Metabolic Pathway Confirmation and Discovery Through ¹³C-labeling of Proteinogenic Amino Acids

JoVE 3583 1/26/2012

Le You¹, Lawrence Page², Xueyang Feng¹, Bert Berla¹, Himadri B. Pakrasi³, Yinjie J. Tang¹

¹Department of Energy, Environmental and Chemical Engineering, Washington University, ²Department of Biology, Washington University, ³Department of Energy, Environmental and Chemical Engineering and Department of Biology, Washington University

¹³C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

June 2011: This Month in JoVE

JoVE 3549 6/01/2011

Aaron Kolski-Andreaco

Identifying Dysregulated Genes Induced by Kaposi's Sarcoma-associated Herpesvirus (KSHV)

JoVE 2078 9/14/2010

Donald Alcendor, Susan Knobel

Department of Microbiology & Immunology and the Center for AIDS Health Disparities Research, Meharry Medical College

Host cell factors play a critical role in the establishment and maintenance of Kaposi's sarcoma (KS). We outline methods to identify host cell factors altered in KSHV-infected DMVEC cells, and in KS tumor tissue. Cellular genes altered by virus will serve as potential target(s) for novel therapeutics.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

JoVE 2288 2/27/2011

Marni J. Falk^1,2, Meera Rao*¹, Julian Ostrovsky*¹, Evgueni Daikhin¹, Ilana Nissim¹, Marc Yudkoff^1,2

¹Department of Pediatrics, The Children's Hospital of Philadelphia, ²Department of Pediatrics, University of Pennsylvania

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

Extraction of High Molecular Weight DNA from Microbial Mats

JoVE 2887 7/07/2011

Benjamin S. Bey, Erin B. Fichot, R. Sean Norman

Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

Magnetic Resonance Spectroscopy of live Drosophila melanogaster using Magic Angle Spinning

JoVE 1710 4/15/2010

Valeria Righi^1,2,3, Yiorgos Apidianakis^2,4, Laurence G. Rahme^2,4, A. Aria Tzika^1,2,3

¹NMR Surgical Laboratory, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, ²Shriners Burn Institute, ³Department of Radiology, Athinoula A. Martinos Center of Biomedical Imaging, Harvard Medical School, ⁴Molecular Surgery Laboratory, Department of Surgery, Massachusetts General Hospital, Harvard Medical School

This technique enables the use of high-resolution magic angle spinning proton MR spectroscopy (HRMAS 1H-MRS) for molecular characterization of live Drosophila melanogaster with a conventional 14.1 tesla spectrometer equipped with an HRMAS probe.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

JoVE 3597 4/15/2012

Monica Mejia¹, Mari D. Heghinian², Alexandra Busch¹, Frank Marí², Tanja A. Godenschwege¹

¹Department of Biological Sciences, Florida Atlantic University, ²Department of Chemistry & Biochemistry, Florida Atlantic University

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

JoVE 1865 3/30/2010

Dor Salomon, Guido Sessa

Department of Plant Sciences, Tel Aviv University

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

JoVE 2516 4/06/2011

Carole A. Farah, Wayne S. Sossin

Neurology and Neurosurgery, McGill University

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

JoVE 2728 5/09/2011

Susan Fotheringham^1,2, Keren Levanon^1,2,3, Ronny Drapkin^1,2,4

¹Department of Medical Oncology, Dana-Farber Cancer Institute, ²Harvard Medical School, Boston, MA, ³Sheba Cancer Research Center, Chaim Sheba Medical Center, ⁴Department of Pathology, Brigham and Women's Hospital

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.