A collaborative clerkship with a focus on rural community health.

The Deans' Rural Primary Care Clerkship was developed through the collaborative efforts of South Carolina's two medical schools. The clerkship provides students an innovative learning experience in rural community medicine through the unique combination of learning opportunities with community-oriented primary care, continuous quality improvement, interdisciplinary health care teams, and cultural competency. Much of students' learning addresses current directives for population health training. The positive experience students are having in these rural, underserved South Carolina communities will help them better understand the rewards and challenges of rural, community-responsive health care.

The aspartic proteinase from the rodent parasite Plasmodium berghei as a potential model for plasmepsins from the human malaria parasite, Plasmodium falciparum.

The gene encoding an aspartic proteinase precursor (proplasmepsin) from the rodent malaria parasite Plasmodium berghei has been cloned. Recombinant P. berghei plasmepsin hydrolysed a synthetic peptide substrate and this cleavage was prevented by the general aspartic proteinase inhibitor, isovaleryl pepstatin and by Ro40-4388, a lead compound for the inhibition of plasmepsins from the human malaria parasite Plasmodium falciparum. Southern blotting detected only one proplasmepsin gene in P. berghei. Two plasmepsins have previously been reported in P. falciparum. Here, we describe two further proplasmepsin genes from this species. The suitability of P. berghei as a model for the in vivo evaluation of plasmepsin inhibitors is discussed.

Biodistribution and radioimmunopharmacokinetics of 131I-Ama monoclonal antibody in atherosclerotic rabbits.

Monoclonal antibodies have been raised against Ama isolated from human and experimental atherosclerotic plaque. 131I-Ama-MoAb in the whole antibody form was injected into normal NZW rabbits and Watanabe hyperlipidemic rabbits. Biodistribution studies showed that atheromatous aortas had a significantly higher (5-7X) uptake of 131I-Ama-MoAb than that of normal aortas. However, 131I-Ama-MoAb was cleared very slowly from atherosclerotic rabbits. As a result, atheromas could not be identified by imaging because of the low target to non-target ratios.

Monomeric human cathepsin E.

Cathepsin E is a homodimer, consisting of two monomers linked by an inter-molecular disulphide bond. The cysteine residue involved is located near to the N-terminus of the mature proteinase. By mutating this residue to alanine, a monomeric form of human cathepsin E was engineered and purified. The activity of the resultant enzyme was not altered significantly (in terms of its ability to hydrolyse two chromogenic peptide substrates; and its susceptibility to inhibition by pepstatin). However, the stability of the mutant enzyme to alkaline pH and to temperature was markedly reduced.

Cellular models of atherosclerosis in the young.

The current interest in vascular lesions formed during childhood and adolescence is both pertinent and timely, as atherosclerosis research has never been more exciting that at the present. A number of attractive hypotheses about the pathogenesis of atherosclerosis have been proposed which provide different perspectives for understanding the disease process. A host of animal and cell models of early lesions are now available for study and new technological advances, such as recombinant DNA techniques and sensitive imaging procedures, will allow these models to be studied in greater depth. Consequently, the next decade should bring new insight on the pathogenesis of atherosclerosis, especially the early lesions of youth.

Use of nile red for the rapid in situ quantitation of lipids on thin-layer chromatograms.

We describe the use of the fluorescent dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, as a general-purpose reagent for the rapid detection and quantitation of a wide variety of lipids and other hydrophobic compounds separated by thin-layer chromatography. After samples are applied to silica gel plates and chromatographed, the plate is briefly dipped into a nile red solution (8 micrograms/ml of methanol-water 80:20, v/v). Background fluorescence of nile red dye adsorbed to the silica gel is then preferentially destroyed by dipping the plate in a dilute aqueous solution of bleach. After drying, lipid bands are visualized under ultraviolet light. Reflectance fluorometry (Ex: 580 nm; Em: 640 nm) is utilized for in situ quantitative analysis of the fluorescence of the lipids on the nile red-stained plate. Neutral lipids, phospholipids, sphingolipids, and fatty acids can be examined, although the nile red fluorescence intensity varies significantly among the lipid classes. Also, staining is stronger for unsaturated lipids than for saturated lipids. The lower detection limit of the assay is 25-100 ng for cholesterol, cholesteryl esters, triacylglycerols, and phospholipids.

Transformation of rabbit arterial smooth muscle cells with simian virus 40.

In vitro studies were carried out to induce viral transformation of vascular smooth muscle cells. Cultured rabbit arterial smooth muscle cells were infected with simian virus 40 (SV 40), and transformed cultures were produced that exhibit altered morphology, increased growth rate and plating efficiency, growth on semi-solid substrate, and chromosomal abnormalities. Nuclear SV 40 T-antigen was detected in all cells of these cultures. Muscle-specific actin was identified by a specific monoclonal antibody suggesting retention of smooth muscle cell characteristics by the transformed cells. Significant cytoplasmic lipid accumulation occurred in transformed cells incubated with beta-very low density lipoprotein, as revealed both by chemical analyses and Nile Red lipid staining of the culture. The transformed smooth muscle cells grow permanently in cell culture. Our investigations show that arterial smooth muscle cells transformed with SV 40 virus exhibit altered phenotypic properties distinct from that of normal arterial smooth muscle cells.

Application of Nile red, a fluorescent hydrophobic probe, for the detection of neutral lipid deposits in tissue sections: comparison with oil red O.

