bluuebunny/crossref_metadata_2025_split huggingface dataset:
Robust and Comprehensive Analysis of 20 Osteoporosis Candidate Genes by Very High-Density Single-Nucleotide Polymorphism Screen Among 405 White Nuclear Families Identified Significant Association and Gene–Gene Interaction
Authors: Xiong Dong-Hai , Shen Hui , Zhao Lan-Juan , Xiao Peng , Yang Tie-Lin , Guo Yan , Wang Wei , Guo Yan-Fang , Liu Yong-Jun , Recker Robert R , Deng Hong-Wen 
Published: September 2006

Abstract
Many “novel” osteoporosis candidate genes have been proposed in recent years. To advance our knowledge of their roles in osteoporosis, we screened 20 such genes using a set of high-density SNPs in a large family-based study. Our efforts led to the prioritization of those osteoporosis genes and the detection of gene–gene interactions.
Introduction: We performed large-scale family-based association analyses of 20 novel osteoporosis candidate genes using 277 single nucleotide polymorphisms (SNPs) for the quantitative trait BMD variation and the qualitative trait osteoporosis (OP) at three clinically important skeletal sites: spine, hip, and ultradistal radius (UD).
Materials and Methods: One thousand eight hundred seventy-three subjects from 405 white nuclear families were genotyped and analyzed with an average density of one SNP per 4 kb across the 20 genes. We conducted association analyses by SNP- and haplotype-based family-based association test (FBAT) and performed gene–gene interaction analyses using multianalytic approaches such as multifactor-dimensionality reduction (MDR) and conditional logistic regression.
Results and Conclusions: We detected four genes (DBP, LRP5, CYP17, and RANK) that showed highly suggestive associations (10,000-permutation derived empirical global p ≤ 0.01) with spine BMD/OP; four genes (CYP19, RANK, RANKL, and CYP17) highly suggestive for hip BMD/OP; and four genes (CYP19, BMP2, RANK, and TNFR2) highly suggestive for UD BMD/OP. The associations between BMP2 with UD BMD and those between RANK with OP at the spine, hip, and UD also met the experiment-wide stringent criterion (empirical global p ≤ 0.0007). Sex-stratified analyses further showed that some of the significant associations in the total sample were driven by either male or female subjects. In addition, we identified and validated a two-locus gene–gene interaction model involving GCR and ESR2, for which prior biological evidence exists. Our results suggested the prioritization of osteoporosis candidate genes from among the many proposed in recent years and revealed the significant gene–gene interaction effects influencing osteoporosis risk.
Bone Density Ligand, Sclerostin, Directly Interacts With LRP5 but Not LRP5G171V to Modulate Wnt Activity
Authors: Ellies Debra L , Viviano Beth , McCarthy John , Rey Jean-Philippe , Itasaki Nobue , Saunders Scott , Krumlauf Robb 
Published: September 2006

