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Heidi Morales,
Published: 1 December 2008
Biological Procedures Online, Volume 10, pp 1-8; https://doi.org/10.1251/bpo137

Abstract:
Activation of lymphocytes in mammals is often quantified by measuring the amount of proliferation during the expansion phase of an immune response. Bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution assays are some of the techniques widely used in mammalian studies of pathogen-induced proliferation and provide a convenient way of quantifying the cellular response. We have extended the use of these proliferation assays to the amphibian Xenopus laevis. We have developed this species as a valuable comparative model to study immunity against a well-known amphibian pathogen, Frog Virus 3 (FV3). Fluorescence activated cell sorting was used to assess the level of BrdU incorporation of lymphocytes in vivo and CFSE dilution in an in vitro activation assay. Both techniques have shown that splenic lymphocytes proliferate specifically upon FV3 challenge. This indicates that common methods for detection of proliferation upon immunologic challenge are easily applied to other vertebrate species, as it highlights the evolutionary conservation of the proliferative nature of immune responses throughout vertebrate phyla.
David Bond, David A. Primrose, Edan Foley
Published: 1 December 2008
Biological Procedures Online, Volume 10, pp 20-28; https://doi.org/10.1251/bpo139

The publisher has not yet granted permission to display this abstract.
Alireza Moeenrezakhanlou, Devki Nandan, Neil E. Reiner
Published: 1 December 2008
Biological Procedures Online, Volume 10, pp 29-35; https://doi.org/10.1251/bpo140

Abstract:
Calcitriol (1α, 25-dihydroxyvitamin D3) induces the expression of CD14 in mononuclear phagocytes. The mechanisms accounting for this have been unclear since the promoter of CD14 does not contain a canonical vitamin D response element (VDRE). Calcitriol has been shown to regulate the activity of the transcription factor Sp-1 and our analysis of the proximal promoter of CD14 indicated the presence of four Sp-1-like binding sequences. To identify which of these sites might be involved in the response to calcitriol, we used a system incorporating an electrophoretic mobility shift assay (EMSA) coupled to Western blot analysis (WEMSA). Using WEMSA, we found that only one of the Sp-1-like binding sequences, located at position −91 to −79 (relative to the transcription start site), bound the transcription factor Sp1. Sp-1 binding to this site was demonstrable using nuclear extracts from control cells. Notably, binding activity was attenuated in nuclear extracts prepared from cells that had been incubated with calcitriol, thus suggesting Sp-1 involvement in calcitriol induction of CD14 expression. Notably, these results show that like EMSA, WEMSA can be broadly applied to aid in the identification of transcription factors involved in regulating gene expression. WEMSA, however, offers a number of distinct advantages when compared with conventional EMSA. Antibodies used for WEMSA often provide less ambiguous signals than those used in EMSA, and these do not have to recognize epitopes under native conditions. In addition, WEMSA does not require the use of labeled oligos, thus eliminating a significant expense associated with EMSA.
Horst Robenek, Nicholas J. Severs
Published: 1 December 2008
Biological Procedures Online, Volume 10, pp 9-19; https://doi.org/10.1251/bpo138

The publisher has not yet granted permission to display this abstract.
David H. Keating, Ana Shulla, Adam H. Klein, Alan J. Wolfe
Published: 1 December 2008
Biological Procedures Online, Volume 10, pp 36-46; https://doi.org/10.1251/bpo141

Abstract:
Acetyl phosphate (acetyl-P) serves critical roles in coenzyme A recycling and ATP synthesis. It is the intermediate of the Pta-AckA pathway that inter-converts acetyl-coenzyme A and acetate. Acetyl-P also can act as a global signal by donating its phosphoryl group to specific two-component response regulators. This ability derives from its capacity to store energy in the form of a high-energy phosphate bond. This bond, while critical to its function, also destabilizes acetyl-P in cell extracts. This lability has greatly complicated biochemical analysis, leading in part to widely varying acetyl-P measurements. We therefore developed an optimized protocol based on two-dimensional thin layer chromatography that includes metabolic labeling under aerated conditions and careful examination of the integrity of acetyl-P within extracts. This protocol results in greatly improved reproducibility, and thus permits precise measurements of the intracellular concentration of acetyl-P, as well as that of other small phosphorylated molecules.
Alexander Unterberger, Jérôme Torrisani,
Published: 1 December 2008
Biological Procedures Online, Volume 10, pp 47-57; https://doi.org/10.1251/bpo142

