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Detecting the Influence of Cell Cycle Regulatory Proteins on Human Telomeres

Maintenance of genome stability depends on an appropriate response to deoxyribonucleic acid (DNA) damage. This response is based on a complex network of signaling pathways that activate numerous processes and ultimately lead to damage repair and cellular survival or cell death. Thus, a r ...

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Monitoring Spindle Assembly and Disassembly in Yeast by Indirect Immunofluorescence

The budding yeast Saccharomyces cerevisiae has become a laboratory favorite for the study of several microtubule-dependent processes, including assembly of the mitotic spindle and spindle positioning. This is owing not only to the pliancy of its genetic system but also to the simplici ...

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Assay of Deoxyhypusine Hydroxylase Activity

The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains an unusual amino acid, hypusine . eIF5A and its hypusine/deoxyhypusine modification are vital for eukaryotic cell proliferation. Hypusine is formed posttranslationally by two en ...

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Identification and Assay of Allosteric Regulators of S-Adenosylmethionine Decarboxylase

Polyamine biosynthesis is extensively regulated in cells by multiple mechanisms, including regulation of enzyme activity posttranslationally. The identified regulatory factors include both small molecules and regulatory proteins, and the mechanisms vary in different ...

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Identification, Assay, and Functional Analysis of the Antizyme Inhibitor Family

Polyamines are small aliphatic polycations present in all living cells. Polyamines are involved in regulating fundamental cellular functions and are absolutely essential for the process of cellular proliferation. Because they fulfill essential cellular functions, their i ...

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Protocols for Studying Antizyme Expression and Function

Antizyme (AZ) is a key molecule in feedback regulation of cellular polyamines. It is induced by polyamines through stimulation of ribosomal frameshifting during its translation. In mammals, AZ is diverged into three paralogs, AZ1–3. Tissue and subcellular distribution are different ...

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Posttranscriptional Regulation of Ornithine Decarboxylase

Activity of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) and intracellular levels of ODC protein are controlled very tightly. Numerous studies have described ODC regulation at the levels of transcription, translation, and protein degradation in normal cel ...

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Heparan Sulfate Proteoglycan-Mediated Polyamine Uptake

The polyamines are polycationic compounds essential for cellular proliferation and transformation. In addition to a well-defined biosynthesis pathway, polyamines are internalized into cells by as yet incompletely defined mechanisms. Numerous reports have shown that effi ...

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Identification and Assays of Polyamine Transport Systems in Escherichia coli and Saccharomyces cerevisiae

Polyamine content in cells is regulated by biosynthesis, degradation, and transport. With regard to transport, uptake and excretion proteins exist in Escherichia coli and Saccharomyces cerevisiae. In E. coli, the uptake systems comprise a spermidine-preferential uptake system c ...

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The Cranial Bone Window Model: Studying Angiogenesis of Primary and Secondary Bone Tumors by Intravital Microscopy

The successful treatment of primary and secondary bone tumors in a huge number of cases remains one of the major unsolved challenges in modern medicine. Malignant primary bone tumor growth predominantly occurs in younger people, whereas older people predominantly suffer from seconda ...

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Genetic and Biochemical Analysis of Protozoal Polyamine Transporters

Polyamines are aliphatic polycations that function in key cellular processes such as growth, differentiation, and macromolecular biosynthesis. Intracellular polyamines pools are maintained from de novo synthesis and from transport of polyamines from the extracellular m ...

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Determination of Acrolein by High-Voltage Capillary Electrophoresis from Oxidized Fatty Acids

Acrolein is an α,β-unsaturated aldehyde with enormous capacity of reaction, occurs in the air like a pollutant, but it is (we know now) an important lipid peroxidation product as well. The compound is one of the several aldehydes produced from fatty acid oxidation, although it is particularly imp ...

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Plasmid DNA Purification Via the Use of a Dual Affinity Protein

Methods are presented for the production, affinity purification and analysis of plasmid DNA (pDNA). Batch fermentation is used for the production of the pDNA, and expanded bed chromatography, via the use of a dual affinity glutathione S-transferase (GST) fusion protein, is used for the captu ...

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Localization of Zona Pellucida Receptors on Live Sperm by Fluorophore-Conjugated Solubilized Zona Pellucida Proteins

The zona pellucida is an extracellular glycoprotein coat that surrounds the oocyte and, in many mammals, provides the first barrier sperm must penetrate for fertilization to occur. The molecular interactions between the sperm and zona pellucida appear to be relatively species-speci ...

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Whole-Cell Recording Using the Perforated Patch Clamp Technique

Many ion channels, particularly potassium channels, are regulated by intracellular substances, such as nucleotides or Ca2+. These modulators are washed out of the cell during standard whole-cell patch clamp recordings, or maintained at a particular concentration if they are includ ...

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Differential mRNA Display: Adaption for In Vivo Studies of Diseased Tissues

This section focuses on the application of differential display in gaining insight into cDNAs upregulated in association with a disease state by using direct in vivo samples. Although the majority of differential display studies have focused on in vitro models, a growing number of laborat ...

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Vascular Smooth Muscle Cells: Isolation, Culture, and Characterization

In blood vessel development (vasculogenesis) smooth muscle cells (SMCs) are derived by differentiation of mesenchymal cells under the influence of mediators secreted by the endothelial cells (ECs) composing newly formed vessels. In angiogenesis, SMCs can be formed in the same way or by p ...

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Immobilization of Enzymes and Cells: Some Practical Considerations

The technology for immobilization of cells and enzymes evolved steadily for the first 25 years of its existence (1), but in recent years it has reached a plateau, if not a slight decline. However, the expansion of biotechnology, and the expected developments that will accrue from advances in genetic ...

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Expression of MMPs andTIMPs in Mammalian Cells

Expression of recombinant matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in mammalian cells is an important step in their functional characterization. Also, transient transfection analysis of promoter constructs driving CAT or luc ...

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Immobilized Metal Ion Affinity Chromatography of Native Proteins

Immobilized metal affinity chromatography (IMAC) is a common place technique in modern protein purification. IMAC is distinct from most other affinity chromatography technologies in that it can operate on a native, unmodified protein without the need for a specialized affinity “tag” ...

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