Effect of fatty acid supplementation on cholesterol and retinol esterification in J774 macrophages

J774 macrophages exposed to medium containing cholesterol-rich phospholipid dispersions accumulate cholesteryl ester. Supplementing this medium with 100 micrograms oleate/ml increased cellular cholesteryl ester contents 3-fold. Cell retinyl ester contents increased 8-fold when medium containing retinol dispersed in dimethyl sulfoxide was supplemented with oleate. These increases were not the result of increases in total lipid uptake by the cells but rather of redistribution of cholesterol and retinol into their respective ester pools. Effective oleate concentration of 15-30 micrograms/ml increased cellular retinyl and cholesteryl ester contents. The effective oleate concentration was reduced to 5 micrograms/ml when the fatty acid/albumin molar ratio was increased. The oleate-stimulated increase in cholesterol esterification was blocked by incubating cells with Sandoz 58-035, a specific inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), indicating that the effect of fatty acid exposure is mediated through changes in ACAT activity. When cholesterol or retinol was added to cells which had been exposed to oleate for 24 h to provide a triacylglycerol store, the cellular contents of cholesteryl or retinyl ester were also significantly increased compared to cells not previously exposed to oleate. The oleate-stimulated increase in the esterification of cholesterol and/or retinol was also observed in P388D1 macrophages, human (HepG2) and rat (Fu5AH) hepatomas, human fibroblasts, rabbit aortic smooth muscle cells and MCF-7 breast carcinoma cells. In addition to oleate, a number of other fatty acids increased retinol esterification in J774 macrophages; however, cellular cholesterol esterification in these cells was increased only by unsaturated fatty acids and was inhibited in the presence of saturated fatty acids. Although the cellular uptake of radiolabeled oleate and palmitate was similar, a significant difference in the distribution of these fatty acids among the lipid classes was observed. These data demonstrate that exogenous fatty acids are one factor that regulate cellular cholesteryl and retinyl ester contents in cultured cells.     

Effect of treatment with glutaraldehyde-fixed myelin basic protein-liposomes on active induction and passive transfer of experimental allergic encephalomyelitis in Lewis rats

Guinea pig myelin basic protein (MBP)-liposomes were prepared and fixed with 0.2% glutaraldehyde (GA). Lewis rats were treated with glutaraldehyde-fixed MBP-liposomes (MBP-L-GA) or with cytochrome-c-liposomes (CYC-L-GA), 7 days before and 7 days after challenge with MBP in CFA. Rats treated with MBP-L-GA, but not with CYC-L-GA, were very well protected against the clinical manifestations of EAE. The protection was better than that obtained after treatment with conventional MBP-liposomes (without glutaraldehyde). Furthermore, when grown in vitro for 72 hr in the presence of MBP, lymphocytes from rats treated with MBP-L-GA and challenged with MBP in CFA exhibited a marked decrease in their ability to transfer EAE to normal syngeneic recipients.     

Seasonally of adult male japanese macaques (Macaca fuscata): Androgens and behavior in a confined troop

Seasonal variations in levels of serum testosterone, dihydrotestosterone (DHT), reproductive behavior, and social behavior were investigated in 12 adult males (5 to 20 + years of age) of the Oregon troop of Japanese macaques (Macaca fuscata). Blood samples were collected at 2- to 4-month intervals, and behaviors were monitored twice weekly over a 15-month period. Significant seasonal variations in levels of testosterone and DHT, and in frequencies of mount series, ejaculations, number of female partners, displays, courtship, and aggression were observed. Seasonal variations in reproductive and social behaviors did not correlate with seasonal variations in androgen levels because seasonal increases in these behaviors followed seasonal increases in the androgens with a 1- to 2-month delay. However, significant correlations between increased androgen levels and the onset of mating activity occur when mean monthly frequencies of mount series are shifted 1 to 2 months earlier to coincide with the rise in serum androgen levels. The frequency of adult male play and male-male mounting increased significantly when androgen levels were low. We suggest that photoperiod changes may function as a proximate cue in male Japanese macaques which induces a state of biological readiness for mating, and the behavioral consequences (i.e., mating) are then dependent upon the presence of receptive females.     

Extracellular matrix components influence the survival of adult cardiac myocytes in vitro

Calcium-tolerant myocytes were isolated from adult rat hearts by collagenase perfusion and plated on various substrates in serum-free medium and their adhesion to various extracellular matrix (ECM) components was determined. The myocytes attached readily to dishes coated with collagen type IV (C-IV), laminin (LN), and to fetal bovine serum (FBS) in a manner dependent on the concentration of the components. Substantially fewer myocytes adhered to dishes coated with fibronectin (FN) or to uncoated plastic dishes. Cells adhered equally well to dishes coated with C-IV, LN and FBS within 1-4 h. However, when examined after 2 weeks in culture it was found that only C-IV and LN could support survival of the attached myocytes, and when cultured on C-IV or LN the myocytes were spread and had formed a dense monolayer. The actin filaments had at this time reorganized linearly along the long axis of the cell and the myocytes contracted spontaneously. Rabbit antibodies were raised against myocyte membranes and their ability to inhibit attachment to ECM components was studied. Purified IgG inhibited attachment to C-IV, while having only a minor effect on attachment to LN. These data are compatible with the presence of a specific cell surface component(s) that interacts with ECM substrates and influences cell shape and possibly thereby influences cellular functions.     

