Increase In Synthetic Rate Research Articles

Adrenal medullary adaptations and cardiovascular regulation after 6-hydroxydopamine treatment in rats

Experiments were conducted to assess the pattern of activation of adrenal medullary adrenaline (ADR) and noradrenaline (NA) chromaffin cells following damage to sympathetic neurons with 6-hydroxydopamine (6-HDA, 100 mg/kg per day for 7 days, s.c.). 6-HDA treatment resulted in a sustained elevation in urinary ADR excretion, suggesting that the adrenal medulla was continuously stimulated. Further evidence of neural stimulation of the adrenal medulla was demonstrated by a persistent elevation of adrenal medullary dopamine (DA) content, an indicator of catecholamine synthesis. The increase in synthetic rate was associated with a significantly augmented medullary NA and ADR content. However, NA content was elevated after only 1 day of treatment, whereas, ADR became elevated after the second day, a finding that suggests a differential activation of the two populations of chromaffin cells. Cardiac and kidney NA contents were reduced by 97 and 87%, respectively, and BP increments to stimulation of the thoracolumbar spinal cord of pithed rats (0.5 to 16 Hz) were attenuated by more than 75% by 6-HDA. Moreover, the residual BP responses were abolished by adrenalectomy. Thus the results indicate that the changes in adrenal CA disposition may be related to a greater reliance upon the adrenal medulla to maintain blood pressure (BP) following damage to sympathetic neurons, and further, the findings are consistent with the possibility that ADR and NA chromaffin cells are separately controlled.

Dynamics of Ubiquitin Pools in Developing Sea Urchin Embryos: (ubiquitin/embryogenesis/proteolysis).

The sea urchin embryo is a closed metabolic system in which embryogenesis is accompanied by significant protein degradation. We report results which are consistent with a function for the ubiquitinmediated proteolytic pathway in selective protein degradation during embryogenesis in this system. Quantitative solid- and solution-phase immunochemical assays, employing anti-ubiquitin antibodies, showed that unfertilized eggs of Strongylocentrotus purpuratus have a high content of unconjugated ubiquitin (ca. 8 × 108 molecules), and also contain abundant conjugates involving ubiquitin and maternal proteins. The absolute content of ubiquitin in the conjugated form increases about 13-fold between fertilization and the pluteus larva stage; 90% or more of embryonic ubiquitin molecules are conjugated to embryonic proteins in hatched blastulae and later-stage embryos. Qualitatively similar results were obtained with embryos of Lytechinus variegatus. The results of pulse-labeling and immunoprecipitation experiments indicate that synthesis of ubiquitin in S. purpuratus is developmentally regulated, with an overall increase in synthetic rate of 12-fold between fertilization and hatching. Regulation is likely to occur at the level of translation, since others have shown that levels of ubiquitin-encoding mRNA remain virtually constant in echinoid embryos during this developmental interval. The sea urchin embryo should be a useful system for characterizing the role of ubiquitination in embryogenesis.

Oilbody Proteins in Microspore-Derived Embryos of Brassica napus

A number of treatments were tested for their ability to affect the synthesis of oilbody proteins in microspore-derived embryos of rapeseed (Brassica napus). Synthesis of the oilbody proteins was determined by [(35)S]methionine incorporation in vivo and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of washed oilbody fractions. Oilbody proteins of approximately 19, 23, and 32 kilodaltons were found to be prominent. These proteins showed differential patterns of regulation. The 19 and 23 kilodalton proteins (oleosins) were greatly enhanced by treatments with abscisic acid, jasmonic acid, and osmotic stress imposed using sorbitol (12.5%). Synthesis of the 32 kilodalton protein was inhibited by abscisic acid and by sorbitol (12.5%), but unaffected by jasmonates. The strong promotion of synthesis of the 19 and 23 kilodalton oilbody proteins appeared to be specific as they are not seen with gibberellic acid treatment or with a stress such as heat shock. Time course experiments revealed that the abscisic acid stimulation of oleosin synthesis is quite rapid (less than 2 hours), reaching a maximum at 6 to 8 hours. The response of the oleosins to abscisic acid is found in all stages of embryogenesis, with a major increase in synthetic rates even in globular embryos on abscisic acid treatment. This suggests that these proteins may accumulate much earlier in embryogenesis than has previously been believed. The 32 kilodalton oilbody-associated protein appears different from the oleosins in several ways, including its distinct pattern of regulation and its unique property, among the oilbody proteins, of undergoing phosphorylation.

Heparin stimulates the synthesis and modifies the sulfation pattern of heparan sulfate proteoglycan from endothelial cells.

