Radioiodinated surface proteins of rabbit trophectoderm cells

Summary We have previously used surface iodination to discriminate between the protein patterns of epithelial cell surfaces in uteri of rabbits receptive (Day 6.5) or nonreceptive (Day 4) to nidation (Ricketts et al. 1984). In this paper, we describe application of the same technique to the trophoblastic surface of rabbit blastocysts collected on the same days of pregnancy. Analysis of labelled proteins by polyacrylamide-gel electrophoresis under denaturing conditions did not reveal qualitative differences between the two days of pregnancy. Scanning densitometry was used to quantitate the area under each protein peak on an autoradiogram; these areas were used as variables in statistical analysis of the protein pattern of individual animals. Quantitative differences between the protein patterns of the two surfaces were detected by canonical variate analysis of the pattern of relative areas of labelled protein peaks. In proteins separated on 7.5% gels, this statistical analysis correctly assigned blastocysts from 8 out of 10 animals to one of two groups according to day of pregnancy. The discrimination was not statistically significant, however, in protein patterns on 12.5% gels, used to give better separation in the lower range of molecular weights. The same analysis in the uterus unequivocally separated the surface iodination patterns from these same days of pregnancy. Thus the changes detected by surface iodination appear to be less pronounced on the trophectoderm than on the uterine epithelium in relation to the time of ovoimplantation.     

Radioiodinated surface proteins of separated cell types from rabbit endometrium in relation to the time of implantation

To investigate changes in surface proteins of uterine cells in relation to the time of implantation, epithelial and stromal cells were isolated from rabbit endometrium and maintained in primary culture for 3 days. Surface-iodination of intact cells was carried out before and after culture, using immobilized Iodogen catalyst. The labeled proteins were analyzed by polyacrylamide gel electrophoresis, followed by autoradiography; peak areas were quantitated by scanning densitometry. Different gestational ages showed no marked qualitative differences in the surface-iodination patterns either of epithelial or stromal cells before or after culture. Quantitative differences between the surface-iodination pattern of epithelial cells from days 4 to 6.5 of pregnancy were revealed by canonical variate analysis of labeled peak areas. Values for individual rabbits clustered according to gestational age, with significant (p less than 0.05) separation of the clusters, although the discrimination was less pronounced for cultured than for freshly isolated cells. Changes involving increases in labeling of a protein of 38000 Mr in fresh cells, and decreases in a protein of 42000 Mr in cultured cells, were evident between day 4 and day 6.5. Thus changes in the surface-labeling pattern of uterine epithelial cells in relation to the time of receptivity for ovoimplantation can be distinguished. The functional significance of these changes remains to be elucidated.     

Impaired proteolytic processing of lysosomal N-acetyl-beta-hexosaminidase in cultured fibroblasts from patients with infantile generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from patients suffering with infantile generalized N-acetylneuraminic acid (NeuAc) storage disease accumulate free NeuAc in a population of lysosomes less dense than those observed in normal fibroblasts (1.035 vs. greater than 1.07 mean density), as assessed by the distribution of lysosomal enzyme activities and NeuAc on Percoll gradients after subcellular fractionation. In the present study, normal and affected fibroblasts were labeled with [35S]methionine, and cell homogenates or subcellular fractions from Percoll gradients were immunoprecipitated with polyclonal antibodies to lysosomal N-acetyl-beta-hexosaminidase (Hex); immunoprecipitated polypeptides were analyzed by SDS-polyacrylamide gel electrophoresis. The synthesis and initial processing of Hex polypeptides were comparable in normal and affected fibroblasts, but mature polypeptides were quantitatively localized in "buoyant" lysosomes of affected cells, along with Hex activity; moreover, mature alpha-chain of Hex was approximately 2 kDa larger than that observed in normal cells. The molecular weight difference was apparently due to impaired proteolytic processing of alpha-chain in affected fibroblasts, since treatment of immunoprecipitated alpha-chain from normal and affected cells with neuraminidase and endo-beta-N-acetylglucosaminidase H failed to resolve the molecular weight difference. The impaired processing was observed to be persistent (after a chase of up to 200 h), but had no apparent effect on the turnover or activity of Hex in affected fibroblasts. The observed proteolytic processing defect may be primary or secondary in infantile NeuAc storage disease.     

