scientificprotocols

Authors: Anju Vasudevan & Pradeep G. Bhide 

Introduction

The anatomy of the brain’s vascular networks is just as complex as that of its neuronal networks. Yet, surprisingly little is known about the ontogeny of cerebral vasculature. Until now, it was believed that brain’s vascular networks developed passively to meet metabolic needs of the rapidly growing nervous tissue (ref.1,2). Although classical studies identified a ventral to dorsal temporal developmental angiogenesis gradient in the telencephalon (ref.3), the sequence of angiogenesis was considered to merely shadow neurogenesis and neuronal maturation. According to current models (ref.4,5) brain vasculature develops in four stages (Fig. 1a), responding to and keeping pace with the rapid onset and progression of neuroepithelial progenitor cell divisions, neurogenesis and gliogenesis. According to this model, blood vessels on the pial surface extend radial branches towards the ventricle (ventriculo-petal branches; stage 1); form new branches up

scientificprotocols

Authors: Kazushi Imai & Yasunori Okada 

Introduction

To detect activities of MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9, use radio-labeled fibrillar collagen (for MMP-1), gelatin (for MMP-2 and MMP-9) and carboxymethylated transferrin (for MMP-3 and MMP-7) substrates for the assays (1), although these can be substituted with commercially available FITC-labeled substrates and fluorogenic synthetic peptides for the MMPs. Zymography using gelatin or casein substrate gels is also useful to monitor gelatinolytic activity of MMP-2 and MMP-9 or caseinolytic activity of MMP-3 and MMP-7 in each column fraction, although zymography is not suitable for calculation of purification fold and recovery of proteinases.

Procedure

Collagenase assay for MMP-1

scientificprotocols

Authors: Françoise Bruyère, Laurence Melen-La, Sarah Berndt, Olivier Peulen, Jean-Michel Foidart & Agnès Noël 

Introduction

Lymphangiogenesis, the formation of new lymphatic vessels, is associated to numerous pathologies (1) and understanding the molecular and cellular basis of this complex process is essential for the development of novel therapeutic strategies. Studies on lymphangiogenesis have been hampered by difficulties in culturing lymphatic capillaries as three-dimensional (3D) structures in vitro that mimic the in vivo features of lymphatic vessels and lymphangiogenesis. The lymphatic ring assay described here phenocopies the different steps of lymphangiogenesis, including the spreading from a preexisting vessel, cell proliferation, migration and differentiation into capillaries. It consists on the adaptation of the aortic ring assay that has proved to be useful to investigate the molecular basis of angiogenesis (2-4). The lymphatic ring model is an ideal assay for testing the activity of

scientificprotocols

Authors: Ry Y Tweedie-Cullen & Magdalena Livingstone-Zatchej 

Introduction

The recent introduction of isobaric peptide tags for relative and absolute quantification (iTRAQ) of proteins in different samples was a major breakthrough in quantitative proteomics. The iTRAQ method is based on the differential covalent labelling of peptides from proteolytic digests with one of four iTRAQ reagents resulting in the incorporation of 144.1 Da to peptide N-termini and lysine residues. Peptides with different tags are indistinguishable by mass but can be differentiated by collision-induced dissociation (CID) (normally applied during MS peptide sequencing) through release of a reporter ion, each of which has a different mass (114.1, 115.1, 116.1 or 117.1 Da). The analysis of the intensity of reporter ions allows the simultaneous sequencing and quantification of labelled peptides.

Reagents

Sample preparation and iTRAQ labelling

scientificprotocols

Authors: Oliver Schilling & Christopher M. Overall 

Introduction

Specificity of numerous proteases is determined by enzyme-substrate interactions in an extended active site cleft. The Schechter and Berger nomenclature (1) designates residues carboxy-terminal to the scissile peptide bond as prime side (P’) and amino-terminal residues as non-prime side (P). P and P’ residues interact with complementary protease subsites called S and S’. While some proteases show very restrictive specificity profiles (e.g. trypsin is specific for lysine and arginine in P1), other proteases display broad specificity profiles involving multiple subsite preferences.

