A point mutation in CD28 distinguishes proliferative signals from survival signals.

Upon interaction with its ligand, B7, CD28 becomes phosphorylated on tyrosines. One tyrosine in particular (Y170 in mouse CD28, Y173 in human CD28) has received much attention. This is because it permits CD28 to recruit SH2-containing signaling molecules, including phosphoinositide 3 kinase, Grb2 and Gads. Using mice we employed a transgenic approach to express a tyrosine-->phenylalanine mutant form of CD28 that uncouples these SH2-mediated interactions from CD28. The CD28 mutant is unable to up-regulate expression of the prosurvival protein Bcl-xL, rendering the T cells more susceptible to radiation-induced death. Nonetheless, this mutated form of CD28 still prevents the induction of anergy and promotes T cell proliferation, interleukin 2 secretion and B cell help. Thus, we describe a single point mutation within the CD28 cytoplasmic domain that uncouples signals required for proliferation and survival.

High pressure liquid chromatographic method for indole in shrimp: development of method and collaborative study.

A collaborative study on the determination of indole in shrimp was conducted in which a high pressure liquid chromatographic (HPLC) method and a spectrofluorometric method were compared with the AOAC gas-liquid chromatographic (GLC) method (18.075-18.078, 13th ed.). In the HPLC method, 10 g shrimp was blended with methanol, an internal standard was added, and the extract was filtered. Indole was separated on an octadecylsilane reverse phase column, using 60% MeOH-H2O, and quantitated with a fluorescence detector (excitation 280 nm, emission 330 nm) by comparing the indole peak height with that of an internal standard, 2-methyl-indole. Recoveries at a 25 micrograms/100 g level averaged 104% with a range of 90-127%, and at a level of 35 micrograms/100 g averaged 102% with a range of 93-112%. In the spectrofluorometric method, 25 g shrimp was extracted with 2% EtOAc-hexane. After several washes, indole was partitioned into a saturated NaCl-MeOH solution and its fluorescence was measured (excitation 280 nm, emission 332 nm). Recoveries at a 25 micrograms/100 g level averaged 93% with a range of 0-255% and at a level of 35 micrograms/100 g averaged 64% with a range of 0-107%. Recoveries obtained by the AOAC-GLC method at a level of 25 micrograms/100 g averaged 96% with a range of 81-116% and at a level of 35 micrograms/100 g averaged 101% with a range of 81-119%. The coefficients of variation were 20, 10, and 64% at a 25 micrograms/100 g level for the GLC method, the HPLC method, and the spectrofluorometric method, respectively. The HPLC method was adopted as official first action for indole levels in shrimp exceeding 1 microgram/100 g.