Abstract:
DNA methyltransferase 1 (DNMT1) is the enzyme responsible for the maintenance of DNA methylation patterns during cell division. DNMT1 expression is tightly regulated within the cell cycle. Our previous study showed that the binding of a protein with an apparent size of approximately 40 kDa on DNMT1 3'-UTR triggered the destabilization of DNMT1 mRNA transcript during G(o)/G(1) phase. Using RNA affinity capture with the 3'-UTR of DNMT1 mRNA and matrix-assisted laser desorption-time of flight tandem mass spectrometry (MALDI-TOF-MS-MS) analysis, we isolated and identified AUF 1 (AU-rich element ARE:poly-(U)-binding/degradation factor) as the binding protein. We then validated the role of this protein in the destabilization of DNMT1 mRNA. In this report, we detail the different approaches used for the isolation, the identification of a RNA binding protein and the validation of its role.
Getu Teressa,
Published: 1 December 2008
Biological Procedures Online, Volume 10, pp 58-65; https://doi.org/10.1251/bpo143

Abstract:
We report a method for studying postsynaptic membrane assembly utilizing the replating of aneural cultures of differentiated skeletal muscle cells onto laminin-coated surfaces. A significant limitation to the current cell culturebased approaches has been their inability to recapitulate the multistage surface acetylcholine receptor (AChR) redistribution events that produce complex AChR clusters found at the intact neuromuscular junction (NMJ). By taking advantage of the ability of substrate laminin to induce advanced maturation of AChR aggregates on the surface of myotubes, we have developed a secondary-plating method that allows more precise analysis of the signaling events connecting substrate laminin stimulation to complex AChR cluster formation. We validate the utility of this method for biochemical and microscopy studies by demonstrating the roles of RhoGTPases in substrate laminin-induced complex cluster assembly.
, Katharina Dittmar
Published: 1 December 2008
Biological Procedures Online, Volume 10, pp 66-73; https://doi.org/10.1251/bpo144

Abstract:
Gene families are widely used in comparative genomics, molecular evolution, and in systematics. However, they are constructed in different manners, their data analyzed and interpreted differently, with different underlying assumptions, leading to sometimes divergent conclusions. In systematics, concepts like monophyly and the dichotomy between homoplasy and homology have been central to the analysis of phylogenies. We critique the traditional use of such concepts as applied to gene families and give examples of incorrect inferences they may lead to. Operational definitions that have emerged within functional genomics are contrasted with the common formal definitions derived from systematics. Lastly, we question the utility of layers of homology and the meaning of homology at the character state level in the context of sequence evolution. From this, we move forward to present an idealized strategy for characterizing gene family evolution for both systematic and functional purposes, including recent methodological improvements.
Steven J Greco, Chunyi Zhou, Jiang-Hong Ye, Pranela Rameshwar#
Published: 27 November 2008
Biological Procedures Online, Volume 10, pp 90-101; https://doi.org/10.1251/bpo147

Abstract:
The present study describes a protocol to generate heterogenous populations of neurotransmitter-producing neurons from human mesenchymal stem cells (MSCs). MSCs are bone marrow (BM)-derived cells which undergo lineage- specific differentiation to generate bone, fat, cartilage and muscle, but are also capable of transdifferentiating into defined ectodermal and endodermal tissues. The purpose of this study is to evaluate the potential of MSCs as an alternative source of customized neurons for experimental neurobiology or other regenerative approaches. Our neuronal protocol utilizes freshly harvested human MSCs cultured on specific surfaces and exposed to an induction cocktail consisting of low serum concentration, retinoic acid (RA), growth factors and supplements. Here we report on the types of neurotransmitters produced by the neurons, and demonstrate that the cells are electrically responsive to exogenous neurotransmitter administration.
, Arméle Guignée, Alain R. Thierry
Published: 22 September 2008
Biological Procedures Online, Volume 10, pp 83-89; https://doi.org/10.1251/bpo146

