis (AQUA). Mean follow‐up time of the cohort was 34.35 months. One hundred and thirty‐five of 150 cases had sufficient tissue for AQUA analysis. In univariate survival analysis, low tumor KLK6 expression was associated with better outcome for overall survival over 3 years (P = 0.019). There was no association between tumor KLK6 expression and progression‐free survival (P = 0.128). In multivariate survival analysis, adjusting for well‐characterized prognostic variables, low tumor KLK6 expression level was one of the most significant predictor variable for overall survival (95% confidence interval, 1.19–3.50; P = 0.009). High tumor KLK6 protein expression is associated with inferior patient outcome in ovarian cancer. KLK6 may represent a promising disease biomarker and therapeutic target in ovarian cancer. (Cancer Sci 2008; 99: 2224–2229)