Developmental changes in collagen and elastin biosynthesis in the porcine aorta

Elastin and collagen are the principal scleroproteins of the aortic wall, and they largely determine its physical and mechanical properties. During perinatal development of the aorta, elastin and collagen accumulate rapidly, being present as inverse gradients by the time of birth. Elastin is most prevalent in the thoracic aorta, decreasing distally, while collagen shows the opposite trend. The present studies have determined the relative and absolute rates of collagen and elastin synthesis in the porcine aorta between 60 days of fetal development (mid-gestation) and 110 days after birth. Although there was measurable elastin synthesis in the upper thoracic aorta at the earliest time evaluated, there was a fourfold increase in relative elastin synthesis (from 4 to 16% of total protein synthesis) between 60 fetal days and birth. Elastin synthesis was maximal in successively distal segments between 1 and 3 weeks after birth. Relative collagen synthesis progressively increased in distal aortic regions between 90 fetal days and 60 days postpartum. Greater than twofold increases over thoracic levels were measured. Both elastin and collagen synthesis largely subsided by 110 days of development. When expressed as absolute rates of protein synthesis, these scleroproteins were maximally expressed in the first 3 postnatal weeks. Elastin mRNA levels were determined with a cloned sheep gene fragment by molecular hybridization. Gradients of elastin message were present at 60 fetal days and at 4 and 14 days after birth, elastin mRNA levels being maximal in the upper thoracic aorta at 14 days after birth. The differentiation of the aortic wall thus follows discrete patterns of phenotypic change which may be coupled to the rheologic stresses accompanying development of the circulatory system.     

Wound tumor virus polypeptide synthesis in productive noncytopathic infection of cultured insect vector cells.

Inoculation of the leafhopper cell line AC-20 with wound tumor virus resulted in a productive noncytopathic infection with no detectable alteration of cellular protein synthesis. Virus-specific polypeptide synthesis, detectable by 8 h postinoculation, increased in a linear fashion, reaching a peak (approximately 10 to 15% of total protein synthesis) by 48 h postinoculation. The rate of viral protein synthesis continued at this level for several days but declined, relative to cellular protein synthesis, as infected cells were passaged. By passage 10, the synthesis of viral polypeptides was reduced to a level approximately 5% of that observed at 48 h postinoculation. Viral protein synthesis was not stimulated by superinfection. Viral antigens and infectious virus persisted in the majority (greater than 90%) of cells in an infected culture even after more than 100 passages. The synthesis of wound tumor virus polypeptides in infected insect vector cells appears to be regulated in a coordinated and selective manner.     

Protein Metabolism in Cultured Plant Tissues: III. Changes in the Rate of Protein Synthesis, Accumulation, and Degradation in Cultured Pith Tissue

During the transition of tobacco (Nicotiana tabacum) pith tissue to callus tissue, there were changes in the composition of the soluble amino acid pools, in the distribution of amino acids between pool and protein, and in the synthesis, accumulation, and degradation of proteins. The size of the leucine pool decreased from 90 nanomoles per gram fresh weight in fresh pith to 20 nanomoles in 24-hour cultured pith, followed by a return to 90 nmoles in pith cultured longer than 5 days. The latter value is the same as that reported for exponentially growing callus cells. Many other pool amino acids changed as dramatically. However, they always approached callus levels after 5 days of culturing. The total amino acid content of pith tissue (the sum of both pool and protein) remained unchanged during culturing. The value for total amino acid content (34 to 42 nanomoles per gram fresh weight) was also similar to that found in callus. The distribution of amino acids between pool and protein did change during culturing. The transition of pith tissue with 88% of its total amino acids free in the soluble pool to callus with 92% of its amino acids in protein was further characterized by changes in protein metabolism. Both protein synthesis and accumulation increased over the first 50 hours in culture to a maximum rate of 45 milligrams protein synthesized gram protein⁻¹ hour⁻¹. After 50 hours in culture, the rate of protein accumulation decreased to equal the rate of fresh weight accumulation (10 mg g⁻¹ hour⁻¹). However, protein synthesis continued at a high rate for several days, suggesting protein degradation was turned on by this time. By 5 days protein synthesis had decreased to a rate similar to that of callus.     