Abstract
We compared and contrasted the mechanism of action for the cysteine knot protein subfamily, Wise and Sost (Sclerostin). Our data suggest that functional interactions between Sost or Wise and LRP5/LRP6 have the potential to regulate bone deposition by modulating the Wnt pathway.
Introduction: The human disease sclerosteosis exhibits an increase in bone mass thought to be caused by hyperactive osteoblasts. Sclerostin, SOST, the gene affected in this disease, has been postulated to exert its activity by functioning as a BMP antagonist. However, recent evidence indicates that SOST is highly related to Wise, which can also modulate the Wnt pathway by binding to LRP5 and LRP6.
Materials and Methods: For this study, we used cell culture to test the BMP and Wnt activity function of both Wise and Sost. In addition, we used Xenopus in vivo Wnt assays along with Xenopus in vitro Wnt assays to support our cell culture results. Epitope tagged cell supernatants containing either Sost or soluble mutant or wildtype LRP5/LRP6 were used for immunoprecipitation. Sost immunoprecipitation results were confirmed in vivo using cell culture. Finally, to support our in vitro data, we co-localized Sost, Wise, LRP5, and LRP6 in mouse long bone sections.
Results: In this study, we report in vitro and in vivo evidence to show that Sost physically interacts with Lrp5 and Lrp6 and inhibits the canonical Wnt signaling pathway. Furthermore, using in vitro and in vivo assays, we showed that a variant of LRP5 (LRP5G171V) known to cause the human high bone mass (HBM) trait and a homologous change in LRP6 (LRP6G158V) abolished protein interactions with Sost. We used variants of Sost amino acids to further identify the contact points between Sost and LRP6. In Xenopus and mammalian cell culture assays, we showed that SOST is able to attenuate Wnt signaling and that this attenuation can be rescued by the addition of α-Sost antibodies or by the introduction of single amino acid substitution that alter its binding to LRP6. Sost differs from Wise in that it is unable to stimulate Wnt signaling. Using immunohistochemistry, we found that Sost and Wise are co-localized to osteoblasts, along with LRP5 and LRP6.
Conclusions: Our data suggest that functional interactions between Sost or Wise and LRPs have the potential to regulate bone deposition by modulating Wnt signaling.
Antibodies to Stem Cell Marker Antigens Reduce Engraftment of Hematopoietic Stem Cells
Authors: Gilner Jennifer B. , Walton William G. , Gush Kimberly , Kirby Suzanne L. 
Published: September 2006

AbstractHematopoietic stem cells (HSCs) have enormous potential for use in transplantation and gene therapy. However, the frequency of repopulating HSCs is often very low; thus, highly effective techniques for cell enrichment and maintenance are required to obtain sufficient cell numbers for therapeutic use and for studies of HSC physiology. Common methods of HSC enrichment use antibodies recognizing HSC surface marker antigens. Because antibodies are known to alter the physiology of other cell types, we investigated the effect of such enrichment strategies on the physiology and lineage commitment of HSCs. We sorted HSCs using a method that does not require antibodies: exclusion of Hoechst 33342 to isolate side population (SP) cells. To elucidate the effect of antibody binding on this HSC population, we compared untreated SP cells with SP cells treated with the Sca-1+c-Kit+Lin− (SKL) antibody cocktail prior to SP sorting. Our findings revealed that HSCs incubated with the antibody cocktail had decreased expression of the stem cell-associated genes c-Kit, Cd34, Tal-1, and Slamf1 relative to untreated SP cells or to cells treated with polyclonal isotype control antibodies. Moreover, SKL antibodies induced cycling in SP cells and diminished their ability to confer long-term hematopoietic engraftment in lethally irradiated mice. Taken together, these data suggest that antibody-based stem cell isolation procedures can have negative effects on HSC physiology.
N-Glycolylneuraminic Acid Xenoantigen Contamination of Human Embryonic and Mesenchymal Stem Cells Is Substantially Reversible
Authors: Heiskanen Annamari , Satomaa Tero , Tiitinen Sari , Laitinen Anita , Mannelin Sirkka , Impola Ulla , Mikkola Milla , Olsson Cia , Miller-Podraza Halina , Blomqvist Maria , Olonen Anne , Salo Hanna , Lehenkari Petri , Tuuri Timo , Otonkoski Timo , Natunen Jari , Saarinen Juhani , Laine Jarmo 
Published: September 2006

Abstract
Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies.
Analysis of Immunotoxicity by Enumeration of Antibody‐Producing B Cells
Authors: Anderson Stacey E. , Munson Albert E. , Meade Barbara J. 
Published: September 2006

AbstractThe plaque‐forming cell (PFC) assay measures the humoral immune response mediated by the concerted actions of antigen‐presenting cells, T lymphocytes, and B lymphocytes. The most common form of the plaque method is used for the detection of murine primary IgM antibodies directed against the T cell–dependent sheep red blood cell (sRBC) surface antigens. Research has shown that the PFC response to sRBC is not only an excellent monitor of the primary effector function of the B cell, but that it may be the most sensitive immune parameter currently available to identify chemical perturbation. Several modifications and variations of the PFC assay are described in detail in this unit.
The Reporter Antigen Popliteal Lymph Node Assay
Authors: Nierkens Stefan , Pieters Raymond 
Published: September 2006