Heparin stimulates 2-3-fold, in a concentration-dependent manner, the synthesis of heparan sulfate secreted by cultured endothelial cells. The increase in synthetic rate takes place immediately after exposure of the cells to heparin, affects only heparan sulfate, and is specific for the endothelial cell. No stimulation by other glycosaminoglycans was observed. Analysis of the disaccharide products formed by the action of heparitinases reveals a higher degree of sulfation of the uronic acid residues in the heparan sulfate of cells exposed to heparin.

Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes.

Lipoprotein lipase (LPL) is the enzyme responsible for hydrolysis of circulating triglyceride-rich lipoproteins and is important for storage of adipocyte lipid. To study the regulation of LPL synthetic rate in adipose tissue, primary cultures of isolated rat adipocytes were pulse-labeled with [35S]methionine, and LPL was immunoprecipitated with an LPL-specific antibody. A pulse-chase experiment identified the cellular and secreted forms of LPL as a 55-57-kDa protein. In the presence of heparin, there was a large increase in secretion of newly synthesized LPL from the cells, although heparin did not stimulate cellular LPL synthetic rate. When cells were exposed to insulin for 2 h, pulse-labeling revealed that insulin stimulated a maximal dose-related increase in LPL synthetic rate of 300% of control. This increase in LPL synthetic rate was observed after an exposure to insulin for as little as 60 min and was accompanied by only a 10-25% increase in total protein synthesis. In addition, insulin had no effect on the turnover of intracellular LPL. Using a cDNA probe for LPL, insulin induced a 2-fold increase in the LPL mRNA. Thus, insulin stimulated an increase in specific LPL mRNA in isolated rat adipocytes. This increase in LPL mRNA then leads to an increase in the synthetic rate of the LPL protein.

Cholesterol biosynthesis and its regulation in dissociated cell cultures of fetal rat brain: developmental changes and the role of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Primary cultures of cells dissociated from fetal rat brain were utilized to define the developmental changes in cholesterol biosynthesis and the role of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the regulation of these changes. Cerebral hemispheres of fetal rats of 15-16 days of gestation were dissociated mechanically into single cells and grown in the surface-adhering system. Cholesterol biosynthesis, studied as the rate of incorporation of [14C]acetate into digitonin-precipitable sterols, was shown to exhibit two distinct increases in synthetic rates, a prominent increase after 6 days in culture and a smaller one after 14 days in culture. Parallel measurements of HMG-CoA reductase activity also demonstrated two discrete increases in enzymatic activity, and the quantitative and temporal aspects of these increases were virtually identical to those for cholesterol synthesis. These data indicate that cholesterol biosynthesis undergoes prominent alterations with maturation and suggest that these alterations are mediated by changes in HMG-CoA reductase activity. The timing of the initial prominent peak in both cholesterol biosynthesis and HMG-CoA reductase activity at 6 days was found to be the same as the timing of the peak in DNA synthesis, determined as the rate of incorporation of [3H]thymidine into DNA. The second, smaller peak in reductase activity and sterol biosynthesis at 14 days occurred at the time of the most rapid rise in activity of the oligodendroglial enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP). These latter observations suggest an intimate relationship of the sterol biosynthetic pathway with cellular proliferation and with oligodendroglial differentiation in developing mammalian brain.

Lesion regression and protein synthesis in rabbits after removal of dietary cholesterol.

This study was conducted to determine whether atherosclerotic lesions regressed in rabbits that were placed on a normal, low cholesterol diet. Rabbits (n = 16) were fed a 2% cholesterol diet for 90 days; nine rabbits were killed, and the remaining seven rabbits were fed a low cholesterol diet for 7 months.. Appropriate noncholesterol-fed controls were used with each group. After 90 days of cholesterol feeding, the rabbits' thoracic aortas showed severe lesions, accompanied by significantly elevated rates of collagen and noncollagen protein synthesis and significantly increased amounts of cholesterol and soluble collagen. Cholesterol-fed rabbits that were fed a normal diet for 7 months had normal serum cholesterol levels, but exhibited extensive aortic lesions with elevated cholesterol content. Biochemically, the rate of aortic collagen synthesis remained significantly elevated above control values; however, the collagen content was not different from controls. This lack of collagen accumulation in the presence of a prolonged increase in synthetic rate was surprising. One possible explanation is that in the diseased aorta there is a rapid turnover of collagen, resulting in a redistribution or remodeling of the connective tissue, rather than an increased accumulation.

Hormonal stimulation in the exocrine pancreas results in coordinate and anticoordinate regulation of protein synthesis.