无血清培养高密度乳猪肝细胞的形态和功能变化

本文研究了无血清培养高密度猪肝细胞的形态和功能变化。将分离的肝细胞以高密度(1×10~7/ml)培养在含激素、多种生长因子和营养成分的无血清培养基中,动态观察培养7天中肝细胞形态、活率、蛋白质合成功能、G-6-Pase活性、安定转化功能及LDH含量;同时以无血清培养低密度(5×10~5/ml)肝细胞作为对照组。研究结果表明:高密度培养的 肝细胞各项功能较低密度培养的肝细胞为低;高密度培养的肝细胞的形态、蛋白质合成功能在培养7天中保持稳定;活率随着培养时间的延长而下降,但均高于90％;安定转化功能在培养第2、3天最强;G-6-Pase活性在培养1天后明显下降,然后维持在较低水平;LDH含量在第1、2、3天较高。     

The influence of early protein-calorie malnutrition on neuronal and glial protein synthesis

The effect of pre- and postnatal undernutrition, produced according to the method of Chow and Lee (3), on the rate of protein synthesis in the brains of rats 11, 21, 34 and 90 days of age was studied by measuring the incorporation ofl-[¹⁴C]valine in vivo andl-[³H]lysine in vitro. Both in vivo and in vitro experiments were performed with high concentration of the precursor to decrease the effects of pool variations and protein degradation. Particular interest was given to the effects of this form of early protein-calorie malnutrition (PCM) on neuronal and glial cells which were isolated from the brains by gradient centrifugation. Brain protein synthesis measured in vivo which showed a peak at 21 days in both animal series, was depressed by PCM at 11 days but stimulated at 34 days of animal age. Small effect was observed in the 90-day-old animals. A similar response as in whole brain was seen for neuronal cells, while glial cells showed a different reaction. Studies of in vitro protein synthesis did not reveal appreciable effects of undernutrition in whole brain. Both neuronal and glial cells showed a moderate but not statistically significant elevation of protein synthesis in animals subjected to early PCM.     

Collagen synthesis in the muscle of developing chick embryos.

Radioactive protein was prepared from the leg muscle of chick embryos, 11, 14, 16 and 17 days old, each injected with radioactive proline and incubated for 30, 60 or 90 min afterwards. The radioactive protein was incubated with collagenase purified by chromatography on a Sephadex G-100 column. Under this condition, only collagen is digested into products soluble in trichloroacetic acid. The relative rate of collagen synthesis was determined by comparing the amount of radioactivity released into the supernatant fraction and that in the residue, by the method of Diegelmann & Peterkofsky [(1972) Dev. Biol. 28, 443--453]. The results show that the rate of collagen synthesis remains at approx. 10% of the rate of synthesis of other non-collagenous proteins during the development of chick embryonic muscle from 11 to 17 days. This suggests that the synthesis of collagen and that of other proteins are co-ordinately regulated at these stages of development.     

The effects of elevated summer temperature and sublethal pollutants (ammonia, low pH) on protein turnover in the gill and liver of rainbow trout (Oncorhynchus mykiss) on a limited food ration.

Protein synthesis, degradation and growth of the liver and gills were determined in juvenile rainbow trout (Oncorhynchus mykiss) fed a limited ration and exposed for 90 days to normal or elevated summer temperatures (+2 degrees above ambient) and either low pH (5.2) in softwater or 70 microM total ammonia in hardwater. The limited ration resulted in low rates of growth (< 0.80% per day) and protein synthesis in all fish. In softwater, whole-body growth was significantly inhibited by elevated temperature but stimulated by low pH, although tissue protein metabolism was generally unaffected by these treatments. There was no significant difference in final size between the groups of fish in hardwater, but liver protein synthesis and degradation were significantly lower at +2 degrees C, the reduction in synthesis being due to an inhibition of both the capacity for protein synthesis, Cs and the RNA translational efficiency, kRNA. Gill protein metabolism was unaffected by the experimental treatments in trout in hardwater. The authors conclude that a global warming scenario would be detrimental to protein synthesis and growth in freshwater fish under conditions of food limitation in summer, and when late summer temperatures approached the upper thermal limit of the species, regardless of food availability.     