AbstractMany chemicals, including drugs and environmental pollutants, may have the intrinsic capacity to stimulate or dysregulate immune responses. These responses may create considerable problems for exposed subjects in terms of development of autoimmunity or hypersensitivity reactions. The popliteal lymph node assay (PLNA) provides a suitable tool to assess the immunostimulating potential of chemicals and might be a potential candidate as a screening tool in immunotoxicological hazard identification. The use of so‐called reporter antigens (RA) in this assay additionally enables differentiation between immunosensitizing (sensitizers), immunostimulating (irritants), and innocent chemicals. In the RA‐PLNA, the compound of interest is injected into the hind footpad together with a nonsensitizing dose of the RA. After 6 to 8 days, RA‐specific responses are monitored in the draining PLN by measuring RA‐specific antibody formation, cytokine secretion, and shifts in immune cell numbers. Hence, this simple and straightforward assay provides immunologically relevant information about the immunomodulating properties of a chemical.
Endoglycosidase and Glycoamidase Release of N‐Linked Oligosaccharides
Authors: Freeze Hudson H. , Kranz Christian 
Published: September 2006

AbstractNearly all proteins entering the lumen of the endoplasmic reticulum (ER) become glycosylated en route to a cellular organelle, the plasma membrane, or the extracellular space. Many glycans can be attached to proteins, but the most common are the N‐linked oligosaccharides. These chains are added very soon after a protein enters the ER, but they undergo extensive remodeling (processing), especially in the Golgi. Processing changes the sensitivity of the N‐glycan to enzymes that cleave entire sugar chains or individual monosaccharides, which also changes the migration of the protein on SDS gels. These changes can be used to indicate when a protein has passed a particular subcellular location. This unit details some of the methods used to track a protein as it traffics from the ER to the Golgi toward its final location.
Dog EEG for Wake‐Promotion Studies
Authors: Parmentier Régis , Bricout Denis , Brousseau Emmanuel , Giboulot Thierry 
Published: September 2006

AbstractDescribed in this unit is a protocol for investigating the wake‐promoting activity of new chemical entities (NCEs) in dog. The experimental approach is based on scoring of sleep/wake stages in animals implanted with a telemetry device for recording EMG and cortical EEG signals. A major advantage of this procedure is that it is conducted in nontethered animals, limiting possible bias and complications encountered with conventional recording systems. In this procedure, polygraphic recording is conducted using four implanted beagles. Results of studies with modafinil, a wake‐promoting agent, are described to demonstrate the utility of this test procedure.
In Vivo Animal Models: Quantitative Models Used for Identifying Antibacterial Agents
Authors: Fernandez Jeffrey 
Published: September 2006

AbstractDetermining the in vivo bactericidal potential of novel agents is critical for selecting drug candidates. Described in this unit are two protocols that measure the amount of bacterial killing under very different conditions. The mouse pouch protocol is a rapid assay that provides efficacy data in an acute infection, whereas the rabbit tissue cage protocol assesses the ability of a compound to eradicate bacteria in a chronic abscess infection. Data from these tests, along with those from other qualitative and quantitative assays, are important for defining antibacterial efficacy in vivo.
In Vitro Antibacterial Resistance Selection and Quantitation
Authors: Young Katherine 
Published: September 2006

AbstractThe study of resistance is vital to the discovery and development of new antibacterial agents. Consider the case where the resistance frequency (the proportion of cells within a population that exhibit a resistance phenotype to the agent under study) of an agent is such that, in an average infection, a mutant resistant to the agent is likely to already exist. This compound will not be useful in the clinic as a single agent, since this mutant can survive its administration, leading to potential clinical failure. This unit provides protocols for determining the frequency and rate of resistance to antibacterial agents, as well as generating resistant mutants for mechanism of action determinations or for use in antibacterial discovery or development programs.
Fluorescence Quenching Methods to Study Lipid‐Protein Interactions
Authors: Carney Joanne , East J. Malcolm , Mall Sanjay , Marius Phedra , Powl Andrew M. , Wright J. Neville , Lee Anthony G. 
Published: September 2006