Abstract:
In this paper, we describe a simple, rapid, specific, sensitive, and reliable method, the FICP method (Fluorescence Immunoassay for Cellular Protein detection) which is readily applicable to the detection of proteins directly on cells cultured in 96-well plates. In order to illustrate this method, we report on the detection of two different proteins, the cell cycle proteins cyclin D1 and p21CIP1/WAF1, in untreated and 2-cyclopenten-1-one treated breast cancer cells. When the FICP method was compared with Western blot procedure, FICP was found to be superior for many characteristics. By using this method, we were able to quantify biological effects of a specific compound on protein levels in non-lysed cells and perform statistical analysis. Therefore, we believe this screening assay could be very useful for detecting poorly expressed proteins and for drug development.
, Christine Neely, Jessica Pfautz, Mindy George-Weinstein
Published: 1 September 2008
Biological Procedures Online, Volume 10, pp 74-82; https://doi.org/10.1251/bpo145

Abstract:
The early chick embryo contains subpopulations of cells that express lineage-specific transcription factors. We have developed protocols to examine the role of these cells during development that involve labeling them for cell tracking purposes and ablating them within the epiblast. The procedures take advantage of the fact that subpopulations of epiblast cells differentially express cell surface antigens recognized by monoclonal antibodies. Embryos are removed from the shell and incubated on the yolk with an antibody. Cells that bind the antibody are either tagged with a fluorescent secondary antibody or lysed with complement. For long-term analyses, embryos are returned to a host shell and placed in an incubator. This method of whole embryo manipulation ex-ovo and incubation in-ovo supports normal development into the fetal period.
Sandra L. Fernandez, David W. Russell, Peter J. Hurlin
Published: 24 December 2007
Biological Procedures Online, Volume 9, pp 84-90; https://doi.org/10.1251/bpo136

Abstract:
Understanding mechanisms of gene regulation has broad therapeutic implications for human disease. Here we describe a novel method for generating human cell lines that serve as reporters of transcriptional activity. This method exploits the ability of recombinant adeno-associated virus to mediate the insertion of exogenous DNA sequences into specific genomic loci through homologous recombination. To overcome the severe size limitation of the rAAV for carrying exogenous DNA, an enhanced green fluorescent protein (EGFP)-Luciferase fusion gene was used as both a selectable marker and gene expression reporter. EGFP was used for selection of correctly targeted alleles by taking advantage of known regulatory conditions that activate transcription of specific genes. Using this method, we describe the generation of primary human fibroblasts that express EGFP-Luciferase under the control of the c-Myc oncogene.
Pierre Antony, Kristen Hoek, Bhaskarjyoti Sarmah, Wasif N. Khan
Published: 24 December 2007
Biological Procedures Online, Volume 9, pp 73-83; https://doi.org/10.1251/bpo135

Abstract:
B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties and limiting cell numbers. We therefore employed mini-scale protocols to assess lipid signaling, particularly that of diacylglycerol and inositol trisphosphate, with as few as 0.5x106 purified early (T1) and late (T2) transitional B cells. Additionally, utilizing flow cytometric techniques, we determined levels of phosphatidylinositol bisphosphate and calcium mobilization in T1 and T2 cells, as well as mature follicular and marginal zone B cells using less than 1x106 primary B cells. Thus, these biochemical and flow cytometric methodologies can be used to analyse signal-induced changes in phosphatidylinositol bisphosphate levels, diacylglycerol and inositol triphosphate production and calcium in each B cell population.
H. Fai Poon, Laila Abdullah, Jon Reed, Sarah M Doore, Cyndi Laird, Venkat Mathura, , Fiona Crawford
Published: 19 December 2007
Biological Procedures Online, Volume 9, pp 65-72; https://doi.org/10.1251/bpo134

