Membrane effects of imidoesters in hereditary stomatocytosis

The marked increase in cation (Na⁺, K⁺) permeability that results in swollen, cup-shaped red cells in the hereditary stomatocytosis syndrome can be corrected in vitro with a bifunctional crosslinking reagent, dimethyl adipimidate (DMA). ⁴⁵Ca influx in intact RBC, ⁴⁵Ca efflux in red ghosts, and ⁴⁵Ca retention in red ghosts are normal and not influenced by DMA. Endocytosis in resealed red ghosts is strikingly impaired but becomes normal if cells are first treated with 2 mM DMA. Protein kinase mediated phosphorylation of membrane proteins by AT³²P–only 20–40% of normal control values in both short-term (5 min) and more extended (60 min) incubations–is not improved by DMA. After reaction of ¹⁴C-DMA with stomatocytes, radiolabel is found associated with phosphatidyl serine and phosphatidyl ethanolamine and is also widely distributed among membrane proteins. Cation permeability of stomatocytes is corrected at DMA concentrations (1 mM) that result in barely detectable crosslinking of aminophospholipids or proteins, suggesting that either crosslinking of a minor component present in only small quantities or intramolecular (rather than intermolecular) crosslinking is responsible for the permeability effects. DMA, whose maximal crosslinking dimension is 7.3–9 Å, is the most effective bifunctional imidoester of those tested. Shorter (dimethyl malonimidate) or longer (dimethylsuberimidate) reagents are either less effective than DMA or totally without effect.     

In situ fluorescence labeling of sheep lung microvascular endothelium

Summary Endothelial cells are intimately involved in a variety of biological processes such as inflammatory disorders, wound healing, and tumor invasion. The finding of endothelial heterogeneity in various tissues has led to major efforts to isolate and culture microvascular endothelial cells in human and animal tissue. In this report we have used phosphatidyl ethanolamine (PE)-labeled liposomes to fluorescently label the sheep lung microvasculature in situ. Using normotensive perfusion pressure, the PE-labeled liposomes did not extravasate into extravascular lung tissue. Mechanical and enzymatic digestion of the lung tissue demonstrated that the PE-labeled liposomes provided a stable label of the vascular lining cells during ex vivo processing. After digestion, the overwhelming majority of the fluorescent label appeared in cellular aggregates. Approximately 80% of these cells demonstrated an in vitro phenotype consistent with microvascular endothelium. A novel monoclonal antibody selective for sheep endothelial cells was developed to confirm the presence of lung endothelium in the fluorescently labeled cellular aggregates. We conclude that in situ fluorescence labeling of vascular lining cells provides an anatomic marker for relevant vascular lining cells and an opportunity to study these cells in vitro.     

Ecological cues, gestation length, and birth timing in African buffalo (Syncerus caffer)

We examined annual variation in the timing of conception andparturition in the African buffalo (Syncerus caffer) and thesynchrony of birth timing with resource cues, using 8 yearsof monthly birth, rainfall, and vegetation data, measured asNormalized Difference Vegetation Index (NDVI). Monthly birthshad the strongest significant correlations with NDVI and rainfalllevels 12 and 13 months in the past, respectively. In addition,the synchrony of current year births corresponds most stronglyto the synchrony of the previous year's NDVI distribution. Becausethe gestation period of buffalo has been estimated to be around11 months, these findings suggest that improved protein levels,occurring approximately a month after the first green flushof the wet season, are either a trigger for conception or conceptionhas evolved to be synchronous with correlated environmentalcues that ensure females enter a period of peak body conditionaround the time of conception and/or parturition. With a gestationperiod of approximately 340 days, parturition occurs to takeadvantage of the period when forage has its highest proteincontent. A comparative analysis of gestation periods withinthe subfamily Bovinae indicates that African buffalo have aprotracted gestation for their body size, which we suggest isan adaptation to their seasonal environment. We also found thatinterannual variation in the birth distribution suggests a degreeof plasticity in the date of conception, and variation in thenumber of calves born each year suggest further synchrony ata timescale longer than a single year.