微生物学技术

The human pathogen Helicobacter pylori causes inflammation in the stomach of infected hosts, leading in some cases to the development of gastric cancer. Several mouse models have been developed to study Helicobacter-induced carcinogenesis with similarities to gastric adenoca ...

Mice used to model helicobacter gastritis should be screened by PCR prior to experimental dosing to confirm the absence of enterohepatic Helicobacter species (EHS) that colonize the cecum and colon of mice. Natural infections with EHS are common and impact of concurrent EHS infection on Hel ...

Animal models of microbial diseases in humans are an essential component for determining fulfillment of Koch’s postulates and determining how the organism causes disease, host response(s), disease prevention, and treatment. In the case of Helicobacter pylori, establishing an anim ...

Processing of tissue and blood must be done in a systematic and controlled fashion in order to optimize results and allow comparison of samples between experiments and between laboratories. Here we present our protocols for blood and tissue processing.

Delayed-type hypersensitivity responses in the skin (in the case of mice, in the foot pad) is used to assess cell-mediated immunity (CMI) in vivo. In the case of CMI to Helicobacter infection, the mice are given an injection of cultured Helicobacter organisms into the hind footpad, and induration is m ...

Immune cells recruited to the infected gastric mucosa can be isolated and used for a variety of purposes. Here we describe methods for the isolation and characterization of gastric lamina propria leukocytes.

Analysis of the immune response of mice to Helicobacter infection has been greatly aided by the use of various deficient mouse strains. Here we present protocols for reconstitution of immune-deficient mice with wild-type immune cells and protocols for analysis of the outcome.

Standardization of bacterial culture is crucial for in vivo experiments addressing Helicobacter/host �interaction. Here we present methods for bacteria culture and infection of mice.

Animal models are essential for in vivo analysis of Helicobacter-related diseases. Transgenic mice and Mongolian gerbil models have been the corner stone of present research focusing on both bacterial virulence factors and host response to infection. Establishing a reproducible ...

Helicobacter pylori CagA and VacA are two critical virulence factors that modulate disease severity in the infected host. The following chapter outlines methods employed to study the effects of these virulence factors on several key signaling pathways in epithelial cells.

Adherence and internalization of Helicobacter pylori into epithelial cells is a recently recognized event in the pathogen’s life cycle.

The precise quantitative determination of the different lipid classes in mutant cells is key to understand the possible role of the respective gene product in lipid homeostasis. In this chapter, we describe methods based on thin-layer chromatography that are employed routinely to dete ...

Pulsed-field gel electrophoresis (PFGE) can be used to separate the 16 budding yeast chromosomes on the basis of size. Here we describe a detailed, practical protocol that will allow a novice to perform informative PFGE experiments. We first describe the culture of yeast prior to analysis, along ...

Two-dimensional gel electrophoresis (2-DE) offers the opportunity of separating several hundred proteins from a total yeast cellular extract. A detailed description is provided here of the different steps required for the separation and visualization of radiolabeled yeast pro ...

Quantitative extraction of lipids from the tissue or microorganism of choice is key to their subsequent analysis. In this chapter, we describe a simple and rapid protocol that relies on glass bead disruption in the presence of organic solvents to quantitatively extract lipids from yeast cel ...

Often preparations of isolated organelles contain other, unwanted, cellular components. For biochemical experiments to determine the localization of newly identified proteins, or to determine the whole set of proteins (or the proteome) from a desired organelle, these unwanted co ...

Peroxisomes are ubiquitous subcellular organelles of eukaryotic cells. As with all organelles, peroxisomes can be purified from cell lysates using a combination of differential centrifugation and density gradient centrifugation. Here, we describe a method for purifying pero ...

Mitochondria fulfill a large variety of metabolic tasks such as respiration, beta-oxidation, heme biosynthesis, ketone-body, or amino acid synthesis. In addition to their metabolic role, mitochondria are also key players in cellular apoptosis and participate in the generation of re ...

The yeast two-hybrid system is a poauwerful molecular genetic tool conceived by Fields and Song (1). The article is a comprehensive set of methods designed to take the reader through a yeast two-hybrid analysis of your favorite gene (YFG). This article details the preparation for a screen, the scre ...

The accurate replication and expression of genetic information is ultimately governed by the interaction of regulatory proteins with specific sites on chromosomes. In recent years, our understanding of how these interactions occur in vivo has advanced considerably, in large part o ...