Abstract:
Recent advances in redox proteomics have provided significant insight into the role of oxidative modifications in cellular signalling and metabolism. At present, these techniques rely heavily on Western blots to visualize the oxidative modification and corresponding two dimensional (2D) gels for detection of total protein levels, resulting in the duplication of efforts. A major limitation associated with this methodology includes problematic matching up of gels and blots due to the differences in processing and/or image acquisition. In this study, we present a new method which allows detection of protein oxidation and total protein on the same gel to improve matching in image analysis. Furthermore, the digested protein spots are compatible with standard MALDI mass spectrometry protein identification. The methodology highlighted here may be useful in facilitating the development of biomarkers, assessing potential therapeutic targets and elucidating new mechanisms of redox signalling in redox-related conditions.
Nitixa Patel, Marianne Castillo, Pranela Rameshwar
Published: 14 December 2007
Biological Procedures Online, Volume 9, pp 56-64; https://doi.org/10.1251/bpo133

Abstract:
In adults, hematopoiesis occurs in bone marrow (BM) through a complex process with differentiation of hematopoietic stem cells (HSCs) to immune and blood cells. Human HSCs and their progenitors express CD34. Methods on hematopoietic regulation are presented to show the effects of the chemokine, stromal-derived growth factor (SDF)-1Î and the neuropeptide, substance P (SP). SDF-1Î production in BM stroma causes interactions with HSCs, thereby retaining the HSCs in regions close to the endosteum, at low oxygen. Small changes in SDF-1Î levels stimulate HSC functions through direct and indirect mechanisms. The indirect method occurs by SP production, which stimulates CD34+ cells, supported by ligand-binding studies, long-term culture-initiating cell assays for HSC functions, and clonogenic assays for myeloid progenitors. These methods can be applied to study other hematopoietic regulators.
Irene Salinas, José Meseguer,
Published: 25 November 2007
Biological Procedures Online, Volume 9, pp 43-55; https://doi.org/10.1251/bpo132

Abstract:
Teleost gut associated lymphoid tissue (GALT) consists of leucocyte populations located both intraepithelially and in the lamina propria with no structural organization. The present study aims to assess different protocols for the isolation of GALT cells from an important fish species in the Mediterranean aquaculture, the gilthead seabream. Mechanical, chemical and enzymatic treatments were assayed. Nylon wool columns and continuous density gradients were used for further separation of cell subpopulations. Light microscopy and flow cytometry showed that the highest density band (HD) consisted of a homogeneous lymphocytic population, whereas the intermediate density band (ID) corresponded to epithelial and secretory cells and some lymphocytes. Respiratory burst activity of total cell suspensions revealed very low numbers of potential phagocytic cells, reflecting results from light microscopy and reports in other teleost species. The present data set up the basis for future functional characterization of GALT in seabream.
Seok Hwee Koo, Tan Ching Ong, Kok Ting Chong, , , Edmund Jon Deoon Lee
Published: 1 November 2007
Biological Procedures Online, Volume 9, pp 18-30; https://doi.org/10.1251/bpo131

Abstract:
We have developed and validated a consolidated bead-based genotyping platform, the Bioplex suspension array for simultaneous detection of multiple single nucleotide polymorphisms (SNPs) of the ATP-binding cassette transporters. Genetic polymorphisms have been known to influence therapeutic response and risk of disease pathologies. Genetic screening for therapeutic and diagnostic applications thus holds great promise in clinical management. The allele-specific primer extension (ASPE) reaction was used to assay 22 multiplexed SNPs for eight subjects. Comparison of the microsphere-based ASPE assay results to sequencing results showed complete concordance in genotype assignments. The Bioplex suspension array thus proves to be a reliable, cost-effective and high-throughput technological platform for genotyping. It can be easily adapted to customized SNP panels for specific applications involving large-scale mutation screening of clinically relevant markers.
Qiwen Deng, Wensheng Luo,
Published: 14 September 2007
Biological Procedures Online, Volume 9, pp 18-26; https://doi.org/10.1251/bpo130