A bottleneck in protease characterization is the determination of consensus cleavage sites as all current techniques provide information on either the prime or nonprime side only−none provide both in the same experiment. Genetic library approaches such as phage display provide cleavable sequences only without identifying the actual scissile bond. Proteome-der

scientificprotocols

Authors: Jun-Xian Shen 

Introduction

The protocol describes quantitative analysis of males’ phonotaxis induced by female calls. This protocol is used to demonstrate that the ultrasonic concave-eared torrent frogs Odorrana tormota inhabiting noise habitats, as a model of acoustic communicating animals, show hyperacute phonotaxis to the female’s courtship calls (1). The acoustic recording and playback systems used have several advantages over the more widely used call or song detection methods: 1) the systems allow continuously recording diverse types of calling with a sampling rate (96-480 kHz); 2) calls can be simultaneously analyzed and displayed using SELENA software; 3) recorded calls can be easily saved into electronic data files and chosen as stimuli, and 4) the stimuli are varied according to experimental need and stably presented through the playback device for auditory electrophysiological or ethological study. The method for quantitative analysis of phonotaxis includes that 1) the scaled

scientificprotocols

Authors: Birgit Obermeier, Reinhard Mentele, Joachim Malotka & Klaus Dornmair 

Introduction

We describe a method for relating oligoclonally expanded B cells at particular body locations to their secreted products, soluble immunoglobulins. The technique makes use of the virtually infinite variability of mature immunoglobulin (Ig) chains, which results from VDJ recombination and somatic hypermutation (SHM). We create patient-specific transcriptome databases by PCR-cloning of Ig transcripts of B cells from the relevant location. In parallel, we analyze the Ig proteome at the location of interest. To this end, we isolate the Ig molecules by affinity chromatography and isoelectric focusing, and analyze them by trypsin digestion and subsequent mass spectrometry. Then the patient-specific Ig proteomes and transcriptomes are compared by searching for the peptides identified by mass spectrometry in the transcriptome databases. We place particular focus on the peptides that carry characteristic amino acids i

scientificprotocols

Authors: Felix H. Lam , David J. Steger & Erin K. O’Shea 

Introduction

This protocol describes a method of assaying nucleosome positions in S. cerevisiae by nuclease protection and quantitative PCR (Q-PCR). Briefly, yeast cells are spheroplasted, treated with micrococcal nuclease, and single-nucleosome length DNA is gel-isolated and quantitated by Q-PCR using overlapping primer pairs tiling the length of a region of interest. In the accompanying Nature paper, this technique is used to map the promoter nucleosome positions of several genes in the phosphate starvation response (PHO) pathway in order to determine the accessibility of Pho4 (transcription factor) binding sites. While this protocol has been used extensively with S. cerevisiae, it should, in principle, be readily adaptable for use in other yeast species.

Reagents

1. 1 M sorbitol – At room temperature.

• 1 M sorbitol – Chilled to 4°C.

scientificprotocols

Authors: Nicolas Manel 

Introduction

Human CD4+ Th-17 cells produce inflammatory cytokines and have been implicated in the development of several inflammatory pathologies. The transcription factor RORgammaT is though to establish Th-17 cell differentiation. Expression of IL-17A is a hallmark of Th-17 cells.

We have used RORgammaT overexpression as an approach to identify factors that induce Th-17 cell differentiation. This led us to the observation that TGF-beta is essential for induction of RORgammaT. However, unknown factors present in serum inhibit Th-17 cell differentiation. Thus, it is important to cultivate cells in serum-free conditions in order to generate Th-17 cells from naive CD4+ T cells. In serum-free conditions, we found that a combination of TGF-beta, IL-1beta and either IL-6, IL-21 or IL-23 is able to induce Th-17 cell differentiation.

Here is described a procedure to generate human Th-17 cells from naive CD4+ T cells isolated from adult or cord blood.

scientificprotocols

Authors: Mark O. Collins, Lu Yu & Jyoti S. Choudhary 

Introduction

Identification of components of protein complexes by mass spectrometry has become an important and powerful approach to understand cell biology. Tandem mass spectrometry can provide unbiased and comprehensive identification of proteins by generating high accuracy mass information for peptide ions in MS1 spectra and further amino acid sequence information is reported as fragment ion information in MS2 spectra. The combination of this data can be used to confidently identify peptides and thus protein matches from sequence databases. The protocol described here is applicable to any protein complex that can be isolated to sufficient purity and quantity, its components separated by gel electrophoresis and sequence databases for the source organism are available. In the referenced Nature Neuroscience paper we used this approach to identify 220 proteins associated with the glutamate receptor NR2 subunit in D. melanogaster. Complexes we