Survival of corneal endothelium following exposure to a vitrification solution

Corneal endothelium, a monolayer of cells lining the inner surface of the cornea, is particularly susceptible to freezing injury. Ice formation damages the structural and functional integrity of the endothelium, and this results in a loss of corneal transparency. Instead of freezing, an alternative method of cryopreservation is vitrification, which avoids damage associated with ice formation. Vitrification at practicable cooling rates, however, requires exposure of tissues to very high concentrations of cryoprotectants, and this can cause damage through chemical toxicity and osmotic stress. The effects of a vitrification solution (VS1) containing 2.62 mol/liter (20.5%, w/v) dimethyl sulfoxide, 2.62 mol/liter (15.5%, w/v) acetamide, 1.32 mol/liter (10%, w/v) propane-1,2-diol, and 6% (w/v) polyethylene glycol were studied on corneal endothelium. Endothelial function was assessed by monitoring corneal thickness during 6 hr of perfusion at 35 degrees C with a Ringer solution supplemented with glutathione and adenosine. Various dilutions of the vitrification solution were introduced and removed in a stepwise manner to mitigate osmotic stress. Survival of endothelium after exposure to VS1 or a solution containing 90% of the cryoprotectant concentrations in VS1 (90% VS1) was dependent on the duration of exposure, the temperature of exposure, and the dilution protocol. The basic dilution protocol was performed at 25 degrees C: corneas were transferred from 90% VS1 or VS1 into 50% VS1 for 15 min, followed by 25% VS1 for 15 min and finally into isosmotic Ringer solution. Using this protocol, corneal endothelium survived exposure to 90% VS1 for 15 min at -5 degrees C, but 5 min in VS1 at -5 degrees C was harmful and resulted in some very large and misshapen endothelial cells. This damage was not ameliorated by using a sucrose dilution technique; but endothelial function was improved when the temperature of exposure to VS1 was reduced from -5 to -10 degrees C. Exposure to VS1 for 5 min at -5 degrees C was well tolerated, however, when the temperature of the first dilution step into 50% VS1 was reduced from 25 to 0 degree C. The large, misshapen cells were not observed under these conditions nor after exposure to VS1 at -10 degrees C. These results suggested that damage was the result of cryoprotectant toxicity rather than osmotic stress. Thus, corneal endothelium survived exposure to two solutions of cryoprotectants, namely, 90% VS1 and VS1, that were sufficiently concentrated to vitrify. Whether corneas can be cooled fast enough in these solutions to achieve vitrification and warmed fast enough to avoid devitrification remains to be determined.     

The composition of the leachate through cropped and uncropped soils in lysimeters compared with that of the rain

Summary The composition of the leachate from undisturbed monolith lysimeters cropped with white clover or meadow fescue or maintained bare was compared with that of the rain falling on them. No nitrogen fertilizer was applied only an initial dressing of phosphorus and potassium. The grass received much more nitrogen from the rain than it lost by leaching whereas the clover lost more than it received. Most of the leached nitrogen was NO₃-N - 92 per cent on the bare soil and 90 per cent on the clover. About 27lb nitrogen per acre (30 kg/ha) per year was drained from the actively growing clover sward rising to about 117lb N/acre/year (131 kg/ha) when the clover died or was removed. Only 2.3lb/ac (2.5 kg/ha) was drained from the actively growing grass sward. It was estimated that the clover fixed at least 270lb N/ac/year (303 kg/ha/year. The rates of leaching of potassium from a grass sward was about 1.7lb/ac/year (1.9 kg/ha) and 0.8 lb (0.9 kg) phosphorus. The quantities were similar for clover. The grass received from the rain more phosphorus and potassium than was leached but only 60 per cent of the calcium and 13 per cent of the magnesium, similar results being obtained with white clover. During the year of establishment of the grass sward there was evidence of loss of gaseous nitrogen (elemental and/or compound) from the soil: subsequently the nitrogen content of the soil slowly increased. Calcium loss from the bare soil with an average rainfall of 26″ (650 mm) was about 100 lb Ca/ac/year (112 kg/ha).     

