生物化学技术

Phosphoinositides are a family of lipid second messengers interlinked by an extensive and highly regulated network of kinases and phosphatases. The modulation of phosphoinositide profiles can regulate numerous cancer-related pathways, including cell survival, cell proli ...

Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze the hydrolysis of phophatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and inositol 1,4,5-triphosphate . PLCε is a recently discovered isoform that has been shown to be activated by members of the ...

Mammalian cells have evolved proteins that can extract single lipids from membranes and sequester them in their hydrophobic cavity. These proteins, collectively known as lipid transport proteins, play essential roles in many aspects of lipid metabolism, lipid signaling, and membr ...

The regulation of phosphoinositide (PI) 3-kinase activities has been linked to many normal and disease-related processes, including cell survival, cell growth and proliferation, cell differentiation, cell motility, and intracellular vesicle trafficking. However, as the fam ...

Signaling lipids are found in specific subcellular membranes, where they recruit and regulate cytosolic proteins and contribute to bilayer structure and dynamics. These interactions are vital for signaling and membrane trafficking pathways and contribute to the organizatio ...

A wide spectrum of intracellular signaling events mediated by up to seven different phosphorylated forms of phosphatidylinositol (PtdIns) occurs in all eukaryotic cells. The activities of multiple, nondegenerate PI kinases and phosphatases control these signaling events. The ...

Members of the protein kinase C family are major effectors of lipid second messengers. We describe three protocols to assess protein kinase C activity in polymorphonuclear leukocytes (neutrophils). These methods are useful to study the activation and function of protein kinase C in these ...

Phospholipase D (PLD) is an enzyme producing phosphatidic acid and choline through hydrolysis of phosphatidylcholine. The enzyme has been identified as a member of a variety of signal transduction cascades and as a key regulator of numerous intracellular vesicle trafficking proces ...

Phosphatidylinositol 3,4,5 trisphosphate is a potent membrane-bound signaling molecule transiently synthesized by phosphoinositide 3-kinase (PI3-kinase) in response to extracellular agonists. PtdIns(3,4,5)P 3 signals need to be strictly controlled. PtdIns(3,4,5)P 3 re ...

This chapter describes an indirect approach to measure PTEN’s lipid phosphatase activity in vivo. PTEN counteracts phosphatidylinositol 3-kinase action in dephosphorylating 3-phosphorylated phosphoinositides. Therefore, PtdIns(3,4,5)P3-dependent activation ...

This chapter describes methods for studying downstream events of the PI3 K/Akt signaling cascade, focusing on the FoxO transcription factors. These approaches also represent alternative means for gauging the phosphoinositide-3 kinase/Akt activity. We describe protocols for t ...

Extensive studies have been performed in order to understand the interaction of receptors with soluble ligands. However, we know very little of the parameters that regulate the interaction of receptors with membrane-bound ligands. Artificial lipid bilayers can be used to mimic cell-to ...

Artificial transcription factors (ATFs) are designed to mimic natural transcription factors in the control of gene expression and are comprised of domains for DNA binding and gene regulation. ATF domains are modular, interchangeable, and can be composed of protein-based or nonpepti ...

Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily alter ...

Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) h ...

We present and discuss results and features related to the synthesis of water-soluble semiconductor quantum dots and their application as fluorescent biomarkers in cancer diagnostics. We have prepared and applied different core-shell quantum dots, such as cadmium telluride-ca ...

Noninvasive whole-body near-infrared fluorescence imaging is now acknowledged as a powerful method for the molecular mapping of biological events in live small animals such as mouse models. With outstanding optical properties such as high fluorescence quantum yields and low phot ...

We describe an alternative to the molecular biology technique of polyacrylamide gel electrophoresis-based Western blotting and immunoprecipitation, which is an extensively used method for separating target proteins from complex cellular mixtures and for identification ...

Molecular beacons have become an important fluorescent probe for sequence-specific DNA detection. To improve the sensitivity and robustness of molecular beacon assays, fluorescent semiconductor quantum dots (QDs) are now being used as the fluorescent moiety for molecular beac ...

DNA combing is a useful strategy for manipulating single DNA molecules and has a wide range of applications in genetics, single molecule studies, and nanobiotechnology. Visualization of combed DNA molecules is usually performed by using DNA binding organic dyes. Such dyes are not suitable ...