Binding of simple carbohydrates and some N-acetyllactosamine-containing oligosaccharides to Erythrina cristagalli agglutinin as followed with a fluorescent indicator ligand

Erythrina cristagalli agglutinin, a dimeric lectin [J. L. Iglesias, et al. (1982) Eur. J. Biochem.123, 247–252] was shown by equilibrium dialysis to be bivalent for 4-methylumbelliferyl-β-d-galactoside. Upon binding to the lectin, this ligand showed a difference absorption spectrum with two maxima (at 322 and 336 nm) of equal intensity (Δ? = 1.2 × 10³m^?1 cm^?1). A similar spectrum with a comparable value of Δ? was obtained with 4-methylumbelliferyl-N-acetyl-β-d-galactosaminide. Binding of methyl-α-d-galactoside, lactose, and N-acetyllactosamine all produced small but equally intense protein difference spectra with a maximum (Δ? = 2.8 × 10² M^?1 cm^?1) at 291.6 nm. Upon binding of N-dansyl-d-galactosamine to the lectin, there was a fivefold increase in fluorescence intensity of this ligand. The association constant for N-dansyl-d-galactosamine was caused by a very favorable ΔS° of the dansyl group without affecting the strictly carbohydrate-specific character of binding. N-Dansyl-d-galactosamine was employed as a fluorescent indicator ligand in substitution titrations. This involved the use of simple carbohydrates, N-acetyllactosamine, and oligosaccharides which occur in the carbohydrate units of N-glycoproteins; the latter were Gal(β → 4)GlcNAc(β1 → 2)Man, Gal(β1 → 4)GlcNAc(β1 → 6)Man, and Gal(β1 → 4)GlcNAc(β1 → 6)[Gal(β1 → 4)GlcNAc(β1 → 2)]Man. The titrations were performed at two temperatures to determine the thermodynamic parameters. In the series N-acetyl-d-galactosamine, methyl-α-d-galactoside, and lactose, ?ΔH° increased from 24 to 41 kJ mol^?1; it increased further for N-acetyllactosamine and then remained unchanged for the N-acetyllactosamine-containing oligosaccharides (55 ± 1 kJ mol^?1). This indicated that the site specifically accommodated the disaccharide structure with an important contribution of the 2-acetamido group in the penultimate sugar. Beyond this, no additional contacts seemed to be formed. This conclusion also followed from considerations of ΔS° values which became more unfavorable in the above series (?23 to ?101 ± 4 J mol^?1 K^?1); the most negative value of ΔS° was observed with N-acetyllactosamine and the three N-acetyllactosamine-containing oligosaccharides.     

Interaction of peanut agglutinin,a lectin specific for nonreducing terminal d-galactosyl residues,with embryonal carcinoma cells

Peanut agglutinin (PNA), a lectin specific for terminal d-galactosyl residues, was found to react with embryonal carcinoma cells, but not with their differentiated derivatives. Receptors for PNA were detectable at the surface of all cells of the quasinullipotent F9 line and on only 50% of the multipotent PCC3/A/1 line. The fraction of the PCC3/A/1 population which expresses the F9 antigen was found to be included in the subpopulation carrying the PNA receptors. PNA⁺ and PNA^? subpopulations of PCC3/A/1 were separated by a PNA-mediated reversible agglutination of PNA⁺ cells with rabbit erythrocytes. These subpopulations were essentially F9⁺ and F9^?, respectively.     

Polyamines in trypanosomatids.

Polyamines were determined by n-butanol extraction and thin-layer chromatography in four trypanosomatids: Trypanosoma brucei (rat infection) and cultures of Crithidia fasciculata, Leptomonas sp., and Trypanosoma mega. All had putrescine and spermidine but no detectable spermine. Putrescine and spermidine levels were quantitated for extracts of leptomonas during the normal growth cycle. Spermidine values peaked 18 h before peak cell populations. Spermidine-putrescine ratios for all organisms were related to the presumed phylogeny of the group.     

Choice of function for mortality analysis: Effective forecasting depends on a minimum parameter representation

For nearly two centuries actuaries, statisticians and demographers have sought a parameter space in which the mortality pattern of a population could be located, that would be linked to the larger space of age-specific rates by an analytical formula. Gompertz, Makeham, Brillinger, Wolfenden, Pollard and others have made contributions to this end. Model or reference tables perform a similar task, usually with fewer parameters, but are less easily manipulated. A common means of reducing dimensionality is to map the original space of mortality rates in five-year groups on a straight line in which are located the expectations of life at age zero (ė₀). A small-space representation is advantageous for filling gaps in data. If the only fact known about a country is the fraction of girls who have a living mother, then a one-dimensional set can be indexed on this to provide the full detail of mortality, assuming the unknown mortality is part of that set. For forecasting one would like the succession of life tables for a given population over past times to be representable by points moving in a simple way through a parameter space—ideally in a straight line—over a succession of calendar years. The simpler the curve, the more realistic is likely to be its projection into the future. The Brass relational method provides a trajectory well suited to extrapolation in a space of only two parameters. Some useful extensions of this have recently been devised.     

Evaluation of storage conditions for refrigerated rabbit skin

An evaluation of various refrigerated (4 °C) storage solutions and conditions was conducted using rabbit skin. Two in vitro methods to assay skin viability are presented: one which directly measures basal cell viability and one which assesses the skin's ability to grow in culture following storage. The superiority of storage in nutrient medium supplemented with fetal bovine serum over conventional storage in saline is clearly demonstrated. Storage in nutrient medium with 10% fetal calf serum resulted in basal cell viabilities which were over 30% higher than viabilities of skin stored by conventional methods in saline. Skin stored in saline failed to grow in culture, while 100% of the cultures of skin stored in medium plus fetal calf serum grew. Although addition of fetal calf serum to the saline improved the basal cell viability, growth in culture occurred only when the skin was stored in a capped tube. Skin stored in medium without serum gave viability results which were not significantly different from the unstored control, but growth rates in culture did differ significantly from the control values. Our study shows that the viability of rabbit skin and its ability to grow in vitro are depressed when the tissue is maintained at 4 °C in saline or in petri dishes, and optimal when refrigerated in nutrient medium supplemented with FBS in a sealed tube.     

Voltage clamp analysis of embryonic heart cell aggregates

The double-microelectrode voltage clamp technique was applied to small spheroidal aggregates of heart cells from 7-d chick embryos. A third intracellular electrode was sometimes used to monitor spatial homogeneity. On average, aggregates were found to deviate from isopotentiality by 12% during the first 3--5 ms of large depolarizing voltage steps, when inward current was maximal, and by less than 3% thereafter. Two components of inward current were recorded: (a) a fast, transient current associated with the rapid upstroke of the action potential, which was abolished by tetrodotoxin (TTX); and (b) a slower inward current related to the plateau, which was not affected by TTX but was blocked by D600. The magnitudes, kinetics, and voltage dependence of these two inward currents and a delayed outward current were similar to those reported for adult cardiac preparations. From a holding potential of -60 mV, the peak fast component at the point of maximal activation (-20 mV) was -185 microA/cm2. This value was about seven times greater than the maximal slow component which peaked at 0 mV. The ratio of rate constants for the decay of the two currents was between 10:1 and 30:1.