Promotion and inhibition of vesicle fusion by polylysine

Polylysine induced rapid aggregation of large unilamellar vesicles composed of phosphatidylcholine-cardiolipin (1:1 molar ratio) but not their fusion. Application of the terbium-dipicolinic acid fusion assay showed that addition of polylysine at nanomolar concentrations enabled a significant lowering of the Ca2+ threshold concentration for vesicle fusion from 9 to 1 mM. Analysis of the kinetics of fusion with a mass-action kinetic model showed that polylysine enhanced significantly the rate of aggregation but affected only slightly the rate of fusion per se. Maximal enhancement of overall fusion rates occurred at a charge ratio (polylysine/cardiolipin) of about 0.5. At larger polylysine concentrations, e.g., at charge ratios greater than 3, polylysine inhibited vesicle fusion.     

The retinoblastoma gene and cell growth control

Rare diseases often provide unique and fascinating insights into the workings of biological machinery. Retinoblastoma is a good example. This hereditary disease occurs rarely (in only one out of 20,000 children), yet it opens a 'window' into the mechanisms that sit at the very center of growth control in cells throughout the body. Children who survive bilateral retinoblastoma often have offspring who are similarly affected. Indeed, transmission of the disease suggests the actions of a simple dominant Mendelian allele of high penetrance. Three years ago the wild-type allele of this gene (termed Rb) was isolated by molecular cloning. With this success has come a rich harvest of information on tumor pathogenesis and the molecular biology of cellular growth control.     

Functional alterations of macrophages in autoimmune MRL-lpr/lpr mice

To assess the role of macrophages (MAC) in the pathogenesis of systemic lupus erythematosus, we investigated functional aspects of peritoneal MAC obtained from autoimmune MRL/MpJ-lpr/lpr (MRL-lpr) mice. MRL-lpr and control C3H/HeN MAC were obtained from untreated mice or mice injected i.p. with 1 ml of 10% sterile peptone 3 days before cell harvest. MRL-lpr mice had significantly more peritoneal cells (MAC and lymphocytes) than did control mice. In endotoxin-free conditions, MRL-lpr MAC were similar to C3H/HeN MAC in their baseline, and IFN-gamma and/or LPS enhanced cytolysis of 3T12 fibrosarcoma tumor cells. Compared with C3H/HeN MAC, MRL-lpr MAC had a significant increase in antibody-dependent cellular cytotoxicity activity against sheep erythrocytes. This enhanced activity was not accompanied by a similar increase in adherence and/or phagocytosis of the same targets. Finally, in response to phorbol myristate acetate stimulation, both resident and peptone-induced MAC from MRL-lpr mice produced significantly more hydrogen peroxide than did those from control mice. These results indicate that MAC from MRL-lpr mice display features of selective "activation", and suggest that MAC or their products may play a role in the pathogenesis of inflammatory disorders seen in autoimmune diseases.     

Sperm-macrophage interaction in the mouse: a quantitative assay in vitro using 111indium oxine-labeled sperm

The role of reproductive tract macrophages in contraception and reproductive failure has become widely recognized. However, in vitro analysis of sperm phagocytosis by macrophages has relied upon a semi-quantitative method of sperm counting that is of limited accuracy and reproducibility. We have developed an assay using murine sperm labeled with 111indium oxine, and results indicate the labeling to be rapid and efficient. Incorporation of 111indium into sperm increased the dose and sperm concentration and reached 90% maximal uptake after 15 min incubation, with maximal uptake occurring at 30 min. No decrease in sperm motility was noted with levels of oxine in excess of those required for significant labeling. Maximal labeling efficiency occurred in phosphate-buffered saline (PBS), with Dulbecco's modified Eagle's medium (DMEM) + 10% adult bovine serum (ABS) producing significantly less uptake. Label dissociation was detectable in PBS at room temperature, but at 37 degrees C in DMEM + 10% ABS, loss of label occurred at a rate of 23.5%/h. Addition of labeled sperm to murine macrophage monolayers under optimal conditions resulted in uptake of 111indium by macrophages, while free label was unincorporated. Results indicated assay specificity for macrophage-limited uptake, with insignificant label uptake by nonphagocytic murine fibroblasts and better sensitivity than sperm counting. Macrophages from Bacillus Calmette-Guerin (BCG)-infected mice resulted in a decrease in sperm uptake. Female macrophages showed greater capacity for sperm uptake than those of the male mouse. These initial studies demonstrated the utility of this model system in enhancing the understanding of sperm-macrophage interaction in the female reproductive tract.     

The effects of a topical PGE2 analogue on global flap ischemia in rats

The present study evaluated the ability of DHV-PGE2ME, a topically effective 16-vinyl prostaglandin E2 analogue, to improve the tolerance of skin flaps to a period of ischemia. DHV-PGE2ME and placebo were applied to bilateral island flaps on 70 anesthetized rats; then the vascular pedicle of each flap was clamped for 10 hours. Treated flaps evidenced significantly better reperfusion, as documented by quantification of fluorescein dye delivery at 90 minutes after clamp release, and they had significantly greater ultimate viability (p less than 0.05, by ANOVA). While less than 3 percent of untreated flaps survived, those treated with 1.75 and 17.5 microgram/cm2 of drug evidenced 76 and 86 percent survival, respectively. Treatment of a given flap did not affect its contralateral mate, since there was no evidence of a systemic effect. Especially since its effect can be limited to the site of application, DHV-PGE2ME should be valuable for the treatment of compromised perfusion in a variety of settings.     

Cellular regulation of iron assimilation

Cells of plants, most microorganisms, and animals require well-defined amounts of iron for survival, replication, and differentiation. The metal is an important component of such processes as synthesis of DNA, RNA, and chlorophyll; electron transport; oxygen metabolism; and nitrogen fixation. Because of the insolubility of iron in aerobic environments at neutral and alkaline pH values, cells have had to devise specific strategies to assimilate the metal. These include (1) development of systems for reducing ferric ions to the more soluble ferrous ions at the cell surface, (2) employment of small carrier molecules (termed siderophores) that have high affinity for ferric ions and receptor proteins for the ferrated molecules, and (3) use of transferrin and other proteins that can transport ferric ions. Excessive amounts of iron are toxic, however, and intracellular storage capacity is limited and efflux mechanisms generally are lacking. Thus, cells have had to develop methods of preventing over-accumulation of the metal. These include use of (1) oxygen to convert ferrous to ferric ions, (2) small molecules that can bind ferrous ions, termed siderophraxes, and (3) proteins that, when combined with ferrous ions, repress the expression of iron transport genes. Often, one organism can prevent growth of neighbors by restricting their access to iron. In other cases, cells assist each other by sharing iron acquisition systems or by restricting influx of excess iron. Homeostatic control of other essential trace metals also is required for optimal cell function. Nevertheless, since iron thus far has received most attention, it serves as the model of mineral metabolism. Moreover, many of the observations made on control of iron metabolism suggest possible applications in prevention and management of plant and animal infections as well as of neoplastic diseases, arthropathy, and cardiomyopathy. This review will focus on (1) problems at the cellular level of iron acquisition, storage, and exclusion; and (2) the strategies devised by cells of plants, microorganisms, and animals to solve these problems.