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Culture of Isolated Human Adipocytes and Isolated Adipose Tissue

Adipose tissue (AT) is no longer considered merely as insulation or padding for human organs. It is an endocrine organ in its own right, which includes composite cells with the ability to differentiate into multiple cell lines. In fact, there is increasing evidence to support the theory that the cau ...

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Primary Culture of Human Renal Proximal Tubule Epithelial Cells and Interstitial Fibroblasts

Renal physiology and pathology are complex systems that are best studied in whole living organisms. This, however, is often restricted by our desire to limit the number of animal experiments undertaken and to replace them with relevant in vitro models that can be used as surrogates for the system u ...

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Isolation, Characterization, and Culture of Normal Human Intestinal Crypt and Villus Cells

The intestinal epithelium is a highly dynamic tissue undergoing constant and rapid renewal. It consists of a functional villus compartment responsible for terminal digestion and nutrient absorption and a progenitor cell compartment located in the crypts that produce new cells. The m ...

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Isolation and Functional Studies of Human Fetal Gastric Epithelium in Primary Culture

Our understanding of gastric epithelial physiology in man is limited by the absence of normal or appropriate cancer cell lines that could serve as an in vitro model. Research mostly relied on primary culture of gastric epithelial cells of animal species, enriched with surface mucous cells, and ...

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Characterization of Genes for Polyamine Modulon

Polyamines are essential for normal cell growth and exist mainly as RNA-polyamine complexes in cells. Thus, effects of polyamines on protein synthesis have been studied. It was found that several kinds of protein synthesis, which are important for cell growth, were enhanced by polyamines at t ...

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Exploring Polyamine Biosynthetic Diversity Through Comparative and Functional Genomics

The existence of multiple, alternative pathways for polyamine biosynthesis, and the presence of alternative polyamine structural analogs, is an indication of the physiological importance of polyamines and their long evolutionary history. Polyamine biosynthesis is modula ...

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Current Status of the Polyamine Research Field

This chapter provides an overview of the polyamine field and introduces the 32 other chapters that make up this volume. These chapters provide a wide range of methods, advice, and background relevant to studies of the function of polyamines, the regulation of their content, their role in disease, a ...

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Posttranscriptional Regulation of Gene Expression in Epithelial Cells by Polyamines

In addition to regulating gene transcription, polyamines also potently modulate gene expression posttranscriptionally. Posttranscriptional gene regulation, which includes processes such as mRNA transport, turnover, and translation, involves specific mRNA sequen ...

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Identification, Chemical Synthesis, and Biological Functions of Unusual Polyamines Produced by Extreme Thermophiles

Unusual long polyamines such as caldopentamine and caldohexamine, and branched polyamines such as tetrakis(3-aminopropyl)ammonium and N 4-aminopropylspermidine were often found in cells of extreme thermophiles and hyperthermophiles belonging to both Bacteria and Arc ...

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Polyamine Block of Inwardly Rectifying Potassium Channels

Polyamine blockade of inwardly rectifying potassium (Kir) channels underlies their steep voltage-�dependence observed in native cells. The structural determinants of polyamine blockade and the structure-activity profile of endogenous polyamines requires speciali ...

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Carcinogenesis Studies in Mice with Genetically Engineered Alterations in Polyamine Metabolism

Polyamines are intimately linked to essential cellular processes that are required for cell growth and �proliferation, and abundant evidence links polyamine metabolism to tumor susceptibility and progression. Intensive efforts over the past 2 decades have yielded numerous mo ...

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Transgenic Rodents with Altered SSAT Expression as Models of Pancreatitis and Altered Glucose and Lipid Metabolism

Depletion of pancreatic acinar cell polyamines in response to activation of polyamine catabolism is associated with the development of acute pancreatitis in experimental rodent models. The disease is characterized by general hallmarks seen also in human pancreatitis, such as acc ...

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Methods for Embryoid Body Formation: The Microwell Approach

Embyroid body (EB) formation is a key step in many embryonic stem cell (ESC) differentiation protocols. The EB mimics the structure of the developing embryo, thereby providing a means of obtaining any cell lineage. Traditionally, the two methods of EB formation are suspension and hanging drop. ...

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Human Embryonic Stem Cell-Derived Mesenchymal Progenitors: An Overview

Mesenchymal stromal/stem cells (MSCs) were originally isolated from bone marrow (BM), but are now known to be present in all fetal and adult tissues. These multipotent cells can be differentiated into at least three downstream mesenchymal lineages that include bone, cartilage, and fat. How ...

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Split-EGFP Screens for the Detection and Localisation of ProteinProtein Interactions in Living Yeast Cells

Proteomics aims to identify and classify the proteins present in a particular cell or tissue. However, we know that proteins rarely function alone and knowledge of which proteins interact with which other proteins is vital if we wish to understand how cells work. The budding yeast, Saccharomyc ...

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Preparation of Fungi for Ultrastructural Investigations and Immunogoldlabelling

Electron microscopic analysis of biological material requires optimal preparation of the samples. This is necessary to prevent degeneration processes and changes of the material during microscopic observation. Both would lead to artefacts. In this chapter, we present methods to p ...

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Evaluation of Antifungal Susceptibility Using Flow Cytometry

Flow cytometry has found wide applications in areas like haematology and immunology, but also presents great potential in microbiology. The susceptibility of clinical isolates of Candida and Cryptococcus to antifungal compounds can be assayed by flow cytometry using fluorescent ...

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Methods to Detect Apoptotic-Like Cell Death in Filamentous Fungi

Fungi are capable of undergoing apoptotic-like cell death, and display many of the characteristic features of apoptosis observed in multicellular organisms. These features include nuclear condensation, DNA fragmentation, translocation of phosphatidylserine from the cy ...

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Live-Cell Imaging of Microtubule Dynamics in Hyphae of Neurospora crassa

Due to the large number of microtubules in the wild-type strain, total internal reflection fluorescence (TIRF) microscopy was used to study cortical microtubule dynamics in leading hyphae of Neurospora crassa expressing β-tubulin-GFP. Detection of plus-end dynamics of individu ...

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Fluorescence In Situ Hybridization for Molecular Cytogenetic Analysis in Filamentous Fungi

Fluorescence in situ hybridization (FISH) is a powerful technology for studying eukaryotic chromosomes and genomes from the combined view of cytogenetics and molecular biology, but its use in filamentous fungi has been limited. In this chapter, we describe protocols to perform three ba ...

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