Elevated Synthesis Rates Research Articles

A High-Turnover Phenotype of Calsequestrin with Elevated Rates of Synthesis and Degradation Replaces Normal Calsequestrin in Heart Failure

Left ventricular hypertrophy involves an increase in the size of cardiomyocytes. A central feature of the phenotype is accelerated protein synthesis, but changes are complex, involving increases in protein translation capacity, efficiency, and turnover. We purified calsequestrin (CSQ), a major protein component of the Ca2+ release protein complex, from frozen heart samples of sham control mongrel dogs, or dogs subjected to either tachycardia-induced or microembolism (ischemia)-induced heart failure. We found that the levels of CSQ protein did not change significantly as a result of either of these two treatments. CSQ recovered from control dog heart tissue was, however, fundamentally different from that present in normal dogs. CSQ undergoes both co-translational addition of an N-linked glycan as well as an unusual co-translational phosphorylation of its C-terminus. In control heart tissue, CSQ exhibited its characteristic polymorphic pattern of post-translational demannosylation and dephosphorylation. Failing hearts, however, was marked by large percentages of CSQ molecules (20-40% of total levels) with Man9,8 glycans, reflecting more newly synthesized protein in rough ER. In addition, there was a significant “retrograde shift” in the average glycan processing of CSQ glycans, with longer-lived CSQ molecules replaced with shorter-lived. Such changes indicate a major degradation and biosynthesis (increased turnover) phenotype for CSQ, perhaps representing changes needed to support increased hypertrophic growth. Two phenotypes are also evident using a SDS gel-based analysis. Taken together with our recent findings that CSQ is synthesized around myonuclei and phosphorylated on its C-terminus during its biosynthesis, we propose that qualitative alterations in CSQ metabolism occur in hypertrophic heart. In this proposed model, increased CSQ synthesis leads to increased CSQ in perinuclear cisternae, while increased CSQ degradation prevents buildup of protein in junctional SR cisternae.

Postnatal ontogeny of skeletal muscle protein synthesis in pigs1,2

The neonatal period is characterized by rapid growth and elevated rates of synthesis and accretion of skeletal muscle proteins. The fractional rate of muscle protein synthesis is very high at birth and declines rapidly with age. The elevated capacity for muscle protein synthesis in the neonatal pig is driven by the high ribosome content and, together with an increased efficiency of the translation process, promotes accelerated protein synthesis rates. Feeding profoundly stimulates muscle protein synthesis in neonatal pigs and the response decreases with age. The feeding-induced stimulation of muscle protein synthesis is modulated by an enhanced sensitivity to the postprandial increase in insulin and amino acids. The developmental decline in the response to insulin and amino acids parallels a marked decrease in the feeding-induced activation of translation initiation factors that regulate the binding of mRNA to the 40S ribosomal complex. The abundance and activation of many known positive regulators of the nutrient- and insulin-signaling pathways that are involved in translation initiation are high, whereas those of many negative regulators are low in skeletal muscle of younger pigs. Thus, the activation and(or) abundance of the positive regulators, such as the insulin receptor, insulin receptor-substrate-1, phosphoinositide-3 kinase, phosphoinositide-dependent kinase-1, protein kinase B, mammalian target of rapamycin, raptor, ribosomal protein S6 kinase-1, eukaryotic initiation factor (eIF) 4E-binding protein 1, and eIF4E associated with eIF4G, are greater in 7-d-old pigs than in 26-d-old pigs. The activation of negative regulators, including protein tyrosine phosphatase-1B, phosphatase and tensin homologue deleted on chromosome 10, protein phosphatase 2A, and tuberous sclerosis complex 1/2, are lower in 7-d-old pigs than in 26-d-old pigs. Thus, the developmental decline in the stimulation of skeletal muscle protein synthesis by insulin and amino acids is due in part to the developmentally related decrease in the activation of the signaling pathways that lead to translation initiation.

Measurement and meaning of apolipoprotein AI and apolipoprotein B plasma levels

Apolipoproteins AI and B are structural components of lipoprotein particles, and also determinants of the metabolic fate of the encapsulated lipid, cholesterol and triglyceride. Development of accurate assays for these apolipoproteins has opened the way for their use as predictors of coronary heart disease risk. Interpretation of AI and apo B levels is best undertaken with background knowledge of the metabolic status of an individual, especially the lipolytic capacity as reflected in the triglyceride concentration. Those with raised triglyceride, in general, not only have an elevated apo B/apo AI ratio, but also apo B-containing lipoproteins with a prolonged residence time and hence ample opportunity for modification and damage. Assessment of apolipoprotein levels is an aid to risk prediction and can be useful in tailoring treatment.

Osteoblast cytoskeletal modulation in response to mechanical strain in vitro

The structural integrity of microfilaments has been shown to be necessary for the signal transduction of mechanical stimuli within osteoblasts. Qualitative and quantitative changes within the cytoskeleton of osteoblasts may therefore be crucial components of the signal transduction processes of these cells in response to mechanical stimulation. Avian osteoblasts were strained with a device that deforms a flexible, cell-laden membrane at a defined frequency and intensity in a uniform biaxial manner. We examined the effects of mechanical strain on the accumulation of protein and the expression of the major cytoskeletal elements and specific integrin-binding (arginine-glycine-aspartic acid) proteins of these cells. Mechanical strain increased the level of total extracellular matrix-accumulated fibronectin by approximately 150% and decreased that of osteopontin by approximately 60% but had no quantifiable effect on the accumulation of beta1 integrin subunit or collagen type I. An examination of the major elements of the cytoskeleton demonstrated that neither the level of actin nor that of the intermediate filament protein vimentin changed; however, the amount of tubulin decreased by approximately 75% and the amount of vinculin, a major protein of focal adhesion complexes, increased by approximately 250%. An analysis of protein synthesis by two-dimensional gel electrophoresis of [35S]methionine-labeled cytoskeletal proteins demonstrated that the changes in the accumulation of vinculin and tubulin resulted from their altered synthesis. Messenger RNA analysis confirmed that the changes in accumulation and protein synthesis observed for vinculin, fibronectin, and osteopontin were controlled at a pretranslational level. Immunofluorescent microscopy demonstrated that mechanical strain led to increased formation and thickening of actin stress fibers, with a commensurate dissociation in microtubules and a clear increase in levels of vinculin at the peripheral edges of the cells. In conclusion, the elevated rate of synthesis and the increased accumulation of vinculin and fibronectin, as well as the increase in the number and size of stress fibers and focal adhesion complexes, suggest that mechanical strain leads to a coordinated change both in the cytoskeleton and in extracellular matrix proteins that will facilitate tighter adhesion of an osteoblast to its extracellular matrix.

