PubMed Health⌕ Search

Biomedical subjects

J S Amenta

Publications and source records attributed to J S Amenta.

At least 37 records · Page 2Linked to original sources
PubMed1984

Deficient lung surfactant apoproteins in amniotic fluid with mature phospholipid profile from diabetic pregnancies.

An enzyme-linked immunoassay to quantitate lung surfactant apoproteins (15 to 250 ng/ml) in human amniotic fluid is described. The immunoassay was used to quantify lung surfactant in 72 samples of amniotic fluid, for which lecithin/sphingomyelin (L/S) ratios, lecithin and phosphatidylglycerol concentrations, and foam stability indices were also available. The results obtained with the immunoassay were in general agreement with those of the other methods. Measurement of the apoproteins, however, may be a better predictor of fetal lung immaturity and of respiratory distress syndrome than the L/S ratio and the concentration of lecithin. This conclusion is based on the data obtained in the analyses of samples of amniotic fluid from four diabetic and five nondiabetic pregnancies, the infants of which developed respiratory distress. In all cases, the apoprotein concentration was less than 2.1 micrograms/ml, which indicated fetal lung immaturity. In six of these cases (one diabetic and five nondiabetic pregnancies), lung immaturity was also predicted on the basis of other tests. However, in three other cases of diabetic pregnancy, the L/S ratio and lecithin concentration falsely indicated lung maturity. In addition to its being an effective predictor of fetal lung maturity in diabetic, as well as nondiabetic, pregnancies, the immunoassay is better suited for clinical use because of its high specificity, sensitivity, and ease of performance.

Amniotic Fluid↗
PubMed1984

Degradative fate of transplanted proteins.

The majority of cell proteins are non-cytosolic and are found in specific extracytosolic cytomorphological sites. Rat liver mitochondria and outer mitochondrial membrane (OMM) vesicles were transplanted homologously into rat hepatocytes and heterologously into rat hepatoma (HTC) cells by polyethylene glycol-mediated organelle--cell or OMM vesicle-cell fusion. The subsequent destructive fate of these non-cytosolic proteins was studied. During culture of hepatocyte monolayers in conditions which give in vivo catabolic rates, the transplanted organelle proteins and monoamine oxidase were degraded at rates similar to in vivo rates, although the transplanted material was not found in the hepatocyte mitochondria. Degradation was preceded by internalization (1-6 h) of the transplanted material and its translocation to a perinuclear, vesicular cytoplasmic position. Prevention of translocation by the disruption of the cytoskeleton inhibited subsequent degradation. In contrast, rat OMM heterologously transplanted into HTC cells was patched, capped and internalized into 'unique' vesicles and degraded 2.5 times faster than in hepatocytes. In both hepatocytes and HTC cells mitochondrial protein degradation was partially susceptible to lysosomotropic agents. The results are discussed in terms of a protein turnover cycle which attempts to coordinate the biochemistry and cell biology of protein synthesis and degradation in eukaryotic cells.

Animals↗
PubMed1984

Sources of error in estimating radioactivity in protein from cell cultures by liquid scintillation counting.

In this study, several common sample preparation techniques for liquid scintillation counting were critically reviewed. It has been shown that techniques such as NaOH or formic acid solubilization of trichloroacetic acid (TCA)-precipitated proteins led to underestimation of the radioactivity by 20-40%; this loss was not corrected by either internal or external standardization. Hydrolysis of the proteins with 6 N HCl or Pronase significantly increased the recovery of the labeled proteins. Also, 10% of the labeled cell proteins remained on the dish when cells were scraped into buffer; these labelled proteins could be recovered either by in situ hydrolysis with Pronase or by solubilization and scraping in 0.3 N NaOH. These techniques increased the recovered radioactivity by 50-60%, allowing quantitative measurements to be made over a 3-day chase period. A possible mechanism and the implications of this observation were discussed.

Animals↗
PubMed1983

Amniotic fluid lecithin, phosphatidylglycerol, L/S ratio, and foam stability test in predicting respiratory distress in the newborn.

