Bile acid profiles in analbuminemia rats

Bile acid profiles in serum, urine and bile of Nagase analbuminemia rats (NAR) and Sprague-Dawley rats (SDR) were examined. Serum bile acid levels in NAR (2.02 + 0.51 micrograms/ml, n = 15, M +/- S.E.) were markedly decreased as compared with those in SDR (20.86 +/- 3.72 micrograms/ml, n = 10). The unbound fraction of acids in serum examined by equilibrium dialysis was about ten times higher in NAR than in SDR. In the profiles of urinary and biliary bile acids in NAR and SDR, as big differences as seen for serum bile acids were not observed. Low bile acid levels in serum of NAR may reflect low bile acid binding capacity of NAR serum because the absence of albumin was thought to be one of the major causes of low bile acid levels in serum of NAR.     

Reduced activity of androgen biosynthesis in the testes of rats with analbuminemia

Steroid metabolism in Nagase Analbuminemia Rats (NAR), a mutant strain established from Sprague-Dawley rats, was studied. NAR are characterized by lack of serum albumin and hyperlipidemia. Total testosterone concentration in the serum of NAR was lower than that of normal rats, while the serum free testosterone, LH and FSH concentrations were similar. The half lives of 14C-labeled testosterone administered intravenously in NAR and normal Sprague-Dawley (SD) rats were 4.4 and 4.1 min, respectively. The plasma clearance rates of testosterone in NAR and normal rats were 34.7 and 39.1 ml/min per kg body weight. On Sephadex G-100 chromatography, a mixture of [3H]testosterone and normal rat serum gave two protein peaks eluted in the void volume and the albumin fraction, and the radioactivity was eluted all in the albumin fraction. In contrast, a mixture of [3H]testosterone and NAR serum gave a single protein peak eluted in the void volume and the radioactivity was mainly eluted with this protein peak. The association constants of testosterone to NAR and normal rat sera were 1.25 and 2.24 X 10(4) M-1. Enzyme activities related to the synthesis of testosterone by the testicular microsomal fractions of NAR and normal rats were examined. The activities of 3 beta-hydroxysteroid dehydrogenase, 5-ene-4-ene isomerase, 17 alpha-hydroxylase, C-17-C-20 lyase and 17 beta-hydroxysteroid dehydrogenase were lower in NAR than in normal rats. The activity for synthesis of testosterone from pregnenolone by the testicular microsomal fraction of NAR was about 40% of that of normal rats. These findings indicate that the low serum concentration of testosterone in NAR is mainly attributable to decreased biosynthesis of testosterone in the testes.     

Receptor-mediated gene delivery in vivo. Partial correction of genetic analbuminemia in Nagase rats

A plasmid (palb3) was constructed containing the structural gene for human serum albumin driven by mouse albumin enhancer-rat albumin promoter elements. Using an asialoglycoprotein-polycation conjugate consisting of asialoorosomucoid coupled to poly-L-lysine, a soluble DNA complex was formed that was capable of targeting specifically to hepatocytes via asialoglycoprotein receptors present on these cells. Groups of Nagase analbuminemic rats were injected with complexed DNA or controls, followed by two-thirds partial hepatectomy to stimulate hepatocyte replication. Using a cDNA probe for the human albumin structural gene, hybridizable sequences were detected in analbuminemic rats treated with complex as determined by Southern blot analysis. Two weeks post-injection, the targeted DNA was found to exist primarily in plasmid form with an average copy number of 1000/diploid cell. Human albumin mRNA was detected by dot-blot hybridization with a specific oligonucleotide cDNA probe and confirmed by RNase protection assay using a vector-specific probe. Circulating human albumin was detected in the serum of palb3-treated Nagase analbuminemic rats by Western blots using an antibody specific for human serum albumin. A time course demonstrated that circulating human albumin was not detectable 24 h after injection, but became measurable at a level of 0.05 micrograms/ml within 48 h and increased in concentration to a maximum of 34 micrograms/ml by 2 weeks post-injection. This level of expression remained stable through 4 weeks after injection and partial hepatectomy.     

Turnover of myelin proteins in mouse brain in vivo.

The incorporation of tyrosine into proteins was measured after the subcutaneous implantation of a pellet of [14C]tyrosine in mice. This method keeps the specific radioactivity of free tyrosine fairly constant and makes it possible to follow incorporation up to a 10-day period. At the end of 10 days most of the protein-bound tyrosine was replaced (i.e. most protein turned over) in lung, liver, heart, kidney and spleen; about half was replaced in brain, one-quarter in muscle. The rate of protein turnover in myelin was approx. 40% of that of whole brain proteins; at 10 days one-fifth of the myelin proteins were replaced. All protein components of myelin measured were in a dynamic state; incorporation decreased in the following order, Wolfgram greater than DM-20 greater than basic greater than proteolipid proteins. The incorporation of tyrosine into each protein fraction was greater in the 0-5-day than in the 5-10-day period, indicating heterogeneity of metabolic rates. The results show that after myelination at least a portion of each protein component of myelin is undergoing significant metabolic turnover. In the adult, myelin components are not stable, but turnover is heterogeneous, and each protein may be compartmentalized. Turnover can be influenced by a variety of factors.     

Turnover studies on proteins of rat liver lysosomes.

