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Authors: Yana Valasatava, Antonio Rosato & Claudia Andreini 

Abstract

In this work, we developed a methodology to perform a systematic classification based on three-dimensional structural similarity of the metal sites contained in metalloproteins. Our definition of metal site extended beyond the metal ion and its aminoacidic ligands by including all the chemical species (aminoacids, nucleotides, exogenous ligands) providing at least one donor atom as well as all any other chemical species within a radius of 5.0 Å. We previously defined this as the Minimal Functional Site of a metalloprotein (MFS), and showed that its characteristics are related to the metalloprotein function. The methodology described here leverages the MetalS2 algorithm, whose total score provides a quantitative measure of structural similarity between pairs of MFS. We used this measure to build clusters of structurally similar MFSs using a two-stage hierarchical clustering algorithm. At the first stage we cluster MFSs identified i

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Authors: Shuyan Wang, Bin Wang & Zhiguo Chen 

Abstract

This protocol describes how to differentiate human induced pluripotent stem cells (iPSCs) to Purkinje cells. Human iPSCs are first differentiated to Neph3+ Purkinje progenitors. To promote maturation of Purkinje progenitors in vitro, a co-culture system is used to enhance the maturation of Purkinje precursors on rat and human fetal cerebellar slices. Furthermore, Purkinje progenitor cells are injected into the cerebellum of newborn immunodeficient mice to test the differentiation ability in vivo.

Introduction

It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) to Purkinje cells. Purkinje neurons are the only output neurons in cerebellum and often afflicted in spinocerebellar ataxias (SCAs) and other medical indications such as ethanol exposure and autoimmune diseases. Obtaining patient-specific Purkinje neurons would offer a valuable tool to model cerebellar diseases in a culture dish for investigating t

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Authors: Dhiman Ghosh & Samir K. Maji 

Abstract

Establishing reproducible aggregation kinetics and amyloid formation of α-synuclein (α-Syn) is of great interest for understanding Parkinson’s disease (PD) pathogenesis. α-Syn, 140 amino acid residues intrinsically disorder protein (IDP), is well known for its inconsistent aggregation behaviour in vitro. Previously different methods/conditions like orbital agitation, usage of small glass beads in plate reader assay were reported to achieve higher reproducibility. In addition to mechanical agitation, here we report the usage of aggregate free low molecular weight (LMW) solution as a starting material for monitoring aggregation pathway of α-Syn. This allowed us to obtain the reproducible fibrillation kinetics of α-Syn at a satisfactory level. This LMW could be used not only for understanding PD pathogenesis but also for screening inhibitors against α-Syn fibrillation and to study many molecular events in vitro.

Introduction

α-Synuclein aggrega

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Authors: Koji Atarashi & Kenya Honda 

Introduction

IL-17-expressing CD4+ cells (TH17 cells) constitute a considerable proportion of lymphocytes in the intestinal lamina propria (LP), even in healthy mice kept under specific pathogen free conditions. TH17 cells are rarely observed in the spleen, mesenteric lymph node, or Peyer’s patches. Germ-free mice or antibiotics (vancomycin and metronidazole)-treated mice show marked reductions in numbers of LP TH17 cells; thus, intestinal commensal bacteria provide a particular environment for the development of LP TH17 cells.

This protocol describes the procedure to isolate LP lymphocytes and to assay for IL-17 expression by intracellular cytokine staining.

Reagents

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Authors: Tomotoshi Marumoto , Dinorah Friedmann-Morvinski , Meng Yang , Robert Hoffman & Inder Verma 

Introduction

Glioblastomas (GBMs) are highly malignant because of their high mitotic activity and prominent invasive characteristic. Animal models are very essential in order to understand the biology of the disease and to find and validate new therapies. Various animal models for GBMs have been reported so far, however each model has strengths and drawbacks.

The transplantation of cultured tumor cells (xenograft or allograft models), often in the immunodeficient recipient animals have been used to model gliomas. While these models are highly reproducible, they do not recapitulate the infiltrative characteristics of glioma, a major cause of lethality (1). Another glioma model in rodents can be generated by treatment with mutagens and histologically resemble gliomas, but the identities of their unique genetic mutations are hard to determine (2). Transgenic models which express oncogenes from a cell

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Authors: Azam Mohammad & George Daley 

Introduction

Protein kinases are one of the largest families of the protein coding genes: constituting some 2% of the expressed proteins, regulate almost all biochemical pathways and phosphorylate up to 30% of the proteome. More than 400 human diseases have been linked, directly or indirectly, to protein kinases. Deregulated tyrosine kinases have been implicated in neoplastic development. Here, we describe biochemical procedures to monitor the activation of the tyrosine kinases (ABL, SRC, PDGFRA, PDGFRB and EGFR). These kinases and its constitutively activated variants were expressed in mammalian cells and kinase activity was measured by tyrosine phosphorylation by immunoblot analysis. We also describe the procedure for cellular transformation of BAF3 cells for IL-3 independent cell proliferation, and the development of leukemia in mice. The combination of these procedures is useful to evaluate the activating mutations in kinases and its potential to develop c

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Authors: Andreas Hejnol 

Introduction

This protocol is based on a in situ hybridization protocol used for embryos of the sea anemone Nematostella vectensis (Martindale et al. 2004).

Reagents

Hybe Buffer (40 mL)

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Authors: Qiang Wang , Jian-Qun Chen & Dacheng Tian 

Introduction

AlignDB is developed to investigate the distribution of single-nucleotide changes around insertion/deletions (indels) in genome comparisons. There are two set of analysis, two-way and three-way, in AlignDB.

The two-way analysis indicates that nucleotide divergence (D) is substantially elevated surrounding indels and decreases monotonically to near-background levels over several hundred bases. D is significantly correlated with both size and abundance of nearby indels. In a comparison of closely related species, the three-way analysis is available. We find that derived nucleotide substitutions surrounding indels occur in significantly greater numbers on the lineage containing the indel than on the one containing the ancestral (non-indel) allele; the same holds within species for single-nucleotide mutations surrounding polymorphic indels.

AlignDB is freely available on request from the authors. The parameters are fully modifiable. The

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Authors: Valentina Gandin , Pier Carlo Marchisio & Stefano Biffo 

Introduction

This procedure describes a method for isolation of murine hepatocytes using a modified two-step perfusion method. This protocol allows to measure the level of protein synthesis in primary hepatocytes after stimulation with growth factor and hormones.

The advantage of this procedure is to easily perform a 35S-metabolic labelling to measure the level of protein synthesis upon stimulation ex vivo.

Reagents

scientificprotocols

Authors: Aubin Penna 

Introduction

This procedure describes how to assess the native quaternary structure of membrane proteins by PAGE using mild solubilization in the non-dissociative detergent perfluoro-octanoic acid (PFO). The number of subunits is deduced from the molecular mass of the preserved homo-multimeric protein complex. The PFO-PAGE technique was first described by Ramjeesingh, M et al. (1999) and applied later to determine the subunit stoichiometry of other channels (e.g. Kedei, N. et al. (2001) and in the present study to Orai and P2X2 expressed in Drosophila S2 cells or human embryonic kidney HEK293 cells).

Procedure

1. Rinse the cells twice with ice-cold phosphate-buffered saline (PBS).

• Harvest the cell pellet in ice-cold PBS supplemented with protease inhibitors and sonicate. Alternatively, solubilize the cells in the presence of 1% NP-40, a mild detergent diluted in PBS, for 20 min at 4°C under agitation and centrifuged at 16,000 × g for 10 min to remove cellular debris