AbstractThis unit describes how fluorescence quenching methods can be used to determine binding constants for phospholipids binding to intrinsic membrane proteins. Reconstitution of a Trp‐containing intrinsic membrane protein with bromine‐containing phospholipids leads to quenching of the Trp fluorescence of the protein; the extent of quenching depends on the strength of binding of the phospholipid to the protein. Protocols are included for the synthesis of bromine‐containing phospholipids from phospholipids containing carbon‐carbon double bonds in their fatty acyl chains and for the reconstitution of membrane proteins into bilayers containing bromine‐containing phospholipids. Details are included on data analysis, including equations and software that can be used for fitting the fluorescence quenching data.
Solution‐Phase Synthesis of Di‐ and Trinucleotides Using Polymer‐Supported Reagents
Authors: Morvan François , Vasseur Jean‐Jacques 
Published: September 2006

AbstractA solution‐phase phosphoramidite synthesis of short oligonucleotides using solid‐supported reagents is described. Polyvinyl pyridinium tosylate is employed as the activator in the coupling step between nucleoside‐3′‐O‐phosphoramidites and 5′‐OH nucleosides. The resulting dinucleotide phosphite triesters are oxidized or sulfurized with polymer‐bound periodate or tetrathionate to phosphotriesters or thiono phosphotriesters, respectively. Removal of the 3′‐O‐levulinyl protection is accomplished with a polymer‐supported hydrazine. In this approach, the excess reagents are removed by filtration, avoiding complicated purifications. All the polymer‐bound reagents used in this method result from salt bridge interactions with the polymers. Consequently, the resins are regenerable.
DNA Synthesis Without Base Protection Using the Phosphoramidite Approach
Authors: Ohkubo Akihiro , Seio Kohji , Sekine Mitsuo 
Published: September 2006

AbstractIn the strategy of DNA synthesis without base protection, a recentO‐selective phosphorylation procedure known as the “activated phosphite method” is used to generate reactive phosphite intermediates fromN‐unprotected phosphoramidite building blocks. This new strategy proved to be advantageous over previous strategies because the cleavage of the N‐P(III) bond, which can be formed by the significant phosphitylation of the nucleobase amino groups, can be omitted. The activated phosphite method provides an attractive route to high‐throughput synthesis of DNA and the synthesis of alkali‐labile modified DNAs such as oligonucleotides containing DNA lesions. This unit presents two convenient methods for the synthesis ofN‐unprotected deoxynucleoside 3′‐phosphoramidite units and detailed procedures for automated synthesis of oligodeoxyribonucleotides from these phosphoramidite units.
Mass Determination of Phosphoramidites
Authors: Harusawa Shinya , Fujitake Mihoyo , Kurihara Takushi , Zhao Zheng‐yun , Lilley David M.J. 
Published: September 2006

AbstractNucleoside phosphoramidites are the most widely used building blocks in contemporary solid‐phase synthesis of oligonucleotides. The accurate molecular weight measurements of such molecules, which are acid‐labile compounds, may be easily determined by mass spectrometry using a matrix system, triethanolamine/NaCl, on a liquid secondary ion mass spectrometer (LSIMS) or fast‐atom bombardment (FAB) MS equipped with a double‐focusing mass spectrometer. The present method rapidly and easily measures the accurate molecular weights of various phosphoramidites as adduct ions [M+Na]+ with an average mass error smaller than 0.4 ppm, allowing determination of the formulas of the phosphoramidites in place of elemental analysis. Further, it was found that intensities of molecular‐related ions could be enhanced to the highest degree by adjustment of the molar ratio of phosphoramidite and NaCl, fixing the amount of triethanolamine on LSIMS, making the present method a powerful tool for identification of phosphoramidites by mass spectrometry.
Chemical Phosphorylation of Deoxyribonucleosides and Thermolytic DNA Oligonucleotides
Authors: Ausín Cristina , Grajkowski Andrzej , Cieślak Jacek , Beaucage Serge L. 
Published: September 2006

