Generating controlled molecular gradients in 3D gels.

A new method for producing molecular gradients of arbitrary shape in thin three dimensional gels is described. Patterns are produced on the surface of the gel by printing with a micropump that dispenses small droplets of solution at controlled rates. The molecules in the solution rapidly diffuse into the gel and create a smooth concentration profile that is independent of depth. The pattern is relatively stable for long times, and its evolution can be accurately described by finite element modeling of the diffusion equation. As a demonstration of the method, direct measurements of protein gradients are performed by quantitative fluorescence microscopy. A complementary technique for measuring diffusion coefficients is also presented. This rapid, flexible, contactless approach to gradient generation is ideally suited for cell culture experiments to investigate the role of gradients of diffusible substances in processes such as chemotaxis, morphogenesis, and pattern formation, as well as for high-throughput screening of system responses to a wide range of chemical concentrations.

Cholesterol esterification as a limiting factor in accumulation of cell cholesterol: a comparison of two J774 macrophage cell lines.

It was previously reported by Tabas et al. that J774 macrophages, unlike mouse peritoneal macrophages, accumulate large amounts of cholesteryl esters when incubated with native low-density lipoprotein (LDL). Comparison of the cell line (designated J774A.2) used in those experiments with its parent line (J774A.1) indicates that it is a variant with a greater rate of cholesterol esterification. This large difference in cholesterol esterification was accompanied by only a small difference in rates of LDL uptake and degradation by the J774A.2 line. The J774A.2 cells have become a variant line through either mutation or selection which has enhanced its susceptibility to foam cell formation by its markedly increased ability to esterify cholesterol.

Acyl coenzyme A:cholesterol acyl transferase in macrophages utilizes a cellular pool of cholesterol oxidase-accessible cholesterol as substrate.

Cholesterol esterification by acyl CoA:cholesterol acyl transferase (ACAT) in macrophages is a key process in atheroma foam cell formation. However, the process of cholesterol substrate delivery to ACAT is not well defined. In this study, J774 macrophages, which form foam cells with native low density lipoprotein (LDL), were labeled with [3H]cholesterol-containing liposomes. Most (80-90%) of the cholesterol label could be converted by cholesterol oxidase to cholestenone, suggesting plasma membrane localization; only 0.6% of the label was in cholesteryl ester (CE). In cells chased for 6 h in medium lacking LDL, the distribution of label was essentially unchanged, whereas in cells chased with LDL, 28% of the label was incorporated into CE concomitant with a decrease in cholestenone label to 50%. [3H]Cholesterol-labeled mouse peritoneal macrophages incubated with acetyl-LDL, and both J774 and mouse peritoneal macrophages incubated with 25-hydroxy-cholesterol, also showed a shift of label from cholestenone to CE. Similar results were found when cellular cholesterol was biosynthetically labeled with [3H]mevalonate. The percentage of cholesterol substrate for ACAT in LDL-treated J774 macrophages which originates from endogenous cellular pools (versus that originating from LDL itself) is approximately 50%. We conclude that upon activation of ACAT in macrophages, there is a novel process whereby a cholesterol oxidase-accessible pool of cellular cholesterol, presumably plasma membrane cholesterol, is translocated to ACAT in the endoplasmic reticulum.