Transformation of Azotobacter vinelandii with plasmid DNA.

Azotobacter vinelandii cells can be transformed at high frequencies with the broad-host-range plasmids pRK2501, RSF1010, and pGSS15, using a modification of the procedure developed by Page and von Tigerstrom (J. Bacteriol. 139:1058-1061, 1979) for chromosomal DNA-mediated transformation. The frequency of transformation per microgram of plasmid DNA per viable cell with pRK2501 and pGSS15 was about 5 X 10(-2) and 2 X 10(-2), respectively. With RSF1010, transformation frequencies ranged from 3 X 10(-4) to 4 X 10(-2). With each plasmid, the frequency of transformation was independent of the phase of the growth cycle. When concentrations of pRK2501 ranging from 0.1 to 51 micrograms of DNA were tested, the frequency of transformation was directly proportional to the amount of DNA. This linear response indicated that, although the uptake of plasmid DNA with this procedure may be inefficient, there is a high probability that once inside a cell the plasmid will be stably maintained. Cells that have been transformed with pRK2501 did not grow well on transforming medium which lacks iron and contains fixed nitrogen. However, on growth medium which contains iron and lacks fixed nitrogen, transformants produced distinctive colonies larger than those of nontransformed cells. Resistance to kanamycin due to transformation by pRK2501 was stably maintained for at least 10 successive generations in the absence of selective pressure. The present protocol should facilitate the molecular cloning of genes in Azotobacter spp.     

Light-induced variation in the growth and dynamics of transplanted ramets of the understory herb,Aster acuminatus

Summary Ramets of the understory herb, Aster acuminatus, were transplanted from two source populations into eight understory garden sites that varied in light and soil moisture levels. Ramet growth, clonal growth, flowering and survivorship were monitored for three growing seasons. Large differences among gardens in ramet growth, clonal growth and flowering developed in the first growing season and increased in the next two years. This variation was positively correlated with garden light level but not at all with soil moisture. Mortality rates were low in all gardens and showed that genets from any particular source could survive over a broad range of environmental conditions. There was no conclusive evidence for any source population differences in the capacity to survive or grow in different environments. The rapid, light-induced responses of transplanted ramets resulted in garden populations very similar in appearance to natural populations experiencing similar light regimes. These results combined with those from other studies of A. acuminatus provide strong evidence for the importance of light in explaining population patterns and dynamics in this species.     

Comparative Hydrolysis of Sphingomyelin and 2-N-(Hexadecanoyl)-Amino-4-Nitrophenyl-Phosphorylcholine by Normal Human Brain Homogenate at Acid and Neutral pH

Hydrolysis of sphingomyelin and 2-N-(hexade-canoyl)-amino-4-nitrophenyl-phosphorylcholine (HDA-PC), a synthetic analogue of sphingomyelin, by acid and Mg-dependent neutral sphingomyelinases was tested with a homogenate of normal human brain cortex. Results demonstrated quite different substrate specificities for these enzymes. Acid sphingomyelinase, which is neither activated by MgCl₂ nor inhibited by EDTA, hydrolyzed both substrates (the hydrolysis ratio of HDA-PC to sphingomyelin is ?2). In contrast, Mg-dependent neutral sphingomyelinase, which is inhibited by EDTA and reactivated by MgCl₂, hydrolyzed only sphingomyelin (the hydrolysis ratio of HDA-PC to sphingomyelin is ?0-0.05). This synthetic substrate seems to be useful for selective determination of acid sphingomyelinase and for avoiding interference of Mg-dependent neutral sphingomyelinase.     

Taxonomic Implications of Spore Fine Structure in Clostridium bifermentans

Thirty-five strains of Clostridium bifermentans were, in most part, culturally homogeneous by conventional taxonomic criteria but were heterogeneous with respect to spore fine structure. Fourteen of the strains produced spores with appendages, distributed among four distinct ultrastructural types. No consistent correlation existed between spore type and other variable properties of these strains. It is proposed, therefore, that these spore appendage-type strains be considered as "varieties" of C. bifermentans and that they should not be designated as new species.     

Sensitivity of Pseudomonas aeruginosa to Normal Serum and to Polymyxin

Strains of Pseudomonas aeruginosa isolated from clinical material were very variable in their sensitivity to the bactericidal action of normal serum mediated by the complement system. Fifty per cent killing end points ranged from 0.015 ml to greater than 0.4 ml. Most of the strains with relatively greater sensitivity to serum were isolated from patients with cystic fibrosis. Immunization of rabbits resulted in antisera with enhanced levels of bactericidal antibody, except with one strain which was resistant to the bactericidal action of normal serum and antiserum. When P. aeruginosa was cultivated at 41 C instead of at 37 C, it was significantly more sensitive to serum and to several antibiotics, thereby implicating fever as a host defense mechanism in Pseudomonas infections. In contrast to their heterogeneity to serum bactericidal activity, the strains were relatively homogeneous in their sensitivity to polymyxin, with no apparent association between their sensitivity to the two antimicrobial agents.