Effect of age and dietary restriction on protein synthesis by isolated kidney cells

The influence of age and food restriction on kidney protein synthesis was studied in Fischer F344 rats. The rate of total protein synthesis by suspensions of kidney cells declined 60% between 4 and 31 months of age. The rate of protein synthesis by kidney cells isolated from 19-month old rats fed a restricted diet (60% of diet consumed by rats fed ad libitum) was 45% higher than the rate of protein synthesis by kidney cells isolated from 19-month old rats fed ad libitum. The excretion of protein in the urine was measured to assess the effect of the age related decline in protein synthesis on kidney function. A dramatic increase in proteinuria was observed with increasing age, and rats fed the restricted diet excreted significantly less protein in the urine than rats fed ad libitum.     

Characterization in primary monolayer culture of separated cell types from rabbit endometrium

Epithelial cells and stromal cells of the rabbit endometrium were separated by successive enzymic digestion of the uterine mucosa. Isolated cell types were obtained in high yield, with good viability, and were maintained in monolayer cultures for up to 2 weeks. Epithelial cells in monolayers appeared as polygonal cells, displayed contact inhibition, and showed the presence of microvilli on the cell surface, with many desmosomes. Stromal cells grew rapidly to confluence, displayed overgrowth, and had a fibroblastic appearance with an absence of junctional complexes between cells. Indirect immunofluorescence showed uteroglobin on the surface of epithelial but not of stromal cells, and only epithelial cells secreted uteroglobin into the medium. These results confirm the identity of the cells and provide biochemical evidence for the epithelial cellular origin of uteroglobin. The method allows the culture of separate endometrial cell types, which retain their morphology and differentiated function in vitro.     

Endocytosis and the adaptive acid hydrolases in Tetrahymena pyriformis GL

Endocytosis of yeast cells by Tetrahymena pyriformis GL for a period of 2.5 h produced changes in cellular acid hydrolases. Acid phosphatase, acid deoxyribonuclease and acid proteinase activities were markedly increased, whereas there was a decrease in acid ribonuclease activity and little change in -glucosidase activity. These alterations do not appear to be due to any alteration in the rates of secretion of these enzymes into the milieu. Evidence is presented that the cellular enzyme increases found upon endocytosis of yeast reflect changes in lysosomal enzymes, because it was shown that the acid phosphatase activity increase resulted in an increased amount of latent enzyme within the cell. The results also support the idea that there are at least 3 distinct populations of lysosomes, in addition to phagolysosomes, present in Tetrahymena pyriformis GL, with different modes of formation. There appears to be a large excess of lysosomes, uncombined with phagosomes, present in these fed cells since latency averaged 66% in broken-cell preparations which contained very few intact phagolysosomes. The phagolysosomal acid phophatase activity cannot account for more than 34% of that present in the cell. The endocytosis of yeast in the presence of growth medium resulted in a marked drop in the rate of cell division as compared to cells growing in the growth medium alone. The results are discussed.     

Mechanism of Prophylaxis by Silver Compounds against Infection of Burns

To clarify tthe mechanism by which local application of silver compounds protects burns against infection, an ion-specific electrode was used to measùre the concentration of silver ions in solutions. By this method it was shown that in burn dressings silver ions were reduced to a very low level by precipitation as silver chloride. The antibacterial effect was found to depend on the availability of silver ions from solution in contact with precipitate. Between 10^-5 and 10^-6 molar silver nitrate solution in water was rapidly bactericidal. The minimal amount of silver nitrate causing inhibition of respiration of skin in tissue culture was about 25 times the minimal concentration of silver nitrate that inhibited growth of Pseudomonas aeruginosa.     

Increased intracellular Ca2+ is necessary for maximal expression of the proto-oncogene c-jun in the Jurkat T-cell line.

The time course and signal-transduction requirements for proto-oncogene c-jun expression in T-cells were investigated. Expression of c-jun mRNA was evident at 30 min after stimulation. Both the activation of Ca2+/phospholipid-dependent kinase as well as an increased intracellular free Ca2+ concentration were necessary for the maximal induction of c-jun mRNA and synthesis of Jun protein 1 h after stimulation.     

Membrane ultrafiltration: concentration of interleukin-3.

A pilot-plant-scale operation was used for studying membrane ultrafiltration and concentration of kiloliter quantities of the lymphokine interleukin-3 with a single set of membranes. Initial use of ammonium sulfate precipitation of interleukin-3 proved erratic in the recovery of biological activity and resulted in corrosion of the processing equipment. Membrane ultrafiltration proved to be effective in enabling control of the degree of concentration and predicting recovery of the biologically active protein.