Reversible trifluoroacetic acid-induced conformational changes in glycophorin as detected by proton nuclear magnetic resonance spectroscopy

High-field (270 MHz) ¹H-NMR has been employed to study the solution conformation of glycophorin A, a sialoglycoprotein which spans the human erythrocyte membrane. Glycophorin A is one of the most fully characterized integral membrane proteins known, making it an excellent model for the study of membrane-bound proteins. This protein consists of three distinct domains: a glycosylated extracellular N-terminus, a hydrophobic intramembranous segment, and a polar cytoplasmic C-terminus. These domains contain aromatic residues which serve as convenient ¹H-NMR conformational probes. The aromatic region of the NMR spectrum of glycophorin A in ²H₂O shows single, well-resolved His and Tyr resonances. No resonances are observed, however, for the Phe residues which are located in or near the hydrophobic domain. These observations suggest that considerable heterogeneity with respect to segmental motions exists within the protein. This is consistent with circular dichroism data showing the intramembranous segment to be completely helical with the extremities of the protein being predominantly random coils. The helix of the hydrophrobic domain is remarkably resistant to conventional denaturing conditions including variations in pH, and temperature, and treatment with guanidine hydrochloride. However, in trifluoroacetic acid, which strongly solvates peptide backbones, there is extensive reversible unfolding of the helical structure as evidenced by the appearance of Phe resonances. Solvent titration experiments indicate that approximately a 1 : 1 volume ratio of trifluoroacetic acid to ²H₂O is required to initiate unfolding of the helix.     

Predators and planariid competitors of the triclad Phagocata vitta (Dugés)

When Phagocata vitta, Crenobia alpina and Polycelis felina were exposed separately to each of seventeen potential invertebrate predators in the laboratory, only two stonefly species, Dinocras cephalotes and Perlodes microcephala, fed on the three triclad species, whilst the trichopteran Rhyacophila dorsalis ate the last two triclads. On exposing pairs of triclad species to D. cephalotes, significantly more P. felina than Ph. vitta were consumed, whereas similar numbers were eaten in each of the other two triclad combinations. Cannibalism and interspecific predation by triclad species were not observed. It is concluded that predation is unlikely to have a major influence in determining the observed distribution and abundance of triclad species in a Welsh study stream which harbours low numbers of effective predators.The de Wit model of competition was used to examine the competitive relationships between Ph. vitta, and C. alpina and P. felina, using chironomids or tubificid worms as food. In mixed cultures of Ph. vitta and P. felina fed on tubificids a stable equilibrium existed within the range of relative densities used in the experiments, whereas Ph. vitta was competitively superior to C. alpina in cultures fed on each of the food types, and to P. felina fed on chironomids. However, in theory, an equilibrium could occur when 10 or 6–7 times as many Ph. vitta as P. felina and C. alpina respectively are in the culture, when intraspecific rather than interspecific competition would become more important. Where the three triclad species coexist in the Welsh study stream, they are in similar numbers. This could imply that food is not limiting, with no consequent interspecific competition, or that the laboratory experiments were too simplistic to allow any interpretation of the field situation.     

Effects of osmotic stress on rabbit corneal endothelium

The effects of osmotic stress on corneal endothelium were investigated by exposing rabbit corneas to anisosmotic conditions, and then perfusing the corneas with isosmotic glutathione bicarbonate Ringer solution for 4 hr at 35 degrees C. During the perfusion, endothelial function was assessed by measuring corneal thickness with a specular microscope. After perfusion, the corneas were prepared for scanning and transmission electron microscopy. Endothelial ultrastructure and function were well maintained after exposure to a wide range of osmolality (0.12-2.7 osmol/kg), but this tolerance of osmotic stress was dependent both on the duration and the temperature of exposure to the anisosmotic conditions. Exposure to an osmolality of 2.7 osmol/kg for 15 min at 23 or 37 degrees C resulted in gross damage to the endothelium when the hyperosmotic agent was sodium chloride. This damage was not the result of increased osmolality per se nor cellular shrinkage because the endothelium tolerated exposure to a sucrose solution of the same osmolality for 15 min at 37 degrees C. The detrimental effect of sodium chloride, however, was mitigated by reducing the temperature of exposure to 0 degrees C or reducing the duration of exposure to 5 min. These results suggest that endothelial cells become more tolerant of high electrolyte concentrations with reducing temperature, and this could be an important factor in the survival of the endothelium in corneal cryopreservation. The results also help to define the limits of osmotic shrinkage and swelling tolerated by endothelial cells. This will be of value in overcoming the detrimental osmotic effects associated with the addition and, in particular, the removal of cryoprotectants.     

A soluble form of the interleukin 4 receptor in biological fluids

Murine biological fluids and murine cell culture supernatants were analyzed for the presence of soluble murine interleukin 4 receptor (sIL4R) with the use of two monoclonal antibodies directed against the receptor. Mouse urine, serum, ascitic fluid, and cell culture supernatants contained varying levels of immunoreactive protein. All of the immunoreactive protein possessed interleukin 4 (IL 4) binding activity. Following partial purification of ascitic fluid a protein was isolated that binds IL 4 with high affinity. This data is consistent with the fact that murine biological fluids contain a soluble version of the murine IL 4 receptor that arises via secretion of the soluble receptor and/or via shedding of the extracellular portion of the full-length receptor from the cell surface.