Abstract:
We developed a rapid mutagenesis method based on a modification of the QuikChange® system (Stratagene) to systemically replace endogenous gene sequences with a unique similar size sequence tag. The modifications are as follows: 1: the length of the anchoring homologous sequences of both mutagenesis primers were increased to 16 – 22 bp to achieve melting temperatures greater than 80°C. 2: the final concentrations of both primers were increased to 5–10 ng/µl and the final concentration of template to 1–2 ng/µl. 3: the annealing temperature was adjusted when necessary from 52°C to 58°C. We generated 25 sequential mutants in the cloned espD gene (1.2 kb), which encodes an essential component of the type III secretion translocon required for the pathogenesis of enteropathogenic E. coli (EPEC) infection. Each mutation consisted of the replacement of 15 codons (45 bp) with 8 codons representing a 24 bp sequence containing three unique restriction endonuclease sites (KpnI/MfeI/SpeI) starting from the second codon. The insertion of the restriction endonuclease sites provides a convenient method for further insertions of purification and/or epitope tags into permissive domains. This method is rapid, site-directed and allows for the systematic creation of mutants evenly distributed throughout the entire gene of interest.
Hailong Wu, Anh Dinh, Yin-Yuan Mo
Biological Procedures Online, Volume 9, pp 9-17; https://doi.org/10.1251/bpo129

The publisher has not yet granted permission to display this abstract.
, Ursula Greferath, Yvette M. Wilson
Published: 20 February 2007
Biological Procedures Online, Volume 9, pp 1-8; https://doi.org/10.1251/bpo128

Abstract:
We have developed a system to visualize functionally activated neurons and their projections in the brain. This system utilizes a transgenic mouse, fos-tau-lacZ (FTL), which expresses the marker gene, lacZ, in neurons and their processes after activation by many different stimuli. This system allows the imaging of activation from the level of the entire brain surface, through to individual neurons and their projections. The use of this system involves detection of neuronal activation by histochemical or immunohistochemical detection of beta-galactosidase (betagal), the product of the lacZ gene. Furthermore, the underlying brain state of the FTL mice determines the basal levels of expression of betagal. Here we describe in detail our protocols for detection of FTL expression in these mice and discuss the main variables which need to be considered in the use of these mice for the detection and mapping of functionally activated neurons, circuits and regions in the brain.
Andrée-Anne Dussault,
Published: 1 December 2006
Biological Procedures Online, Volume 8, pp 1-10; https://doi.org/10.1251/bpo114

The publisher has not yet granted permission to display this abstract.
Published: 1 December 2006
Biological Procedures Online, Volume 8, pp 36-43; https://doi.org/10.1251/bpo116

Abstract:
Microtubule inhibitors such as Vinblastine and Paclitaxel are chemotherapy agents that activate the mitotic spindle checkpoint, arresting cells in mitosis and leading to cell death. The pathways that connect mitotic arrest to cell death are not well characterized. We developed a mammalian cell-based cDNA cloning method to isolate proteins and protein fragments whose expression inhibits colony formation in the presence of microtubule inhibitors. Understanding how these proteins impact cellular responses to microtubule drugs will lead to better understanding of the biochemical pathways connecting mitotic arrest and cell death in mammalian cells and may provide novel targets that can enhance microtubule inhibitor-mediated chemotherapy.
Bobbie Ann Austin, Cassandra M. James, Peter Härle,
Published: 1 December 2006
Biological Procedures Online, Volume 8, pp 55-62; https://doi.org/10.1251/bpo118

The publisher has not yet granted permission to display this abstract.
Sanhita Ray,
Published: 1 December 2006
Biological Procedures Online, Volume 8, pp 69-76; https://doi.org/10.1251/bpo120

Abstract:
Chromatin immunoprecipitation (ChIP) technique allows detection of proteins that bind to chromatin. While this technique has been applied extensively in cell-based studies, its tissue-based application remains poorly explored. We are specifically interested in examining estrogen-dependent transcriptional mechanism in respect of recruitment of estrogen receptor-alpha (ERalpha), a ligand-activated transcription factor, to uterine gene promoters in mice. Recent gene-array studies, utilizing ERalpha knock-out vs. wild-type mice, have revealed that estrogen regulates numerous uterine genes temporally and most importantly via ERalpha during the phase-II response, including three well characterized genes viz., lactoferrin (Ltf), progesterone receptor (Pgr) and cyclinD1 (Ccnd1). Here, utilizing systematic ChIP studies, we demonstrate endogenous recruitment of ERalpha to above uterine gene promoters following estradiol-17beta (E(2)) injection in mice.
Matthew P. Badtke, Feng Cao,
Published: 1 December 2006
Biological Procedures Online, Volume 8, pp 77-86; https://doi.org/10.1251/bpo121

