Toolbox/Lecture 11

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WIKIPEDIA BIO154/254: Molecular and Cellular Neurobiology

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Contents


Lecture 11 Model Systems

Mouse gustatory system

Mouse olfactory system

Lecture 11 Techniques

Two-bottle preference test

Wild-type mice typically prefer sweet water over plain water. A two-bottle preference test showed that Sac mutant mice are less sensitive to sucrose than wild-type mice. Genetic mapping indicated that the gene for Sac lies on a locus close to the gene encoding T1R3. This test cleanly showed that a T1R3 transgene rescue provides wild-type sensitivity to sucrose in Sac mutant mice.

Calcium imaging

Calcium imaging is a scientific technique designed to reflect the calcium status of a particular tissue or medium. In calcium imaging a substance called Fura is used to bind to calcium. When Fura binds to calcium after being exposed to fluorescent light, it fluoresces. The Fura-Ca complex affects the wavelength typically associated with unbound Fura and the resulting fluorescence can be detected by a camera adapted (usually through a microscope) for fluorescent light imaging. A computer-generated image is thus created which can be analyzed according to intensity, which reflects calcium status in the given medium or tissue.

G15

G15 is able to bind promiscuously to 7 transmembrane proteins so that they can be easily labeled. G15 was utilized to identify that T1R2 and T1R3 recognize sugars together as a functional heterodimer receptor.

Fura-2

Fura-2 is a ratiometric fluorescent dye which binds to free intracellular calcium. Fura-2 is excited at 340 nm and 380 nm of light, and the ratio of the emissions at those wavelengths is directly correlated to the amount of intracellular calcium. The use of the ratio allows freedom from a myriad of confounding factors, such as ambient light, making Fura-2 one of the most preferred tools to quantify calcium levels.

Ace K

Acesulfame potassium is a calorie-free artificial sweetener, also known as Acesulfame K or Ace K, and marketed under the trade names Sunett and Sweet One.

Chemically, acesulfame potassium is the potassium salt of 6-methyl-1,2,3- oxathiazine-4(3H)-one 2,2-dioxide. It is white crystalline powder with molecular formula of C4H4KNO4S and molecular weight of 201.24.

Acesulfame K is 180-200 times sweeter than sucrose (table sugar), as sweet as aspartame, about half as sweet as saccharin, and one-quarter the sweetness of sucralose. Like saccharin, it has a slightly bitter aftertaste, especially at high concentrations.

The studies that purport to show safety have been challenged by a number of individuals and organizations, most notably the Center for Science in the Public Interest in the USA. They claim that the existing studies are inadequate (despite being peer-reviewed), that there are flaws in the research protocols, dosing, and time length of the studies, and that as a result the carcinogenicity of acesulfame K may not be properly understood. In particular they note that there have not been long-term human studies, so they doubt the studies which show that acesulfame is rapidly absorbed and then excreted unchanged (i.e., not metabolized by the human body) are representative of the long-term. Currently, the scientific community's official position is that acesulfame K is safe to consume, which is the view put forth on the sweetener industry's public relations website, IFIC.

Inosinate

Inosinate (IMP) is a nucleotide that increases the effect of L-glutamate in the gustatory system. This nucleotide synergism is characteristic of umami.

Cycloheximide

Cycloheximide is chemical that inhibits protein synthesis in eukaryotic cells. In is made by the bacteria Streptomyces griseus, and functions by interfering with peptidyl transferase in the 60S ribosome. When this chemical is added, transcriptional elongation is arrested, and protein synthesis stops. Cycloheximide is a commonly used reagent in biological research to inhibit protein synthesis in in vitro tissue culture experiments. The effects of the agent are rapid and can be reversed simply by replacing the cell culture media.

Diphtheria toxin (DTA)

Diphtheria toxin (DTA) is an exotoxin secreted by Corynebacterium diphtheriae, the pathogen bacterium that causes diphtheria. DTA is a single polypeptide chain of 535 amino acids consisting of two subunits linked by disulfide bridges. Binding to the cell surface of the less stable of these two subunits allows the more stable part of the protein to penetrate the host cell. It catalyzes the ADP-ribosylation and inactivates the eucaryotic elongation factor-2 (eEF2). It does so by ADP-ribosylating the unusual aminoacid diphtamide. In this way, it acts as a DNA translational inhibitor. The lethal dose for humans is about 0.1 μg/kg of pure protein.

Enzyme digestion of PCR

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