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1.  A method to generate human mesenchymal stem cell-derived neurons which express and are excited by multiple neurotransmitters  
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.
doi:10.1251/bpo147
PMCID: PMC2683550  PMID: 19461957
Mesenchymal stem cells; Gamma-aminobutyric acid
2.  A rapid microwell fluorescence immunoassay for cellular protein detection  
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.
doi:10.1251/bpo146
PMCID: PMC2683549  PMID: 19461956
Fluoroimmunoassay; Drug Evaluation
3.  Tracking and ablating subpopulations of epiblast cells in the chick embryo  
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.
doi:10.1251/bpo145
PMCID: PMC2683548  PMID: 19461955
Chick Embryo; Staining and Labeling
4.  Characterizing gene family evolution  
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.
doi:10.1251/bpo144
PMCID: PMC2683547  PMID: 19461954
genomics; evolution, molecular; phylogeny; sequence homology
5.  Cell culture-based analysis of postsynaptic membrane assembly in muscle cells  
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.
doi:10.1251/bpo143
PMCID: PMC2683546  PMID: 19461953
Acetylcholine receptors;; Muscle; Agrin; Laminin; Synapse
6.  A method for purification, identification and validation of DNMT1 mRNA binding proteins 
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 ~40 kDa on DNMT1 3’-UTR triggered the destabilization of DNMT1 mRNA transcript during Go/G1 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.
doi:10.1251/bpo142
PMCID: PMC2591025  PMID: 19048127
DNA Methylation; RNA-Binding Proteins
7.  Optimized two-dimensional thin layer chromatography to monitor the intracellular concentration of acetyl phosphate and other small phosphorylated molecules 
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.
doi:10.1251/bpo141
PMCID: PMC2275044  PMID: 18385806
Phosphoric Acid Esters; Chromatography, Thin Layer
8.  Identification of a Calcitriol-Regulated Sp-1 Site in the Promoter of Human CD14 using a Combined Western Blotting Electrophoresis Mobility Shift Assay (WEMSA) 
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.
doi:10.1251/bpo140
PMCID: PMC2275043  PMID: 18385805
Electrophoretic Mobility Shift Assay; Transcription Factors; Antigens, CD14
9.  Quantitative Evaluation of Signaling Events in Drosophila S2 Cells 
Drosophila activates a robust defense response to gram-negative bacteria through the Immune deficiency (Imd) pathway. Imd signaling proceeds through c-Jun N-terminal Kinase (JNK), NF-kB and caspase modules. The individual signaling modules act in a highly coordinated manner to yield a stereotypical response to infection. While considerable attention has focused on NF-kB-mediated antimicrobial activities, more recent studies have highlighted the involvement of JNK signaling in the Imd pathway response. JNK signaling occurs in a transitory burst and drives the expression of a number of gene products through the AP-1 transcription factor. In this report, we describe a simple method for the quantification of JNK activation by Western blot analysis or directly in tissue culture plates.
doi:10.1251/bpo139
PMCID: PMC2275046  PMID: 18385808
RNA interference; immunity; drosophila
10.  Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique 
Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to freeze-fracture cytochemistry that have been developed and tried, the most successful is the technique termed freeze-fracture replica immunogold labeling (FRIL). In this technique, samples are frozen, fractured and replicated with platinum-carbon as in standard freeze fracture, and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure. Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed.
doi:10.1251/bpo138
PMCID: PMC2275045  PMID: 18385807
Freeze Fracturing; Microscopy, Immunoelectron; Immunogold Techniques
11.  In vivo and in vitro techniques for comparative study of antiviral T-cell responses in the amphibian Xenopus 
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.
doi:10.1251/bpo137
PMCID: PMC2275042  PMID: 18385804
xenopus laevis; ranavirus; T-Lymphocytes

Results 1-11 (11)