Amniotic fluid phospholipids (lecithin, sphingomyelin, phosphatidylglycerol) were extracted and separated by the method of Gluck and associates. In addition, these lipids were quantified against standards that were concurrently developed in three adjacent thin layer chromatography (TLC) channels. A log-log transformation of the reflectance and concentration values provided rectilinear plots for quantification. A foam stability assay (FS) of 0.48 or greater was associated with high values of L/S ratio and lecithin concentration, and values of 0.44 or less almost always were associated with low values. Lecithin values of 12 micrograms/mL or more were associated with L/ S ratios of 2.0 or greater. Phosphatidylglycerol was detected consistently only when lecithin levels were 40 micrograms/mL or greater and L/S ratios 4.0 or greater. Case reviews indicated that: (1) FS test of 0.48 almost always was associated with pulmonary maturity in the newborn; (2) that a pulmonary maturity index combining the lecithin concentration and L/S ratio was a better predictor of pulmonary maturity in the newborn then either one alone; (3) that phosphatidylglycerol added little to this discrimination; and (4) that these assays showed no significant interpretative differences between diabetic and non-diabetic mothers.

Amniotic Fluid↗
PubMed1982

Control of cell protein catabolism in rat liver. Effects of starvation and administration of cycloheximide.

1. The loss of liver protein occurring in rats starved for 24 h was largely prevented by the administration of repeated doses of cycloheximide, an inhibitor of protein synthesis. Similar effects were produced on tubulin, a 'fixed' liver protein. 2. Starvation accelerated, whereas cycloheximide markedly lowered, the rate of protein radioactivity decay after labelling with [3H]valine or [14C]bicarbonate, indicating that changes in catabolic rates played an important role in the above regulations of liver protein mass. 3. The total activity of several lysosomal hydrolases showed little change in livers of starved rats, but a marked progressive decline developed after the administration of cycloheximide, particularly in the activities of cathepsins B, D and L as well as acid ribonuclease. There was no evidence that these changes might be due to endogenous inhibitors (at least for cathepsin B activity, which fell to less than 30% of the control values) or enzyme leakage into the bloodstream; rather, plasma beta-galactosidase and beta-N-acetylglucosaminidase activities fell progressively during the cycloheximide treatment. 4. Endogenous proteolytic rates, measured in vitro by incubating subcellular preparations from livers prelabelled in vivo with [3H]valine, were markedly decreased in cycloheximide-treated animals. 5. The osmotic fragility of hepatic lysosomes, appreciably enhanced in starved animals, after cycloheximide treatment was found to be even lower than in fed controls. 6. The present data are consistent with the view that in starved animals the loss of liver protein is mostly accounted for by increased breakdown, due, in part at least, to enhanced autophagocytosis. 7. Cycloheximide largely counteracted these effects of starvation, altering the liver from being 'poised' in a proteolytic direction to a protein-sparing condition. The present data suggest that, besides suppression of the autophagic processes, a decrease in the lysosomal proteolytic enzyme system may also play a role in this regulation, and they seem to provide further circumstantial evidence for the existence of co-ordinating mechanisms between protein synthesis and degradation.

Animals↗
PubMed1982

Liquid-chromatographic estimation of saturated phospholipid palmitate in amniotic fluid compared with a thin-layer chromatographic method for acetone-precipitated lecithin.

The "high-performance" liquid-chromatographic method we report for measuring palmitate derived from saturated phospholipids in amniotic fluid is sensitive and precise and possesses a wide dynamic range. Palmitate from nonsurfactant sources is largely excluded. The procedure involves oxidation of the chloroform/methanol-extracted lipids with osmium tetroxide, precipitation of the resulting phosphatides with cold acetone, mild alkaline hydrolysis, derivatization of the liberated fatty acids to phenacyl esters, and reversed-phase chromatography. The method is no less convenient or more time-consuming than measurement of the lecithin/sphingomyelin ratio by thin-layer chromatography. The clinical usefulness of the procedure is indicated by results of a correlative study of 26 amniotic fluids evaluated by "lung profile" tests in a second laboratory. A value for dipalmitoyl lecithin of 13 mg/L or greater indicates fetal lung maturity.