The publisher has not yet granted permission to display this abstract.
Published: 1 December 2006
Biological Procedures Online, Volume 8, pp 163-163; https://doi.org/10.1251/bpo124

Abstract:
This paper was originally published in Biological Procedures Online (BPO) on March 23, 2006. It was brought to the attention of the journal and authors that reference 74 was incorrect. The original citation for reference 74, “Stanford V. Biosignals offer potential for direct interfaces and health monitoring. Pervasive Computing, IEEE 2004; 3(1):99–103.” should read “Costanza E, Inverso SA, Allen R. ‘Toward Subtle Intimate Interfaces for Mobile Devices Using an EMG Controller’ in Proc CHI2005, April 2005, Portland, OR, USA.”
Rebecca L. Dryer,
Published: 1 December 2006
Biological Procedures Online, Volume 8, pp 44-54; https://doi.org/10.1251/bpo117

Abstract:
The Chromatin Immunoprecipiation (ChIP) provides a powerful technique for identifying the in vivo association of transcription factors with regulatory elements. However, obtaining meaningful information for promoter interactions is extremely challenging when the promoter is a member of a class of highly homologous elements. Use of PCR primers with small numbers of mutations can limit cross-hybridization with non-targeted sequences and distinguish a pattern of binding for factors with the regulatory element of interest. In this report, we demonstrate the selective in vivo association of NF-kappaB, p300 and CREB with the human Igamma1 promoter located in the intronic region upstream of the Cgamma1 exons in the immunoglobulin heavy chain locus. These methods have the ability to extend ChIP analysis to promoters with a high degree of homology.
Jianrun Xia, Song Hon H. Kim, Susan Macmillan,
Published: 1 December 2006
Biological Procedures Online, Volume 8, pp 63-68; https://doi.org/10.1251/bpo119

Abstract:
Live cell fluorescence microscopy using fluorescent protein tags derived from jellyfish and coral species has been a successful tool to image proteins and dynamics in many species. Multi-colored aequorea fluorescent protein (AFP) derivatives allow investigators to observe multiple proteins simultaneously, but overlapping spectral properties sometimes require the use of sophisticated and expensive microscopes. Here, we show that the aequorea coerulescens fluorescent protein derivative, PS-CFP2 has excellent practical properties as a blue fluorophore that are distinct from green or red fluorescent proteins and can be imaged with standard filter sets on a widefield microscope. We also find that by widefield illumination in live cells, that PS-CFP2 is very photostable. When fused to proteins that form concentrated puncta in either the cytoplasm or nucleus, PSCFP2 fusions do not artifactually interact with other AFP fusion proteins, even at very high levels of over-expression. PSCFP2 is therefore a good blue fluorophore for distinct three color imaging along with eGFP and mRFP using a relatively simple and inexpensive microscope.
Min Lin, Wei Zhao,
Published: 1 December 2006
Biological Procedures Online, Volume 8, pp 164-174; https://doi.org/10.1251/bpo125

Abstract:
How the power required for bird flight varies as a function of forward speed can be used to predict the flight style and behavioral strategy of a bird for feeding and migration. A U-shaped curve was observed between the power and flight velocity in many birds, which is consistent to the theoretical prediction by aerodynamic models. In this article, we present a general genetic model for fine mapping of quantitative trait loci (QTL) responsible for power curves in a sample of birds drawn from a natural population. This model is developed within the maximum likelihood context, implemented with the EM algorithm for estimating the population genetic parameters of QTL and the simplex algorithm for estimating the QTL genotype-specific parameters of power curves. Using Monte Carlo simulation derived from empirical observations of power curves in the European starling (Sturnus vulgaris), we demonstrate how the underlying QTL for power curves can be detected from molecular markers and how the QTL detected affect the most appropriate flight speeds used to design an optimal migration strategy. The results from our model can be directly integrated into a conceptual framework for understanding flight origin and evolution.
, , Frances M. Van Dolah
Published: 1 December 2006
Biological Procedures Online, Volume 8, pp 175-193; https://doi.org/10.1251/bpo126