Nile red is a phenoxazone dye that fluoresces intensely, and in varying color, in organic solvents and hydrophobic lipids. However, the fluorescence is fully quenched in water. The dye acts, therefore, as a fluorescent hydrophobic probe. We utilized this novel property of nile red to develop a sensitive fluorescent histochemical stain for tissue lipids. Nile red was prepared by boiling Nile blue A under reflux for 2 hr in 0.5% H2SO4, and extracting the product into xylene. For staining, the purified dye is dissolved in 75% glycerol (1-5 micrograms/ml) and applied to frozen tissue sections. Tissue lipids then fluoresce yellow-gold to red, depending on their relative hydrophobicity. Using sections of liver and aorta from a cholesterol-fed rabbit, we assessed the value of Nile red as a stain for neutral lipids by comparing the staining pattern obtained with that produced by oil red O, a commonly used dye for tissue cholesteryl esters and triacylglycerols. In the cholesterol fatty liver, Nile red staining was comparable to that of oil red O. In contrast, Nile red staining of rabbit aortic atheroma revealed ubiquitous lipid deposits not observed with oil red O staining. These latter results suggest that Nile red can detect neutral lipid deposits, presumably unesterified cholesterol, not usually seen with oil red O or other traditional fat stains.

Spectrofluorometric studies of the lipid probe, nile red.

We found that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, can be applied as a fluorescent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry (J. Cell. Biol. 1985. 100: 965-973). To understand the selectivity of the staining, we examined the fluorescence properties of nile red in the presence of organic solvents and model lipid systems. Nile red was found to be both very soluble and strongly fluorescent in organic solvents. The excitation and emission spectra of nile red shifted to shorter wavelengths with decreasing solvent polarity. However, the fluorescence of nile red was quenched in aqueous medium. Nile red was observed to fluoresce intensely in the presence of aqueous suspensions of phosphatidylcholine vesicles (excitation maximum: 549 nm; emission maximum: 628 nm). When neutral lipids such as triacylglycerols or cholesteryl esters were incorporated with phosphatidylcholine to form microemulsions, nile red fluorescence emission maxima shifted to shorter wavelengths. Serum lipoproteins also induced nile red fluorescence and produced spectral blue shifts. Nile red fluorescence was not observed in the presence of either immunoglobulin G or gelatin. These results demonstrate that nile red fluorescence accompanied by a spectral blue shift reflects the presence of nile red in a hydrophobic lipid environment and account for the selective detection of neutral lipid by the dye. Nile red thus serves as an excellent fluorescent lipid probe.

Nile red: a selective fluorescent stain for intracellular lipid droplets.

We report that the dye nile red, 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one, is an excellent vital stain for the detection of intracellular lipid droplets by fluorescence microscopy and flow cytofluorometry. The specificity of the dye for lipid droplets was assessed on cultured aortic smooth muscle cells and on cultured peritoneal macrophages that were incubated with acetylated low density lipoprotein to induce cytoplasmic lipid overloading. Better selectivity for cytoplasmic lipid droplets was obtained when the cells were viewed for yellow-gold fluorescence (excitation, 450-500 nm; emission, greater than 528 nm) rather than red fluorescence (excitation, 515-560 nm; emission, greater than 590 nm). Nile red-stained, lipid droplet-filled macrophages exhibited greater fluorescence intensity than did nile red-stained control macrophages, and the two cell populations could be differentiated and analyzed by flow cytofluorometry. Such analyses could be performed with either yellow-gold or red fluorescence, but when few lipid droplets per cell were present, the yellow-gold fluorescence was more discriminating. Nile red exhibits properties of a near-ideal lysochrome. It is strongly fluorescent, but only in the presence of a hydrophobic environment. The dye is very soluble in the lipids it is intended to show, and it does not interact with any tissue constituent except by solution. Nile red can be applied to cells in an aqueous medium, and it does not dissolve the lipids it is supposed to reveal.

Lipoprotein-heparin-fibronectin-denatured collagen complexes enhance cholesteryl ester accumulation in macrophages.

The sequestration of low-density lipoprotein (LDL) by components of the vascular extracellular matrix has long been recognized as a contributing factor to lipid accumulation during atherogenesis. The effects, however, that components of the extracellular matrix might have on LDL catabolism by scavenger cells have been little investigated. For these purposes we have prepared insoluble complexes of LDL, heparin, fibronectin, and denatured collagen (gelatin) and examined their effects on lipid accumulation, LDL uptake and degradation, and cholesteryl ester synthesis in mouse peritoneal macrophages. The results of these experiments have demonstrated that the cholesteryl ester content of macrophages incubated with a particular suspension of LDL, heparin, fibronectin, and collagen complexes is four- to fivefold that of cells incubated with LDL alone. The uptake of complexes containing 125I-LDL is rapid; however, in contrast to either endocytosed 125I-LDL or 125I-acetyl LDL, the degradation of complex-derived LDL is impaired. In addition, the uptake of complex-derived LDL stimulates the incorporation of [14C]oleic acid into cholesteryl oleate, however, the stimulation was a small fraction of that observed in cells incubated with acetyl LDL. Ultrastructurally, macrophages incubated with LDL, heparin, fibronectin, and collagen complexes did not contain many lipid droplets, but rather their cytoplasm is filled with phagosomes containing material similar in appearance to LDL-matrix complexes. These results indicate that components of the extracellular matrix can alter the catabolism of LDL by scavenger cells, suggesting that they may play a role in cellular lipid accumulation in the atherosclerotic lesion.