Protein degradation rates in regions of the central nervous system in vivo during development.

The rate of protein degradation was estimated in several regions of rat brain at various ages by subtracting the rate of accumulation of protein from the rate of synthesis. The rate of degradation in cerebral hemisphere, which was 1.3%/h at 2 days of age, declined steadily with age, approaching the synthesis rate is about 30 days of age (0.8%/h). Degradation rates in the pons medulla, mid-brain and spinal cord were of a similar order to that in the cerebral hemisphere. The cerebellum had an exceptionally high rate of degradation in young rats, 1.9%/h at 2 days of age, which complemented its high rates of synthesis and accumulation. The degradation rate in the young was 2-2.5 times the rate in older rats and was approx. 65% of the synthesis rate during the more active phase of growth. The rapid accumulation of protein in the nervous system during the first week post partum was accompanied by high rates of breakdown, and was the result of a relatively small difference between that high rate of degradation and an even higher synthesis rate.     

Age-correlation of protein utilization to saccharide intake

Male Wistar rats aged 30, 90, 150 and 360 days were fed ad libitum on diets with an optimum protein and fat content for their respective ages and an increasing saccharide content. Net protein utilization (NPU) was determined from the body nitrogen and protein intake values and the course of gluconeogenesis in the liver was measured by specific phosphoenolpyruvate carboxykinase (PEPCK) activity. According to the growth curve for the standard diet, animals aged 30 and 90 days have a high growth rate (3.245 g/day), 150-day-old rats grow more slowly (1.856 g/day) and 360-day-old animals put on scarcely any weight at all (277 mg/day). In 30-day-old rats, NPU attains maximum values in the presence of a 36% saccharide content in the diet, in 3- and 5-month-old animals in the presence of 51% saccharides and in one-year-old animals in the presence of 41% saccharides in their food. The course of gluconeogenesis also corresponds to these values. PEPCK activity in the youngest age group is greatest in the presence of 31% saccharides in the food, at 90 days it is stimulated in the presence of 31-46% saccharides, at 150 days the decisive concentration is 41 and 46% and at one year proteins are used for saccharide synthesis in diet with a 31 and 36% saccharide concentration. For optimum saccharide values, PEPCK activity is reduced in every age group; together with the maximum NPU values, this indicates that proteins are used for growth and building of the organism at an early age and for the renewal of tissues and organs and maintenance of the organism in adulthood.     

Butachlor influence on selected metabolic processes of plant cells and tissues

Time- and concentration-course studies were conducted to determine the effects of butachlor (N-[butoxymethyl]-2-chloro-2′,6′-diethylacetanilide) on photosynthesis, protein synthesis, RNA synthesis, and lipid synthesis using isolated leaf cells of red kidney bean (Phaseolus vulgaris L.). At the 2-h incubation period, butachlor inhibited photosynthesis, protein synthesis, RNA synthesis, and lipid synthesis 99, 99, 96, and 81% respectively at 100 μM, and 0, 19, 17, and 40% respectively at 10 μM. At 100 μM and 15-, 30-, and 60-min incubations, RNA synthesis was inhibited 20, 76 and 90% respectively, and lipid synthesis 35, 48, and 62% respectively; photosynthesis and protein synthesis were inhibited over 90% at all of these time periods. The effects of 50 μM butachlor on protein and RNA synthesis in rice (Oryza sativa L.) and barnyardgrass (Echinochloa crusgalli L.) root and shoot segments were also investigated. Protein synthesis was inhibited in both species and to a greater degree in roots (81–90%) than in shoots (55–65%). RNA synthesis was inhibited 33% in barn-yardgrass roots but not significantly in barnyardgrass shoots or either organ of rice.     

