Carboxyfluorescein as a probe for liposome-cell interactions effect of impurities,and purification of the dye

Impurities in 5(6)-carboxyfluorescein can affect phospholipid vesicle stability and apparent rates of carboxyfluorescein transfer into cells. Thorough purification and characterization of the dye are thus important to many applications with vesicles and/or cells. The dye can be purified by adsorption chromatography on a hydrophobic gel, following treatment with activated charcoal and precipitation from ethanol-water. The 5- and 6-carboxy-isomers can be separated from each other (though for most purposes it is not necessary to do so) by synthesis, crystallization, and hydrolysis of the diacetate derivatives. Purification is monitored by thin-layer and high pressure chromatography.     

Energetics of mice in stable and unstable social conditions: Evidence of an air-borne factor affecting metabolism

The metabolic rates of laboratory mice were compared in three conditions: isolated mice, mice paired together over six days (stable groups), and mice paired with strange partners daily (unstable groups). Stable pairs had 15% lower metabolic rates than either isolated or unstable pairs. In other experiments when two mice were placed in separate metabolic chambers and connected together via an air flow, the metabolic rate of the recipient in the series was 35% lower than the donor. The data suggest that a ‘factor’ produced by the donor mouse was passed via the air supply into the recipient's chamber.     

The plasma membrane of Saccharomyces cerevisiae. Isolation and some properties.

The isolation of Saccharomyces cerevisiae plasma membrane was carried out after hypotonic lysis of yeast protoplasts treated with concanavalin A by two independent methods: a, at low speed centrifugation and b, at high speed centrifugation in a density gradient. Several techniques (electron microscopic, enzymic, tagging, etc.) were used to ascertain the degree of purification of the plasma membranes obtained. The low speed centrifugation technique as compared with the other method gave a higher yield of plasma membranes with a similar degree of purification. Analysis of the yeast plasma membrane of normally growing cells by sodium dodecyl sulphate polyacrylamide gel electrophoresis showed at least 25 polypeptide bands. Twelve glycoprotein bands were also found, and their apparent molecular weights were determined. Treatment of the protoplasts with cycloheximide resulted in a significant decrease in the carbohydrate and protein content of the plasma membrane. The electrophoretic pattern of the plasma membrane of cycloheximide-treated cells showed a redistribution of the relative amounts of each protein band and a drastic reduction in the number of Schiff-positive bands. The isoelectric point of the most abundant proteins was low (pI 4) or lower than expected from previous data. A large part of the mannosyl transferase activity found in the cell (80%) was associated with the internal membranes, the remaining activity (20%) was located in the plasma membrane preparation. Part of the mannosyl transferase activity of the cells is located at the plasma membrane surface. Invertase (an external mannoprotein) is found in both the plasma and internal membranes, and as the specific activity dropped significantly following cycloheximide treatment of the cells, it is suggested that these membranes systems are the structures for the glycosylation of a precursor invertase and its subsequent release into the periplasmic space. Other transferase found in the plasma membrane preparation transfers glucose residues from UDPglucose to a poly(alpha(1 leads to 4) polymer identified as glycogen.     

Transdetermination of Drosophila imaginal discs cultured in vitro

Imaginal discs of Drosophila melanogaster undergo transdetermination when cultured in vivo in the abdominal cavity of adult female hosts. We report here that leg discs cultured in vitro, in a recently developed system, also undergo transdetermination. Whether cultured in vivo or in vitro, leg discs produce a similar range of specific transdetermined structures. Moreover, in comparison to discs cultured in vivo, the discs cultured in vitro exhibit a similar correlation between the amount of growth and the total frequency of transdetermination.     

Genetic Analysis of Two Allelic Temperature-Sensitive Mutants of DROSOPHILA MELANOGASTER Both of Which Are Zygotic and Maternal-Effect Lethals

After fertilization, the development of a zygote depends upon both gene products synthesized by its maternal parent and gene products synthesized by the zygote itself. To analyze genetically the relative contributions of these two sources of gene products, several laboratories have been isolating two classes of mutants of Drosophila melanogaster: maternal-effect lethals and zygotic lethals. This report concerns the analysis of two temperature-sensitive mutants, OX736hs and PC025hs, which were isolated as alleles of a small-disc mutant, l(3)1902. These alleles are not only zygotic lethals, but also maternal-effect lethals. They have temperature-sensitive periods during larval life and during oogenesis. Mutant larvae exposed continuously to restrictive temperature have small discs. One-or two-day exposures to the restrictive temperature administered during the third larval instar lead to a homeotic transformation of the midlegs and hindlegs to the pattern characteristic of the forelegs. Mutant females exposed to the restrictive temperature during oogenesis produce eggs that can develop until gastrulation, but do not hatch.--The existence of these mutants, and one that was recently described by another group, implies that there may be a class of genes, heretofore unrecognized, whose products are synthesized during oogenesis, are essential for embryogenesis and are also synthesized during larval stages within imaginal disc cells.     

Long chain fatty acid binding to human plasma albumin.

The binding of six physiologically important long chain fatty acids to defatted human plasma albumin was measured at 37 degrees in a calcium-free Krebs-Ringer phosphate buffer, pH 7.4. The data were analyzed in terms of multiple stepwise equilibria. With the saturated acids, the magnitude of the equilibrium (association) constants, Ki, increased as the chain length increased: laurate smaller than myristate smaller than palmitate smaller than stearate. Oleate was bound more tightly than stearate; by contrast, linoleate was bound less tightly than stearate. The equilibrium constants, K1 through K12, ranged from 2.4 times 10-6 - 3.5 times 10-3 m-1 for laurate to 2.6 times 10-8 - 3.5 times 10-5 m-1 for oleate. Successive values of Ki decrease for each of the acids, indicating that major cooperative binding effects do not occur over the physiological range of fatty acid concentrations. In no case could the Ki be segregated into distinct classes, suggesting that any grouping of albumin binding sites is somewhat arbitrary. The results were inconclusive concerning whether premicellar association of unbound fatty acid occurs. Although corrections for premicellar association produced very little change in the Ki values for myristate, they raised the Ki for palmitate and stearate by 300 to 700 per cent. A sigmoidal relationship was obtained when the logarithm of Ki was plotted against chain length for the saturated fatty acids containing 6 to 18 carbon atoms, indicating that the binding energy is not simply a statistical process dependent only on the fatty acid chain length. This selectivity that albumin contributes to the binding process may be due to varying degrees of configurational adaptability of its binding sites as the fatty acid increases in length.     

Age influence on the lipolytic effect of glucagon in geese.

The lipolytic effect of glucagon was measured in vitro with adipose tissue of "young" (4-8 wk) and "old" (over 1 yr) geese. The response of the young geese tissue was about twice that observed with tissue of old geese, for glucagon concentrations of 0.05, 0.5, and 5.0 mug/ml. Our estimates indicate that the number of adipose cells per g of adipose tissue of young geese was three times that of the old geese tissue. This suggests that the greater lipolytic response to glucagon, observed in young geese adipose tissue, may possibly be due to its greater cellularity, rather than to a greater lipolytic response of the individual adipocyte. The lipolytic effect of glucagon in vivo, for each of the doses between 1.0 and 20.0 mug/kg, was significantly greater in the old than in the young geese. The slope of the linear equation relating log10 of glucagon dose and elevation of plasma FFA 5 min after injection, was significantly greater for the old than for the young geese. In the goose, therefore, the influence of age on the adipokinetic effect of glucagon appears to be mediated by factors operating in the whole animal, more than by changes in the adipose cell itself. A slower removal rate of circulating FFA by the old geese, could be one of these factors.