Acetone↗
PubMed1980

Evidence of heterogeneity of protein-turnover states in cultured cells.

Previous studies on L-cell cultures [Amenta & Sargus (1979) Biochem. J. 182, 847--859] have suggested: (a) that degradation of slow-turnover proteins occurs in a distinct cell state (D-state); (b) that cells randomly enter the D-state with a first-order transition constant, rapidly degrade cell protein, and return to a quiescent G0-state. In the present study we have tested the hypothesis that the putative D-state exists as a substate within A-state (non-replicating) fibroblasts. Rat-embryo fibroblasts were prelabelled with [14C]leucine and [3H]thymidine, 'chased' for 24 h, and then placed in fresh growth medium containing either vinblastine (10 microM) or colchicine (25 microM) for three successive 24 h periods. Cells trapped in mitosis were separated from the residual non-replicating cells and rates of protein synthesis, degradation and net accumulation were measured in both populations. We observed that significant protein degradation occurred only in the non-replicating population, although both populations showed equally high rates of protein synthesis induced by fresh growth medium. These data support the hypothesis that degradation of slow-turnover protein is heterogeneous, occurring only in A-state cells. A model that proposes a separate D-state within G0-phase successfully accounts for these observations and previous reports on this cell line [Amenta, Sargus & Baccino (1978) J. Cell. Physiol. 97, 267--283] showing no differences in degradation of the slow-turnover protein pool in growth-stimulated and stationary-phase fibroblast cultures.

Animals↗
PubMed1980

Role of lysosomes in protein turnover: catch-up proteolysis after release from NH4Cl inhibition.

Cultured rat embryo fibroblasts, when placed in media with 10% serum containing 20 mM NH4Cl, show an inhibition of protein degradation and, concurrently, an accumulation of numerous, large vacuoles, partially filled with cellular debris. Cells placed in a serum-free media exhibit an enhanced degradation of cell protein, which is also inhibited by NH4Cl. When these cells are removed from media containing NH4Cl and placed in fresh media, the material accumulated in these vacuoles is rapidly and quantitatively released to the media in both an acid-soluble and acid-insoluble from. NH4Cl inhibits rapidly and specifically the lysosomal proteolytic mechanism, and is without effect on the basal turnover mechanism. The lysosomal proteolytic mechanism accounts for approximately 25% of protein turnover, and, at least in low density cultures, can be stimulated to levels which account for more than half of the protein turnover in the cell. The major pathway for the degradation of fast turnover proteins appears to be separate from lysosomal mechanism.

Ammonium Chloride↗
PubMed1980

Protein synthesis and degradation in growth regulation in rat embryo fibroblasts: role of fast-turnover and slow-turnover protein.

Cultured rat embryo fibroblasts, when stimulated to grow by the addition of fresh medium containing 10% serum, showed an increase in synthesis of slow-turnover proteins while maintaining a uniform degradation rate for these proteins. Slow-turnover proteins with a half-life of 2.4 days accounted for approximately 95% of the cell protein, while the remaining protein could be described in terms of two fast-turnover pools. When we labeled cells to limiting levels over a period of 4 days, the fast-turnover pools became undetectable; with 2-hour labeling periods, however, 25% of the label entered the fast-turnover pools. Fibroblasts, stimulated to grow by fresh growth medium, showed proportionate and coordinate increases in synthesis of both fast-turnover and slow-turnover proteins during the growth period, both returning to baseline levels on reaching the new steady state. No changes could be detected in degradation of either pool during growth. Fibroblasts placed in a serum-free medium showed a decrease in cellular protein and an increased degradation of slow-turnover proteins, while degradation of fast-turnover proteins remained unchanged. We conclude that the slow-turnover protein pool forms the bulk of the cell proteins and turns over at a fairly constant rate. Growth stimulation is effected almost entirely by stimulation of protein synthesis in this pool, while decreasing cellular protein growth is a result of enhanced degradation within this pool.

Animals↗
PubMed1979

Mechanisms of protein degradation in growing and non-growing L-cell cultures.