Abstract:
Quantitative real-time PCR (qPCR) is a commonly used validation tool for confirming gene expression results obtained from microarray analysis; however, microarray and qPCR data often result in disagreement. The current study assesses factors contributing to the correlation between these methods in five separate experiments employing two-color 60-mer oligonucleotide microarrays and qPCR using SYBR green. Overall, significant correlation was observed between microarray and qPCR results (rho=0.708, p0.80 consistently observed when quality scores are applied.
Jose M. Mellado-Gil,
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 162-171; https://doi.org/10.1251/bpo113

Abstract:
Pancreatic β-cell apoptosis is known to participate in the β-cell destruction process that occurs in diabetes. It has been described that high glucose level induces a hyperfunctional status which could provoke apoptosis. This phenomenon is known as glucotoxicity and has been proposed that it can play a role in type 1 diabetes mellitus pathogenesis. In this study we develop an experimental design to sensitize pancreatic islet cells by high glucose to streptozotocin (STZ) and proinflammatory cytokines [interleukin (IL)-1β, tumor necrosis factor (TNF)-α and interferon (IFN)-γ]-induced apoptosis. This method is appropriate for subsequent quantification of apoptotic islet cells stained with Tdt-mediated dUTP Nick-End Labeling (TUNEL) and protein expression assays by Western Blotting (WB).
Jason S. T. Deveau, , Bernard Grodzinski
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 31-40; https://doi.org/10.1251/bpo103

Abstract:
We describe an improved, efficient and reliable method for the vapour-phase silanization of multi-barreled, ion-selective microelectrodes of which the silanized barrel(s) are to be filled with neutral liquid ion-exchanger (LIX). The technique employs a metal manifold to exclusively and simultaneously deliver dimethyldichlorosilane to only the ion-selective barrels of several multi-barreled microelectrodes. Compared to previously published methods the technique requires fewer procedural steps, less handling of individual microelectrodes, improved reproducibility of silanization of the selected microelectrode barrels and employs standard borosilicate tubing rather than the less-conventional theta-type glass. The electrodes remain stable for up to 3 weeks after the silanization procedure. The efficacy of a double-barreled electrode containing a proton ionophore in the ion-selective barrel is demonstrated in situ in the leaf apoplasm of pea (Pisum) and sunflower (Helianthus). Individual leaves were penetrated to depth of ∼150 µm through the abaxial surface. Microelectrode readings remained stable after multiple impalements without the need for a stabilizing PVC matrix.
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 48-59; https://doi.org/10.1251/bpo105

Abstract:
Mature osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a short life span. Recently, we have demonstrated that osteoclast apoptosis is regulated by ERK activity and Bcl-2 family member Bim. In this paper, we summarize the methods we used to study osteoclast apoptosis in vitro and in vivo. Using adenovirus and retrovirus vectors, we were able to introduce foreign genes into osteoclasts and examine their effects on osteoclast survival in vitro. In addition, we established the modified methods for in situ hybridization and BrdU labeling of bone sections from mice to study osteoclast survival in vivo. The detailed methods described here could be useful for studying the biological process in bone.
Robin M. Ricke,
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 60-69; https://doi.org/10.1251/bpo106

Abstract:
The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE and Western blot analysis. Unlike techniques that separate chromatin and non-chromatin interacting proteins by centrifugation, this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover, the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes.
Corinne Jud, Isabelle Schmutz, Gabriele Hampp, Henrik Oster,
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 101-116; https://doi.org/10.1251/bpo109

The publisher has not yet granted permission to display this abstract.
Jack M. Gallup, Kenji Kawashima, Ginger Lucero, Mark R. Ackermann
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 70-92; https://doi.org/10.1251/bpo107