24-h intravenous caerulein infusion studies in the rat were combined with in vitro amino acid incorporation studies followed by high-resolution separation of proteins by two-dimensional isoelectric focusing and SDS gel electrophoresis to study the extent to which persistent changes in the biosynthesis of exocrine pancreatic proteins are regulated by cholecystokinin-like peptides. Beginning in the third hour of optimal hormone infusion at 0.25 microgram kg-1 h-1, changes were observed in the synthetic rates of 12 proteins, which progressed over the course of the 24-h study. Based on coordinate response patterns, exocrine proteins could be classified into four distinct groups. Group I (trypsinogen forms 1 and 2) showed progressive increases in synthetic rates reaching a combined 4.3-fold increase over control levels. Group II (amylase forms 1 and 2) showed progressive decreases in synthesis to levels 7.1- and 14.3-fold lower than control levels, respectively. Group III proteins (ribonuclease, chymotrypsinogen forms 1 and 2, procarboxypeptidase forms A and B, and proelastase 1) showed moderate increases in synthesis, 1.4-2.8-fold, and group IV proteins (trypsinogen 3, lipase, proelastase 2, and unidentified proteins 1-4) did not show changes in synthesis with hormone stimulation. Regulation of protein synthesis in response to caerulein infusion was specific for individual isoenzymic forms in the case of both trypsinogen and proelastase. The ratio of biosynthetic rates of trypsinogen forms 1 + 2 to amylase forms 1 + 2 increased from a control value of 0.56 to 24.4 after 24 h of hormonal stimulation (43.5-fold increase). Biosynthetic rates for an unidentified protein (P23) with an Mr = 23,000 and isoelectric point of 6.2 increased 14.2-fold, and the ratio of synthesis of P23 to amylase 2 increased 200-fold during caerulein infusion. During hormone stimulation the anticoordinate response in the synthesis of pancreatic glycosidases (decreased synthesis) and serine protease zymogens (increased synthesis) explain previous observations that showed little change in rates of total protein synthesis under similar conditions.

Estrogen-induced creatine kinase in the reproductive system of the immature female rat.

An increase in the biosynthetic rate of the brain-type isozyme of creatine kinase (CKBB, first described in the uterus as the "estrogen-induced protein") was found in the ovary, vagina and estrogen receptor-rich regions of the brain (preoptic area, anterior hypothalamus and median eminence), one hour after injection of 5 micrograms of estradiol-17 beta into 25-28 day-old rats. The increase in synthetic rate in the ovary, detected by 35S methionine incorporation, peaked at 1h, but still remained higher than in control ovaries at 6 h and was reflected in a longer-term increase in ovarian CK specific activity after 4 daily injections. Both ovary and vagina, similarly to brain, contained exclusively the BB isozyme of CK. These findings suggest that the entire female reproductive system can respond to estrogen by a rapid induction of CKBB.

The biosynthesis of deoxyribonucleic acid during the life-span of the mosquito.

Previous results from this laboratory [Kao et al, (1976) Biochem. J. 154, 471-480] demonstrated that a low-molecular-weight DNA species, "sDNA", present in high concentrations during rapid larval growth of the yellow-fever mosquito, Aedes aegypti, may be an intermediate in DNA replication. We have now determined the life-span profiles of synthetic rates and the steady-state contents of sDNA and total DNA by radiometric and biochemical procedures and by using mosquitoes grown and maintained under defined and controlled conditions, including axenic culture and synchronization of stages. 2. Our results showed exponential increases in DNA contents and synthetic rates which paralleled the changes in weight during larval growth. Of special importance was the finding that in rapidly-growing larvae and newly-emerged adult mosquitoes high contents and rates of synthesis of sDNA were correlated with increases in total DNA/organism, which suggests that sDNA content may be an index of DNA replication. In senescent mosquitos a twofold increase in the synthetic rate of bulk DNA ("pDNA") without an increase in sDNA may reflect repair synthesis. 3. The significance of this investigation is that it may be the first quantitative and systematic description of the synthetic rates and contents of both DNA-replication intermediates and total DNA in an axenic, eukaryotic organism rather than a specific cell type. Equally important is that these findings describe DNA changes during growth and also during aging through senescence in an age-characterized organism.