L-cells prelabelled with [14C]leucine and [3H]thymidine were placed in either fresh growth medium (minimal essential medium with 10% serum) or stepdown medium (minimal essential medium) for 3 days. The 14C/3H ratio remained constant in the growing cultures and decreased in the stationary-phase cultures, indicating no protein turnover in growing cultures and a degradative rate of 0.6%/h in the stationary-phase cultures. Media analysis, however, indicated that 14C-labelled proteins were being degraded at approx. 1.2%/h in growing cultures and 1.7%/h in stationary-phase cultures. Additional studies indicated that a subpopulation of L-cells in the monolayer, comprising approx. 20--30% of the total, were lost in the original processing procedure. Experiments in which recoveries approached 100% by fixation of the monolayer in situ indicated that a protein-degrading subpopulation accounted for all the observed proteolysis in the growing cultures. Proteolysis in these cultures was only partially inhibited with NH4Cl, indicating that only a small part of the protein degradation was occurring in an activated lysosomal-autophagic system. NaF produced a more effective inhibition of proteolysis, but we were not able to distinguish whether this effect was on an ATP-requiring basal-turnover mechanism or a direct effect on unregulated activity of proteinases in the cell hyaloplasm. However, NH4Cl inhibited the proteolysis induced when cells were placed in stepdown medium, suggesting that the induced proteolysis was occurring via the autophagic system. We conclude that L-cells exist in at least two states with respect to protein degradation: (a) a subpopulation that is actively replicating and does not degrade cellular proteins, and (b) a second subpopulation of cells, derived from the preceding one, which degraded most of their labelled proteins, are not capable of further replication, and are not sedimented in an iso-osmotic EDTA buffer solution. In addition, proliferating L-cells, when placed in stepdown medium, begin to degrade cell protein through a mechanism involving autophagolysosomes.

Ammonium Chloride↗
PubMed1978

Role of the vacuolar apparatus in augmented protein degradation in cultured fibroblasts.

Rat embryo fibroblasts, grown in Eagle's MEM with 10% serum, showed a rapid increase in autophagic vacuoles when placed in MEM with 0-1% serum. Concurrent with this response, degradation of cellular proteins showed a 2-fold increase. We did not find any increases in cathepsin D, beta-glucuronidase, beta-galactosidase, and beta-glucosidase, or proteolytic activity of cell homogenates at pH 3.7 towards endogenous substrates. Homogenates prepared in 250 mM sucrose at pH 7.0 showed a 40% increase in protein breakdown. These data support the hypothesis that the induced increase in proteolysis, characteristic of cells placed in a nutritionally deficient medium, is effected by an activated vacuolar apparatus (lysosomes and autophagic vacuoles). We suggest, however, that this mechanism is distinct from normal protein turnover in the cell, but can be rapidly induced by appropriate alterations in the cellular environment. Finally, this induced proteolytic mechanism is not dependent upon an increase in lysosomal enzymes, but rather a structural alteration within the cell which effects a transfer of cellular proteins into the vacuolar apparatus.

Cathepsins↗
PubMed1978

Inhibition of basal protein degradation in rat embryo fibroblasts by cycloheximide: correlation with activities of lysosomal proteases.

Rat embryo fibroblasts were grown in medium containing 14C-leucine and 3H-thymidine. After a 24-hour chase in nonlabeled medium, cultures were placed in either fresh growth medium or medium containing 10-20 microgram/ml cycloheximide. Cell monolayers were processed at daily intervals for three days. Four hours prior to processing, cultures were placed in fresh medium and the accumulation rate of trichloroacetic acid soluble 14C in the media assayed. Cycloheximide effects a progressive decrease in the fractional degradation rate of the labeled cell protein, primarily during the first 24 hours. The specific activities of cathepsin D, cathepsin B, and neutral protease correlate closely with the fractional degradation rate. Other lysosomal hydrolases show little change during this period. The activities of the lysosomal proteases approach a new steady state which is correlated with the new steady state level of protein synthesis. A model is proposed which relates the rate of protein breakdown in the cell to the level of protein synthesis. The data also suggests the possibility that subpopulations of high turnover and low turnover cells exist in these cultures.

Animals↗