Abstract:
We describe a new approach for reliably isolating one-step real-time quantitative RT-PCR-quality RNA from laser captured cells retrieved from frozen sections previously subjected to immunofluorescent immunohistochemistry (IF-IHC) and subsequently subjected to fluorogenic one-step real-time RT-PCR analysis without the need for costly, time-consuming linear amplification. One cell's worth of RNA can now be interrogated with confidence. This approach represents an amalgam of technologies already offered commercially by Applied Biosystems, Arcturus and Invitrogen. It is the primary focus of this communication to expose the details and execution of an important new LCM RNA isolation technique, but also provide a detailed account of the IF-IHC procedure preceding RNA isolation, and provide information regarding our approach to fluorogenic one-step real-time RT-PCR in general. Experimental results shown here are meant to supplement the primary aim and are not intended to represent a complete scientific study. It is important to mention, that since LCM-RT-PCR is still far less expensive than micro-array analysis, we feel this approach to isolating RNA from LCM samples will be of continuing use to many researchers with limited budgets in the years ahead.
, Maryssa Canuel, Jibin Zeng, Carlos R. Morales
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 17-25; https://doi.org/10.1251/bpo101

Abstract:
To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs.
Michele A. Wozniak,
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 144-161; https://doi.org/10.1251/bpo112

Abstract:
Several pathological and disease conditions can alter the mechanical properties of the extracellular matrix (ECM). Conversely, some diseases may arise from changes in the density or rigidity of the ECM. This necessitates the use and development of in vitro models to understand how both biophysical and biochemical signals regulate complex cellular behaviors. T47D breast epithelial cells will differentiate into duct-like tubules when cultured in a floating three-dimensional (3D) collagen gel, but not a 3D collagen gel that is left attached to the culture dish. This paper details several protocols we have developed for analyzing breast cell biology in 3D matrices, including culturing cells in 3D collagen gels, immunostaining cellular structures, and performing biochemical procedures directly from cells embedded in collagen gels.
Motomu Watanabe, Yayoi Fujioka-Kaneko, Hisayuki Kobayashi, Mamoru Kiniwa, Michihiko Kuwano,
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 41-47; https://doi.org/10.1251/bpo104

The publisher has not yet granted permission to display this abstract.
Sergio Encarnación, Magdalena Hernández, Gabriel Martínez-Batallar, Sandra Contreras, Maria Del Carmen Vargas, Jaime Mora
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 117-135; https://doi.org/10.1251/bpo110

The publisher has not yet granted permission to display this abstract.
Kelly E. Corcoran, Prem S. Patel, Pranela Rameshwar
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 8-16; https://doi.org/10.1251/bpo100

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, Stephen K. Wikel
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 93-100; https://doi.org/10.1251/bpo108

The publisher has not yet granted permission to display this abstract.
Published: 1 January 2005
Biological Procedures Online, Volume 7, pp 1-7; https://doi.org/10.1251/bpo99

Abstract:
RNA Interference has rapidly emerged as an efficient procedure for knocking down gene expression in model systems. However, cross-reactivity, whereby multiple genes may be simultaneously targeted by a single short interfering RNA (siRNA), can potentially jeopardize correct interpretation of gene function. As such, it is essential to test the specificity of a siRNA prior to a full phenotypic analysis. To this end, we have adapted a reporter-based assay harnessing the sensitivity of luciferase activity to provide a quantitative readout of relative RNAi efficacy and specificity. We have tested different siRNAs directed against Thymosin beta4 (Tbeta4); determined their effectiveness at silencing Tbeta4 and have both excluded off-target silencing of the Tbeta4 homologue Thymosin beta10 (Tbeta10) and demonstrated partial knockdown of Tbeta10 despite significant (12/23; 52%) sequence mismatch. This assay system is applicable to any RNAi study where there is a risk of targeting homologous genes and to the monitoring of off-target effects at the genome level following microarray expression profiling.
Melanie MelanieOhi, Ying Li, Yifan Cheng,
Published: 1 January 2004
Biological Procedures Online, Volume 6, pp 23-34; https://doi.org/10.1251/bpo70

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