CAML regulates Bim-dependent thymocyte death.

Appropriate control of apoptosis during T lymphocyte differentiation is critical for destruction of T cells bearing potentially autoreactive or useless immuno-receptors and for survival of those T cells bearing antigen receptors that may recognize foreign proteins. Despite the well-established importance of thymocyte survival, the exact signals regulating thymocyte apoptosis have not been fully elucidated. Here, we show that thymocytes lacking the endoplasmic reticulum protein calcium-modulating cyclophilin ligand (CAML) failed to undergo normal T-cell development and exhibited dramatically increased rates of apoptosis. In vitro, CAML-deficient thymocytes accumulated high levels of reactive oxygen species (ROS) and underwent abnormally accelerated death in response to several cytotoxic stimuli, including treatment with etoposide, cytokine deprivation, or Fas ligation. Although neither p53 deletion nor loss of Fas rescued the survival and continued development of CAML-deficient thymocytes, removal of the pro-apoptotic BH3-only Bcl-2 family member Bim significantly restored their survival. This work reveals CAML to be a critically important regulator of ROS- and Bim-dependent thymocyte death.

Chemical reactivity, cytotoxicity, and mutagenicity of chloropropanones.

Studies were conducted to assess the in vitro toxicity of three chloropropanones: monochloropropanone (MCP), 1,1-dichloropropanone (1,1-DCP), and 1,3-dichloropropanone (1,3-DCP). Chloropropanones reacted directly with reduced glutathione (GSH) in sodium phosphate buffer at pH 7.4. All chloropropanones were cytotoxic to suspensions of male rat hepatocytes in a concentration range of 0.5-10 mM. Cytotoxicity was preceded by rapid decline in cellular GSH levels. Mutagenic potencies among the chloropropanones in Salmonella typhimurium bacteria differed greatly. 1,3-DCP was mutagenic in the nanomole range, 1,1-DCP was weakly mutagenic in the micromole range, and MCP was not mutagenic. Mutagenicity of the dichloropropanones was evident without metabolic activation. These results suggest that the three chloropropanones may, in part, be directly cytotoxic but only 1,3-DCP and 1,1-DCP are directly mutagenic.

Analysis of polysorbate and its polyoxyethylated metabolite.

Polysorbates are used as emulsifiers in a number of pharmaceuticals and have been implicated as the possible toxic agent in the neonatal vitamin supplement, E-Ferol. In the investigation of the toxicity of this compound, it was necessary to find a method to separate and quantitate polysorbate and its polyoxyethylated metabolite from biological fluids. A high-performance liquid chromatography method was developed which combines the use of a 500 A mu Styragel size exclusion column with an ammonium cobaltothiocyanate complexation column and detection at 620 or 320 nm. The detection limit is approximately 5 micrograms. The method was used to demonstrate that polysorbate was metabolized in vitro by hepatocytes and that the urinary metabolite in humans is comparable to that produced by the rat.

Determination of polysorbate in ascites fluid from a premature infant.

A method is described for the determination of poly(oxy-1,2-ethanediyl)oligomers in body fluids using ammonium cobaltothiocyanate complexation in conjunction with high pressure liquid chromatography and visible spectrophotometry. Analysis of peritoneal fluid from a baby given E-ferol, a vitamin E supplement, revealed levels as high as 100 micrograms/mL polysorbate.

Human conjunctival surface mucins: a quantitative study of normal and diseased (KCS) tissue.

The architecture and distribution of surface mucins were studied qualitatively and quantitatively by transmission electron microscopy in ruthenium red stained biopsies of the human conjunctiva. Six control specimens were compared with three from early keratoconjunctivitis sicca (KCS) and with three specimens from severe KCS cases. The area of the ruthenium red/OsO4/mucin reaction product on the conjunctival epithelial surface was measured by image analysis and the values were expressed in micron2 per micron length of epithelium. The total area of surface mucins was differentiated from the narrow zone of mucins in close contact with the microplicae. The value for total mucins in two cases of early KCS was higher (0.4 micron2/micron) than the control range (0.1-0.3 micron2/micron) due to the presence of large clumps of mucins on the surface. The values for microplical mucins in early and severe KCS were within the control range, but were an overestimate owing to the presence of abnormal structures, e.g. vesicles, and abnormal clumps on the surface. This morphometric technique has limitations, but with appropriate material it might be a useful tool for the identification of conjunctival surface mucins.