The turnover of rat liver lysosomal proteins was studied by a double isotope-labeling technique. The cellular fractions investigated included soluble lysosomal proteins, lysosomal membrane proteins, highly purified lysosomal beta-glucuronidase, and for comparison, microsomal proteins and soluble cytoplasmic proteins. Both "normal" lysosomes and Triton WR-1339-filled lysosomes (tritosomes) were studied, with similar results. It was found that (a) the turnover rate of lysosomal proteins, of both the soluble and membranous compartments, was very similar to that of the proteins of the microsomal and soluble cytoplasmic fractions, and (b) the turnover rate of lysosomal proteins was asynchronous. The latter conclusion was based on two lines of evidence: (a) lysosomal beta-glucuronidase had a distinctly slower turnover rate than the average rate of the soluble lysosomal proteins, and (b) subunits of the proteins of the soluble lysosomal fraction as separated by sodium dodecyl sulfate. Sephadex G-200 gel filtration showed different rates of degradation.     

Interaction of histones with human serum proteins.

The authors describe the interactions of whole calf thymus histone and its fractions with the human serum proteins. Immunoelectrophoretic analysis revealed two types of interactions: 1) a decrease in the electrophoretic mobility of a whole, immunochemically uniform fraction, and 2) a decrease in the electrophoretic mobility of only some molecules of such a fraction. In this association, the arginine-rich histone fraction (F3) was found to be the most active. The findings are important for interpreting the results of biological treatments.     

Turnover of canavanine-containing proteins inSaccharomyces cerevisiae

Saccharomyces cerevisiae grown for 2 h in the presence of 0.5 mmol/L canavanine in a synthetic medium with ethanol as the sole carbon source (OEC) exhibited a slowing down of protein synthesis for 3–4 h after a shift to fresh ethanolbased medium containing 1.0 mmol/L arginine (OEA) in comparison with untreated cells grown on OEA. The change of carbon source from ethanol to glucose (OGA) after growth in the OEC medium resulted in an even deeper decline of protein synthesis. The degradation of canavanine-containing proteins in cells pregrown and labelled in an OEC medium after transfer to OEA was more rapid than in the OGA medium. The initial rate of protein degradation during the first hour in the OGA medium was less than 1%/h whereas in the OEA medium it reached almost 10%/h. The fraction of proteins with high turnover (half-life 0.46 h) constituted 8.3% on OEA, while during subsequent growth on OGA it was only 0.75% with a half-life of 0.12 h.     

Turnover of proteins in the extreme thermophileThermus flavus

Thermus flavus contained a limited fraction of short-lived protein when growing in a complex medium; the residual proteins were stable. When incubated in buffer, the residual protein fraction was also degraded. The extent of the short-lived protein fractions was increased by increasing the temperature.     

Turnover of the surface proteins and the receptor for serum asialoglycoproteins in primary cultures of rat hepatocytes

The turnover of plasma membrane proteins in primary rat hepatocyte cultures was examined by following the loss of polypeptides labeled in situ by lactoperoxidase-catalyzed iodination using 125I and 131I. Most plasma membrane proteins had similar rates of degradation, having a half-life of approximately 85 h. By in situ labeling via lactoperoxidase-catalyzed iodination, as well as metabolically labeling cells with L-[35S]methionine, the asialoglycoprotein receptor, a plasma membrane constituent, was identified and shown to exist in three forms which were structurally related. The turnover of receptor on the cell surface was examined by following the loss of iodinated cell surface receptor, while the turnover of total cellular receptor, including both surface and internally localized receptor was assayed by following the loss of receptor labeled metabolically with [35S]methionine. The turnover rate in both cases was approximately 20 h. Receptor-mediated endocytosis of asialoglycoproteins had no effect on the turnover of the plasma membrane proteins or receptor. Based on estimates of the rate of metabolism of the asialoglycoprotein ligand relative to the turnover rate of the receptor, we conclude each molecule of receptor can deliver about 1,000 molecules of ligand to the lysosome to be degraded.     

Electrodecantation of serum proteins.

The sedimentation of albumin under the action of the electric and gravitational fields was determined as a function of time in discontinuous experiments in a rectangular cell, using serum with the albumin fraction stained blue. It was shown that even under the influence of strong electric fields, the upper boundary of the albumin layer fell no further than the mid-point of the cell. In continuous single-stage separation of gamma-globulin from other serum proteins, only about half the gamma-globulins can be obtained from the solution because it remains homogeneously distributed throughout the solution and is only free from albumin and other proteins in the upper half of the cell. In experiments with continuously operated triangular cells, the process was optimized to give gamma-globulin of 97.5% purity in a yield of 80%, at serum flow-through rates of up to 0.5 l/h in a block composed of 40 cells.     

Cytoskeletal proteins of the cell surface in Tetrahymena : II. Turnover of major proteins

The turnover of surface membrane-associated cytoskeletal proteins has been studied in Tetrahymena. These proteins undergo rapid turnover and appear to be in equilibrium with precursor pools within the cytosol. Carefully controlled pulse-chase experiments, alone and in combination with cycloheximide, have shown that labeled proteins accumulated in the cytoskeleton after they were no longer being synthesized within the cell. The movement of subunits from the cytoskeleton to the cytosol was also demonstrated using tubulin isolated from these two compartments within the cell.