Effect of level of nutrition on onset of puberty and conception rates of zebu heifers

Sixty zebu heifers were divided into three equal groups and reared on isocaloric diets but on different levels of protein. The protein levels were 19.17% (high), 13.37% (medium; NRC recommendations) and 8.3% (low). All animals were examined weekly per rectum for the presence of follicles and corpora lutea and also checked daily for standing heat. The presence of a mature corpus luteum was considered to indicate the attainment of puberty. Body weight and pelvic dimensions were taken at biweekly intervals. Intact bulls were introduced to heifers after they reached puberty and once they reached 200 kg live weight. Pregnancy diagnosis was carried out at 60, 90 and 120 days following introduction of bulls. Mean age at puberty for heifers in the high protein group (570.4 days), medium protein group (640.8 days) and low protein group (704.2 days) differed significantly (P<0.05). body weight at puberty for heifers in the high, medium and low protein groups was 207.1, 187.0 and 161.7 kg, respectively (P<0.05). Pelvic size at puberty was not influenced by the nutritional level. Conception rates of heifers in the three groups were influenced by level of nutrition. Pregnancy rates for the high protein group (58.8%), medium protein group (27.8%), and low protein group (16.7%) by 90-day post-breeding period were significantly different (P<0.05), but body weight at conception between the groups did not differ significantly. The results show that increasing the protein level in the diet is a means of improving the reproductive performance of zebu cattle.     

Regulation of myofibrillar protein turnover during maturation in normal and undernourished rat pups

The study tested the hypothesis that a higher rate of myofibrillar than sarcoplasmic protein synthesis is responsible for the rapid postdifferentiation accumulation of myofibrils and that an inadequate nutrient intake will compromise primarily myofibrillar protein synthesis. Myofibrillar (total and individual) and sarcoplasmic protein synthesis, accretion, and degradation rates were measured in vivo in well-nourished (C) rat pups at 6, 15, and 28 days of age and compared at 6 and 15 days of age with pups undernourished (UN) from birth. In 6-day-old C pups, a higher myofibrillar than sarcoplasmic protein synthesis rate accounted for the greater deposition of myofibrillar than sarcoplasmic proteins. The fractional synthesis rates of both protein compartments decreased with age, but to a greater degree for myofibrillar proteins (-54 vs. -42%). These decreases in synthesis rates were partially offset by reductions in degradation rates, and from 15 days, myofibrillar and sarcoplasmic proteins were deposited in constant proportion to one another. Undernutrition reduced both myofibrillar and sarcoplasmic protein synthesis rates, and the effect was greater at 6 (-25%) than 15 days (-15%). Decreases in their respective degradation rates minimized the effect of undernutrition on sarcoplasmic protein accretion from 4 to 8 days and on myofibrillar proteins from 13 to 17 days. Although these adaptations in protein turnover reduced overall growth of muscle mass, they mitigated the effects of undernutrition on the normal maturational changes in myofibrillar protein concentration.     

Cell growth of skeletal muscle as an indicator of protein requirements of the adult rat

Skeletal muscle growth, muscle nucleic acids and muscle protein synthesis capacity, were measured to evaluate the protein requirement of adult rats. Wistar rats were fed on diets containing 4%, 10% or 20% casein + D,L-methionine. All diets were provided for 21 days beginning at 90 days of age. Body weight, food efficiency and net weight change increased as the casein content of the diet increased. Muscle DNA, RNA and RNA/protein were lost, but protein and protein/DNA increased on the 4% and 20% protein diet. This fact involves an aplasia phenomenon although the hypertrophic growth is maintained. Alterations of the insulin and GH plasma levels were observed. These findings indicate that for adult rats the 4% and 20% protein diets are not adequate for the period of adult maintenance.     

Developmental program of PGK-1 and PGK-2 isozymes in spermatogenic cells of the mouse: Specific activities and rates of synthesis

The specific activities and synthesis of the ubiquitous isozyme, PGK-1, and the testis-specific isozyme, PGK-2, have been quantitated and localized in spermatogenic cells of the mouse. There is a fivefold increase in total PGK specific activity between immature and adult testes which begins at approximately 30 days of age, coincident with the appearance of late-middle stage spermatids. The increase in total PGK is entirely due to the appearance and increase of the PGK-2 isozyme. Rates of PGK synthesis were measured by labeling testicular cells in vitro with [³H]leucine and purifying the PGK isozymes. When total testicular cells were examined, PGK-2 synthesis was detectable after 22 days of age at very low levels and increased in older testes to a level of 0.5% of total protein synthesis. PGK-1 synthesis remained relatively constant at all ages at a level 100-fold lower (0.005%). Testicular cells were separated into highly enriched fractions of particular spermatogenic stages by centrifugal elutriation. The PGK-1 synthesis rates were, again, very low and not significantly different between the various spermatogenic stages. PGK-2 synthesis was low to nondetectable in pachytene spermatocytes, increased to 0.07% in early spermatids and represented 0.7% of total protein synthesis in late spermatids. This increased rate of PGK-2 synthesis appears to require an increase in the amount of PGK-2 mRNA in late spermatids, cells in which no active RNA synthesis is detectable.     