AbstractThe phosphorylating reagent bis[S‐(4,4′‐dimethoxytrityl)‐2‐mercaptoethyl]‐N,N‐diisopropylphosphoramidite is prepared in three steps from commercial methyl thioglycolate and diisopropylphosphoramidous dichloride. The phosphorylating reagent has been used successfully in the solid‐phase synthesis of deoxyribonucleoside 5′‐/3′‐phosphate or ‐thiophosphate monoesters and oligonucleotide 5′‐phosphate/‐thiophosphate monoesters. Bis[S‐(4,4′‐dimethoxytrityl)‐2‐mercaptoethyl]‐N,N‐diisopropylphosphoramidite has also been employed in the construction of a thermolytic dinucleotide prodrug model to evaluate the ability of the reagent to produce thermosentive oligonucleotide prodrugs under mild temperature conditions (∼25°C) for potential therapeutic applications.
Production of Monoclonal Antibodies
Authors: Yokoyama Wayne M. , Christensen Michelle , Santos Gary Dos , Miller Diane 
Published: September 2006

AbstractThis unit describes the production of monoclonal antibodies beginning with immunization and cell fusion and selection. Support protocols are provided for screening primary hybridoma supernatants for antibodies of desired specificity, establishment of stable hybridoma lines, cloning of these B cell lines by limiting dilution to obtain monoclonal lines, and preparation of cloning/expansion medium. An alternate protocol describes cell fusion and one‐step selection and cloning of hybridomas utilizing a semi‐solid methylcellulose‐based medium (ClonaCell®‐HY).
Preparation and Coculture of Neurons and Glial Cells
Authors: Viviani Barbara 
Published: September 2006

AbstractThe close relationship between neurons, glia, astroglia, and astrocytes plays a relevant role in modulating a neurotoxic effect and propagating the consequent degenerative event in the brain. After exposure to a neurotoxicant, glial cells are often activated to release substances responsible for the propagation of the damage. the production of such mediators is the result of both neural death and communication between microglia and astrocytes. Because they are simple and highly controlled, cocultures of different cells of the nervous system are a valuable tool in the investigations of these complex relationships. This unit describes protocols to set up neuron‐glia and microglia‐astrocyte sandwich cocultures, in which the selected cell populations grow on two different surfaces. This system has the advantage that the two cell populations can be separated at any point in the exposure without disrupting the cells integrity and organization
Assays for Regulated Exocytosis of Mast Cell Granules
Authors: Blank Ulrich , Rivera Juan 
Published: September 2006

AbstractMast cells are important effectors in innate and adaptive immune responses. They contain numerous secretory granules filled with inflammatory mediators in their cytoplasm. Exocytosis of granular content does not take place until the cell receives an appropriate stimulus such as the aggregation of IgE antibody bound to high‐affinity IgE receptors by specific antigen. This process is therefore referred to as regulated exocytosis. A characteristic of mast cell exocytosis is that it does not involve release of a few individual granules, but rather, a large fraction of the granular content is released due to compound exocytosis, implicating the occurrence of granule‐to‐granule and granule‐to‐plasma membrane fusion. This unit describes assays that measure the release of granular content from mast cells. They include in vitro colorimetric‐, radiolabel‐, or antibody detection–based assays for substances stored in the granules. Given that animal models for basophil and mast cell activation are being used with increasing frequency, the unit also includes protocols to measure exocytosis of mast cell granule content in vivo.
An Introduction to Recognizing Functional Domains
Authors: Stormo Gary D. 
Published: September 2006

AbstractThis unit provides an overview of issues involved in domain recognition in protein and DNA sequences. It opens with a discussion of the two primary methods of domain representation, namely consensus sequences and alignment matrices (e.g., the log‐odds matrix). The unit continues with a brief overview of some of the resources available for identifying functional domains in nucleotide sequences (e.g., TRANSFAC). In addition, it reviews databases such as Pfam, InterPro and Blocks, which are available for protein analysis.