An adherent cell model to study different stages of apoptosis.

Apoptosis in the classical thymocyte model occurs very rapidly making it difficult to study the intermediate steps in the process. An alternative adherent cell model is characterized and proposed in this paper. HT29 cells treated with a teniposide were collected at various times for morphological and biochemical assessments. Large DNA breaks (450-500, 350-400, 100-200 kb) were observed in these cells between 6 and 24 h. The larger DNA breaks appeared initially and in progression such that the smaller DNA break of 100-200 kb became apparent by 24 h. These changes in DNA corresponded with an increase in cell diameter and a gradual rounding and detaching of cells from each other but not from the tissue culture plates. The smallest DNA break of 23-50 kb appeared at 48 h and persisted throughout the 96 h of incubation. DNA ladders of 180- to 200-bp oligomers were also observed between 48 and 96 h and these coincided with the presence of small floating cells. Changes in cell adherence after teniposide treatment have permitted the consistent isolation of cells in four distinct morphological and biochemical stages of apoptosis: (1) "preapoptotic," (2) "swelling," (3) "rounding," and (4) "floating." The main advantages of this adherent cell model are: (1) apoptosis occurs very slowly (minimum of 48 h) permitting the observation of progressive changes; (2) cells from four stages of apoptosis can be used to study the sequence of events of other biochemical and genetic factors involved in the process; and (3) extracellular matrix proteins are present in this model so their participation in apoptosis, if any, can be determined.

A novel very high molecular mass protein located in the membrane skeleton.

A monoclonal antibody generated from a mouse immunized with L6 rat myoblast cells was found to react with a major 700-kDa band and a minor 500-kDa band in immunoblots. Immunofluorescence microscopy demonstrated a submembranous location in tissue sections and an exclusion from stress fiber regions in cultured cells. Further, permeabilization of cultured cells with nonionic detergent prior to fixation changed the diffuse pattern of fluorescence to a web. These findings are characteristic of membrane skeletal proteins. In muscle tissue, the protein was much more abundant in fast twitch fibers and was found in internal locations as well as at the membrane. The protein could be solubilized in the absence of detergents and, hence, is not transmembrane. Although initially discovered in myoblast cells, the protein is present in a variety of tissue types, including brain, kidney, heart, liver, and lung. Pulse-chase labeling of the two bands suggested that the 500-kDa band was not a breakdown product of the 700-kDa protein. The protein appears to be a previously undiscovered membrane skeletal constituent for which the name "endossin" is proposed.

Iron catalyzed oxidation of trout diets and its effect on the growth and physiological response of rainbow trout.

Two experiments were conducted to determine 1) the effect of iron supplementation and the quality of fish oils on dietary lipid peroxidation and 2) the concurrent effects of diet rancidity and iron overload on the growth and physiological response of rainbow trout. Semi-purified diets supplemented with graded levels of iron (0-6250 mg/kg diet as ferrous sulphate) were fed to trout for 12-36 weeks. The malonaldehyde (MA) concentration of the test diets increased as the iron levels in the diets increased indicating that iron catalyzed lipid oxidation was occurring. However, when ethoxyquin was added to the oils, the increase in dietary MA level was significantly reduced. Fish oils with an initial high peroxide value were more susceptible to iron-catalyzed lipid oxidation. The concurrent effects of diet rancidity and iron overload (greater than 86 mg/kg) led to the development of unique histopathological signs, poor growth and high mortalities in the trout. In contrast, when diet rancidity was low (less than 10 µg MA/g diet), the toxic level of dietary iron was greater than 1380 mg/kg diet. The concentration of iron in trout tissues, and the hematocrit and hemoglobin concentrations increased as dietary iron levels increased and were not affected by the degree of diet rancidity.