Biological activity of tRNA in rat liver during cycloheximide-blocked translation

The aminoacylation of tRNA was investigated with respect to protein synthesis in the rat liver. No correlation was found between the 85-90% inhibition of protein synthesis 2 h after cycloheximide injection and aminoacylation level of some tRNAs both in vivo and in vitro. A decrease in aminoacylation (28%) was established only for lysine. During the recovery phase of protein synthesis 12 and 24 h after cycloheximide treatment the aminoacylation maximal level of mixture with 14C amino acids, 14C leucine, 14C glutamic acid was unchanged.     

Estimation of growth potential by measurement of tissue protein synthetic rates in feeding and fasting rainbow trout, Salmo gairdnerii Richardson

Protein synthesis in liver, gill and muscle tissue was measured in vivo by constant infusion of ¹⁴C-tyrosine in fed and fasted freshwater rainbow trout, Salmo gairdnerii , at 12° C. Synthesis rates (percentage of tissue protein synthesized per day) were 15-17% in liver, 4–5% in gill and 0.38% in muscle of fed fish. Liver and gill synthesis rate showed no significant change in fish that had been without food for 15 days, whereas muscle protein synthesis fell to 0.09%. The greater susceptability of muscle protein synthesis to fasting, possibly results from the greater proportion of synthesis retained as growth in this tissue. Growth rates indicate little change in protein turnover in the muscle but increased protein degradation with fasting. The difference between fed and fasted synthesis rates in muscle may be used as a measurement of potential growth rate for a particular species.     

Differentiated inhibition of DNA, RNA and protein synthesis in L1210 cells by 8-methoxypsoralen

The synthesis of DNA, RNA and protein was measured in L1210 cells following treatment with 8-methoxypsoralen in combination with long wavelength ultraviolet irradiation. The results show that the DNA synthesis is strongly inhibited (approximately 95%) at 200 ng/ml reaching a minimum within 2 hours while RNA synthesis is only weakly affected at this concentration (approximately 40% inhibition). At 2 micrograms/ml the RNA synthesis is inhibited approximately 90%. Even at this concentration only a moderate effect is seen on the protein synthesis. These results strongly indicate that the phototoxic action of 8-methoxypsoralen is primarily due to inhibition of DNA synthesis.     

Beta adrenergic receptor repopulation of C6 glioma cells after irreversible blockade and down regulation

C6 glioma cells possess beta adrenergic receptors coupled with adenylate cyclase which can be irreversibly blocked by bromoacetylaminomethylpindolol (Br-AAM-pindolol), a beta adrenergic antagonist. With 1 microM Br-AAM-pindolol, more than 80% of beta adrenergic receptors, labeled by (3H)-dihydroalprenolol [3H)-DHA), were blocked. After this blockade, new beta adrenergic receptors were synthesized only during cell division. However, at cell confluency when the cell number was constant, turnover of beta adrenergic receptors was barely detectable. Cycloheximide (1 microgram/ml) inhibited cell growth as well as reappearance of beta adrenergic receptors. A 90% loss of beta adrenergic receptors in C6 glioma cells was obtained after down-regulation for 15 h with 10 microM isoproterenol, a beta adrenergic agonist. After removal of the agonist, recovery of beta-adrenergic-sensitive adenylate cyclase was complete within 2 to 3 days, whereas beta adrenergic receptors reached 90% of control value within 6 days. The half-life of the receptor recovery was 2 to 3 days. Pretreatment of C6 glioma cells by Br-AAM-pindolol and subsequent cell exposure to isoproterenol indicated that down regulation and recovery of unblocked beta adrenergic receptors did occur; however isoproterenol did not accelerate the biosynthesis of beta adrenergic receptors. The recovery of both biological response and beta adrenergic receptor occupancy was restored both in the presence or absence of cycloheximide (1 microgram/ml), a concentration which blocked 90% of protein synthesis. Our results suggest that reappearance of beta adrenergic receptors in C6 glioma cells, following isoproterenol-induced down regulation, was not due to synthesis of new receptors but to recycling of the beta adrenergic receptors.     