An alteration in the kinetic properties of a cholesterogenic enzyme in fowlpox virus infection

Cholesterogenesis in the chorioallantoic membrane (CAM) of the chick embryo during fowlpox virus infection has been studied by a variety of techniques. Analysis of the sterol content of the CAM epithelium revealed an elevation in the sterol content on the fourth day postinfection, but not on the third day. The increase was due primarily to the accumulation of sterol ester. That the increased level of sterols is due to an increase in synthetic rate is suggested by the observation that the activity of the enzyme usually considered to be rate limiting in cholesterogenesis, 3-hydroxy,3-methylglutaryl coenzyme A (HMG-CoA) reductase, becomes elevated above control values at about this same time period, i.e., between 3 and 4 days postinfection. The ability to synthesize sterol from labeled mevalonate is lower in the infected tissue than in the control. These data indicate that the HMG-CoA reductase reaction may be a primary site of action in the stimulation of cholesterogenesis in fowlpox virus infection. An examination of the kinetic properties of the HMG-CoA reductase from infected CAM epithelium suggested that two forms of the enzyme are present in infected cells, one form identical to the control enzyme- and an altered form. This conclusion was based on the biphasic nature of the saturation curve of the enzyme for HMG-CoA. Other kinetic properties such as thermal denaturation and pH titration also revealed differences between the enzymes from control and infected tissues.

Effect of Thyroid Hormone on Polypeptide Chain Assembly Kinetics in Liver Protein Synthesis in Vivo

Abstract The time constant for assembly and release of polypeptide chains in the process of protein synthesis in rat liver in vivo has been determined by a new technique which is independent of most sources of variation and error in amino acid incorporation experiments. In this method the ratio of incorporation of 14C-amino acids into completed soluble proteins to that in total protein (including ribosome-bound chains) is measured as a function of short incubation periods (15 to 105 s) after hepatic portal vein injection to obtain tc, the average polypeptide chain assembly time. For normal male Long-Evans rats, ages 2 to 2½ months, tc in liver in vivo is 1.16 ± 0.16 min. Induction of hyperthyroidism by three injections of triiodothyronine (40 µg/100 g body weight) over 72 hours led to a value of tc = 0.92 ± 0.08 min, representing a 27% increase in synthetic rate. Surgically thyroidectomized rats showed a markedly longer time for polypeptide chain assembly, tc = 1.92 ± 0.22 min, corresponding to a 39% depression in protein synthetic output compared with sham-operated and normal controls of the same age. Almost complete recovery of protein synthetic rate in thyroidectomized rats was achieved by replacement doses of triiodothyronine (20 µg/100 g body weight) given daily for 2 days prior to assay. The results are discussed in relation to the mechanism of control of the protein synthetic pathway, the anabolic action of the thyroid hormones, and other methodology which has been used in attempts to estimate protein synthetic capacity.

The effect of testosterone on the synthesis of mouse kidney mitochondrial and microsomal proteins in vivo and in vitro.

Castration of 8–week—old male mice resulted in a decrease in kidney wt to a plateau level about 65–70% of normal within 14 days. This wt loss was accompanied by decreases in the rate of incorporation of radioactive amino acids in vivo into mitochondrial and microsomal proteins as well as a decrease in the rate of incorporation of radioactive amino acids into protein by isolated mitochondria in vitro. Rates of incorporation both in vivo and in vitro reached minimal values (60–65% of normal) approximately 8 days after castration and appeared to recover spontaneously, reaching normal or near normal levels by the 3rd week following castration. The rate of degradation of mitochondrial and microsomal proteins isolated from liver and kidney was determined by measuring the rate of disappearance of L—arginine—guanidino–14C radioactivity from the protein of the two fractions. The small changes found in the protein half lives of the two fractions in either organ following castration suggested that the decrease in kidney wt following castration could be attributed primarily, if not entirely, to the decreased synthetic rate. Administration of testosterone to castrate animals during the period of depressed protein synthesis (days 6–14 after castration) resulted in an increase in synthetic rate within 11–18 hr when tested in vitro and within 14 hr when tested in vivo. (Endocrinology91: 125, 1972)

Histochemical studies on proteins of the milkweed bug after juvenile hormone treatment

1. 1. Hemolymph from the fifth instar milkweed bugs was separated by disc gel electrophoresis into eleven bands. 2. 2. A number of histochemical stains were applied to these gels in an attempt to identify band 8 which showed an increase in synthetic rate after juvenile hormone treatment. This band was found to be acid phosphatase. 3. 3. Histochemical procedures for acid phosphatase were applied to tissue sections and whole mounts of various organs of the milkweed bug in an attempt to localize the site of increased synthesis. 4. 4. The testes, fat body, midgut and brain were positive for acid phosphatase; however, no quantitative difference could be seen between the bugs treated with juvenile hormone and those not treated.