Effects of hypoxia and anoxia on the ex vivo release of prostaglandins from mouse cortical slices.

Arachidonic acid is transiently accumulated in the brain as a result of a variety of pathological conditions. The synthesis and release of some of its metabolites, namely, prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) from cortical slices of mice were studied following exposure to 6 min of hypoxia (7% O2), 45 s of anoxia, and 5 min-4 h of reoxygenation following anoxia. Hypoxia induced a slight increase in the rate of TXB2 release and a slight decrease in the rate of PGE2 release, whereas 6-keto-PGF1 alpha was unaffected. Anoxia (45 s) followed by reoxygenation induced a transient increase in the release of PGE2 and of 6-keto-PGF1 alpha with a return to the normal rate at 30 min and 2 h of recovery, respectively. However, the rate of TXB2 synthesis and release reached its peak (twofold increase) after 1 h and remained significantly higher than the control rate even after 4 h of normal air breathing. Our results demonstrate that hypoxia and anoxia, even of short duration, selectively trigger the activity of thromboxane synthetase and that this elevated rate of synthesis and release persists long after normal oxygen supply is restored. We suggest that enhanced thromboxane synthesis, with normal prostacyclin levels, might have a role in the pathophysiology of ischemic cell damage.

Intra-blood-brain barrier IgG synthesis in cerebral cysticercosis.

Patients with central nervous system cysticercosis show elevated binding of cerebrospinal fluid (CSF) IgG to homogenized cysticercus. To determine whether any of the CSF IgG was the result of de novo intra-blood-brain barrier (BBB) synthesis, CSF and serum samples from six patients were examined for elevated rate of synthesis and oligoclonal bands. Five of the six patients had increased intra-BBB IgG synthesis rate and four of these patients also had oligoclonal IgG bands present in the CSF that were absent in the serum. These results demonstrate intra-BBB IgG synthesis similar to that observed in other infectious and inflammatory diseases of the central nervous system.

Developmental changes in fatty acid synthesis in interscapular brown adipose tissue of lean and genetically obese (ob/ob) mice.

Fatty acid synthesis was measured in vivo with 3H2O in interscapular brown adipose tissue of lean and genetically obese (ob/ob) mice. At 26 days of age, before the development of hyperphagia, synthesis in brown adipose tissue was higher in the obese than in the lean mice; synthesis was also elevated in the liver, white adipose tissue and carcass of the obese mice. At 8 weeks of age, when hyperphagia was well established, synthesis remained elevated in all tissues of the obese mice, with the exception of brown adipose tissue. Elevated synthesis rates were not apparent in brown adipose tissue of the obese mice at 14 days of age, nor at 35 days of age. These results demonstrate that brown adipose tissue in ob/ob mice has a transitory hyperlipogenesis at, and just after, weaning on to a low-fat/high-carbohydrate diet. Once hyperphagia has developed, by week 5 of life, brown adipose tissue is the only major lipogenic tissue in the obese mice not to exhibit elevated rates of fatty acid synthesis; this suggests that insulin resistance develops much more rapidly in brown adipose tissue than in other lipogenic tissues of the ob/ob mouse.

Possible regulation of macromolecular biosynthesis in mammalian cells by a novel dinucleoside polyphosphate (HS3) produced during step-down growth conditions.

AbstractShortly after the withdrawal of L‐glutamine from the growth medium of Chinese hamster ovary (CHO) cells, the rate of synthesis of a bizarre dinucleoside polyphosphate, HS3, increased by 5‐ to 6‐fold. This elevated rate of synthesis was maintained for six hours before it gradually declined to basal level 22 hours later. The pool size of HS3 increased and decreased coincidentally with rate changes. Withdrawal of L‐isoleucine did not affect HS3 biosynthesis. A glycine, adenosine, thymidine (GAT−) auxotroph of CHO cells accumulated HS3 when adenosine, not glutamine, was withdrawn. Replenishment of either glutamine (“wild type” cells) or adenosine (GAT− cells) caused an immediate depletion of HS3 intracellularly.When HS3 accumulated in CHO cells, DNA and RNA synthesis decreased and, vice versa. A similar correlation was not seen for protein synthesis. But, inhibition of protein synthesis by either puromycin or cycloheximide, and of RNA biosynthesis by actinomycin D facilitated HS3 depletion in L‐glutamine starved cells.Mutant CHO cells that are deficient in purine salvage metabolism, HGPRT− (hypoxanthine‐guanine phosphoribosyltransferase) failed to deplete their accumulated HS3 when fed with hypoxanthine, whereas the “wild type” CHO cells responded accordingly. The available data suggest that HS3 metabolism is connected with de novo and salvage pathways of nucleotide biosynthesis, and may play a crucial role in regulating nucleic acid metabolism in CHO cells under conditions of nutritional stress.