Time course of the effect of catabolic doses of corticosterone on protein turnover in rat skeletal muscle and liver.

The time course of the response of protein synthesis in muscle and liver to catabolic doses of corticosterone (10 mg/day per 100 g body wt.) was studied in vivo in growing rats over a 12-day period. The rate of protein synthesis in muscle and liver and the rate of actomyosin synthesis in muscle were measured by the phenylalanine-flooding technique, and 3-methylhistidine (N tau-methylhistidine) synthesis was measured by injection of labelled histidine. 3-Methylhistidine concentrations in tissue free pools and urinary excretion were also measured to compare directly with the rate of muscle protein degradation determined as the difference between synthesis and growth each day during the treatment. The overall rate of protein synthesis in muscle fell gradually over the first 4 days, reaching a rate after 5 days that was 36% of the initial rate, and this lower rate was then maintained for the following week. This decrease in the overall rate was accompanied with changes in the relative rate of synthesis in muscle proteins, since during the first 4 days there was a disproportionate decrease in the rate of actomyosin synthesis, and specifically 3-methylhistidine synthesis. In the latter case the synthesis rate was decreased to only 4% of its initial rate after 4 days. These changes in protein synthesis in muscle were accompanied by a transient increase in the rate of protein degradation, which was more than doubled on days 2 and 3 of treatment but which returned to the original rate on day 5, and a similar pattern of response was indicated by urinary 3-methylhistidine excretion, which also exhibited a transient increase. Thus in this case 3-methylhistidine excretion and measured rates of protein degradation in muscle do correlate. The transient effects of the glucocorticoids on degradation compared with the sustained effect on synthesis suggest that these two responses are achieved by different mechanisms. The hepatic size and protein mass were increased by the treatment, and protein synthesis was well maintained until after 12 days, when the rate was suppressed. Although the fractional synthesis rate was transiently increased for 24 h, it is argued that the enlarged liver most likely reflects a decrease in protein degradation resulting from the increased amino acid supply to the liver. This would result from the cessation of muscle growth while dietary supply was maintained.     

Relationship between histidyl-tRNA level and protein synthesis rate in wild-type and mutant Chinese hamster ovary cells.

A preliminary investigation was carried out to determine how conditional lethal mutants affected in particular aminoacyl-tRNA synthetases may be used to study the role of tRNA charging levels in protein synthesis. The relationship between rate of protein synthesis and level of histidyl-tRNA in wild-type cultured Chinese hamster ovary cells was determined using the analogue histidinol to inhibit histidyl-tRNA synthetase activity. This response was compared with that obtained using a mutant strain with a defective histidyl-tRNA synthetase that phenotypically shows decreased rates of protein synthesis at reduced concentrations of histidine in the growth medium. The approach used was based on measuring the histidyl-tRNA levels in live cells. The percentage charging was estimated by comparing [14C]histidine incorporated into alkali-labile material in paired samples, one of which was treated with cycloheximide, five minutes before terminating during the incubation, to produce maximal aminoacylation. Wild-type cells under histidinol inhibition exhibited a sensitive, sigmoidal relationship between the level of histidyl-tRNA and the rate of protein synthesis. A decrease in the relative percentage of acylated tRNA (His) from 46% to 35% elicited a large reduction in the rate of protein synthesis from 90% to 30% relative to untreated cells. An unpredicted result was that the relationship between protein synthesis and histidyl-tRNA in the mutant was essentially linear. High acylation values for tRNA (His) were associated with rates of protein synthesis that were not nearly as high as in wild-type cells. These findings suggest that the charging charging levels of tRNA (His) isoacceptors could play a regulatory role in determining the rate of protein synthesis under conditions of histidine starvation in normal cells. The mutant appears to be a potentially useful system for studying the pivotal role of tRNA charging in protein synthesis, assuming that the altered response in the mutant is caused by its altered synthetase.