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Bayamon, Puerto Rico

The Universidad Central del Caribe is a private non-profit university in Bayamon, Puerto Rico offering graduate studies and professional certifications in health science. It was founded in 1976 in the municipality of Cayey, but since 1990 all its facilities have been integrated into one campus at the grounds of the Dr. Ramón Ruiz Arnau University Hospital in Bayamón. Wikipedia.

Coleman W.L.,Lehigh University | Bykhovskaia M.,Central University of the Caribbean | Bykhovskaia M.,Lehigh University
Molecular and Cellular Neuroscience | Year: 2010

To understand how the presynaptic proteins synapsin and Rab3a may interact in the regulation of the synaptic vesicle cycle and the release process, we derived a double knockout (DKO) mouse lacking both synapsin II and Rab3a. We found that Rab3a deletion rescued epileptic-like seizures typical for synapsin II gene deleted animals (Syn II(-)). Furthermore, action potential evoked release was drastically reduced in DKO synapses, although spontaneous release remained normal. At low Ca2+ conditions, quantal content was equally reduced in Rab3a(-) and DKO synapses, but as Ca2+ concentration increased, the increase in quantal content was more prominent in Rab3a(-). Electron microscopy analysis revealed that DKO synapses have a combined phenotype, with docked vesicles being reduced similar to Rab3a(-), and intraterminal vesicles being depleted similar to Syn II(-). Consistently, both Syn II(-) and DKO terminals had increased synaptic depression and incomplete recovery. Taken together, our results suggest that synapsin II and Rab3a have separate roles in maintaining the total store of synaptic vesicles and cooperate in promoting the latest steps of neuronal secretion. © 2010 Elsevier Inc.

Schikorski T.,Central University of the Caribbean
Methods in Molecular Biology | Year: 2010

A modern electron microscopic approach to the investigation of the structural organization of proteins and subcellular structures demands the use of molecular genetic techniques. The successful implementation of genetic techniques is closely tied to a reporter gene such as the green fluorescent protein (GFP). Although GFP has been widely used for light microscopy, it has many limitations for use in electron microscopy. In the search for a reporter gene for electron microscopy, interest in the use of horseradish peroxidase (HRP) DNA has recently increased, and several studies already have proven the feasibility of HRP expression in mammalian cells. Here, we describe a protocol that uses a HRP chimera to label the endoplasmic reticulum of HEK cells. © Springer Science+Business Media, LLC 2010.

Bykhovskaia M.,Central University of the Caribbean
Seminars in Cell and Developmental Biology | Year: 2011

Synaptic vesicles are organized in clusters, and synapsin maintains vesicle organization and abundance in nerve terminals. At the functional level, vesicles can be subdivided into three pools: the releasable pool, the recycling pool, and the reserve pool, and synapsin mediates transitions between these pools. Synapsin directs vesicles into the reserve pool, and synapsin II isoform has a primary role in this function. In addition, synapsin actively delivers vesicles to active zones. Finally, synapsin I isoform mediates coupling release events to action potentials at the latest stages of exocytosis. Thus, synapsin is involved in multiple stages of the vesicle cycle, including vesicle clustering, maintaining the reserve pool, vesicle delivery to active zones, and synchronizing release events. These processes are regulated via a dynamic synapsin phosphorylation/dephosphorylation cycle which involves multiple phosphorylation sites and several pathways. Different synapsin isoforms have unique and non-redundant roles in the multifaceted synapsin function. © 2011 Elsevier Ltd.

Schikorski T.,Central University of the Caribbean
Methods in Molecular Biology | Year: 2010

Cells communicate via endo- and exocytosis with their environment and neighboring cells. At synapses of the nervous system, fast exocytosis is coupled to fast endocytosis, which forms the basis for neurotransmitter release. The introduction of the unique fluorescent FM dyes allowed the monitoring of fast synaptic vesicle exo-endocytic cycling during live imaging sessions and after photoconversion of FM dyes into an electron-dense diaminobenzidine polymer synaptic vesicle cycling can be studied in the electron microscope. This protocol describes FM dye labeling of synaptic vesicles of cultured hippocampal neurons and photoconversion of the fluorescent synaptic vesicles for analysis in the electron microscope (EM). © Springer Science+Business Media, LLC 2010.

Schikorski T.,Central University of the Caribbean
Methods in Molecular Biology | Year: 2010

The detection of proteins with antibodies that are conjugated to gold particles has been a major asset to cell biology and the neurosciences, and knowledge about the subcellular location of antigens has formed the basis for many hypotheses regarding protein function. Many protocols have been developed since the introduction of colloidal gold to immunocytochemistry. The two most widely used techniques, however, are based on transmission electron microscopy and consist of either immunolabeling before the specimens are embedded in resin (pre-embedding immunogold labeling) or immunolabeling after embedding in resin (post-embedding immunogold labeling). The following protocol describes a pre-embedding procedure that gives reliable results with all antibodies that produce adequate staining as observed with a light microscope. This procedure results in almost perfect preservation of the ultrastructure. The procedure employs thick sectioning using a vibratome, permeabilization of membranes with Triton X-100, and immunolabeling with fluorescently conjugated Nanogold antibodies, followed by gold enhancement and embedding for electron microscopy. We also discuss some limitations inherent to pre-embedding immunogold labeling. © Springer Science+Business Media, LLC 2010.

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