Rate Of Total Protein Synthesis Research Articles

Oral Administration of Leucine Stimulates Ribosomal Protein mRNA Translation but Not Global Rates of Protein Synthesis in the Liver of Rats

The objective of the current study was to examine the role of the branched-chain amino acid (BCAA) leucine in the regulation of hepatic protein synthesis and ribosomal protein (rp) mRNA translation in vivo. Food-deprived (18 h) male rats (200 g) were orally administered saline (control) or 270 mg leucine, isoleucine or valine and killed 1 h later. Administration of any BCAA resulted in enhanced phosphorylation of eukaryotic initiation factor (eIF) 4E-binding protein-1 (4E-BP1) compared with controls. However, leucine was the most effective at stimulating phosphorylation of 4E-BP1 as well as the 70-kDa ribosomal protein S6 kinase (S6K1). Despite these effects on components of the translation initiation process, there were no differences in total protein synthesis rates among treatment groups. The distribution of rp (S4, S8, L26) and non-rp (albumin, beta-actin) mRNAs across sucrose density gradients showed that the preponderance of hepatic rp mRNAs in control rats was unloaded from polysomes. Of the BCAA, only leucine was the most effective in causing a shift in the distribution of rp mRNA to polysomes compared with controls. Non-rp transcripts remained mainly polysome-associated under all conditions. These results suggest that leucine is most effective among the BCAA in its ability to stimulate translation of rp mRNA in liver. Furthermore, the translation of rp mRNA is disjointed from rates of total protein synthesis in liver and related to the degree of S6K1 phosphorylation.

Mechanism‐related changes in the gene transcription and protein synthesis patterns of Haemophilus influenzae after treatment with transcriptional and translational inhibitors

High-resolution two-dimensional gel electrophoresis of pulse-labeled Haemophilus influenzae extracts allows for the separation and quantification of more than five hundred protein spots. We have determined the changes in the protein synthesis patterns triggered by treatment with inhibitors of transcription, Rifampicin (Rif) and translation, Chloramphenicol (Chl), Erythromycin (Ery), Fusidate (Fus), Puromycin (Pur), Kanamycin (Kan), Streptomycin (Str), and Tetracycline (Tet) relative to the total protein synthesis rate. More than 200 spots changed in intensity under at least one condition. With the exception of the aminoglycosides, Kan and Str, all inhibitors triggered a clear increase in the synthesis rates of ribosomal proteins and RNA polymerase subunits. Northern analysis of rpoA, rpoB, rpoC, and six ribosomal protein genes indicated induction of transcription as well as antitermination as part of the mechanism of the regulation of gene expression. Total RNA synthesis was increased after exposure to Chl, Ery, Fus, and Tet, whereas Str had no effect. Rif led to an almost complete shutdown of RNA synthesis. Exposure to Chl, Ery, Fus, Rif, and Tet resulted in a decrease in the concentration of the stringent factor, guanosine 5',3'-bis-diphosphate (ppGpp) whereas Str again had no effect. Thus, as in Escherichia coli, the response of H. influenzae to translational inhibitors appears to be mediated by the regulatory nucleotide ppGpp.

Mechanism-related changes in the gene transcription and protein synthesis patterns ofHaemophilus influenzae after treatment with transcriptional and translational inhibitors

High-resolution two-dimensional gel electrophoresis of pulse-labeled Haemophilus influenzae extracts allows for the separation and quantification of more than five hundred protein spots. We have determined the changes in the protein synthesis patterns triggered by treatment with inhibitors of transcription, Rifampicin (Rif) and translation, Chloramphenicol (Chl), Erythromycin (Ery), Fusidate (Fus), Puromycin (Pur), Kanamycin (Kan), Streptomycin (Str), and Tetracycline (Tet) relative to the total protein synthesis rate. More than 200 spots changed in intensity under at least one condition. With the exception of the aminoglycosides, Kan and Str, all inhibitors triggered a clear increase in the synthesis rates of ribosomal proteins and RNA polymerase subunits. Northern analysis of rpoA, rpoB, rpoC, and six ribosomal protein genes indicated induction of transcription as well as antitermination as part of the mechanism of the regulation of gene expression. Total RNA synthesis was increased after exposure to Chl, Ery, Fus, and Tet, whereas Str had no effect. Rif led to an almost complete shutdown of RNA synthesis. Exposure to Chl, Ery, Fus, Rif, and Tet resulted in a decrease in the concentration of the stringent factor, guanosine 5',3'-bis-diphosphate (ppGpp) whereas Str again had no effect. Thus, as in Escherichia coli, the response of H. influenzae to translational inhibitors appears to be mediated by the regulatory nucleotide ppGpp.

Sphingosylphosphorylcholine induces a hypertrophic growth response through the mitogen-activated protein kinase signaling cascade in rat neonatal cardiac myocytes.

The sphingolipid metabolites, sphingosine (SPH), SPH 1-phosphate (S1P), and sphingosylphosphorylcholine (SPC), can act as intracellular as well as extracellular signaling molecules. These compounds have been implicated in the regulation of cell growth, differentiation, and programmed cell death in nonmyocytes, but the effects of sphingolipid metabolites in cardiac myocytes are not known. Cultured neonatal rat cardiac myocytes were stimulated with SPH (1 to 10 micromol/L), S1P (1 to 10 micromol/L), or SPC (0.1 to 10 micromol/L) for 24 hours to determine the effects of sphingolipid metabolites on the rates of protein synthesis and degradation. Stimulation with SPC led to an increase in the total amount of protein, an accelerated rate of total protein synthesis, and a decrease in protein degradation in a dose-dependent manner. However, S1P had little effect and SPH had no effect on total protein synthesis. In addition, stimulation with SPC led to a 1.4-fold increase in myocardial cell size and enhanced atrial natriuretic factor gene expression. Pretreatment of the cardiac myocytes with pertussis toxin or PD98059 attenuated the SPC-induced hypertrophic growth response. Further, stimulation with SPC increased phosphorylation of mitogen-activated protein kinase (MAPK) and stimulated MAPK enzyme activity. Finally, endothelin-1 stimulated the generation of SPC in cardiac myocytes. The observation that SPC induces a hypertrophic growth response in cardiac myocytes suggests that SPC may play a critical role in the development of cardiac hypertrophy. The effects of SPC could be mediated, in part, by activation of a G protein-coupled receptor and a MAPK signaling cascade.

Mitochondrial protein synthesis in rainbow trout (Oncorhynchus mykiss) heart is enhanced in sexually mature males but impaired by low temperature

Throughout the life cycle of the rainbow trout (Oncorhynchus mykiss), the heart exhibits periods of enhanced growth. Two such instances are cardiac enlargement associated with sexual maturity in males and heart growth at seasonally low environmental temperatures. Heart growth includes a parallel increase in the number of mitochondria. These natural models of heart growth have been exploited to study protein synthesis directed by the mitochondrial genome. Methods were developed to assess protein synthesis in mitochondria isolated from the heart of rainbow trout. Protein synthesis was assessed by tracking the incorporation of l-[2,6-(3)H]phenylalanine into trichloracetic-acid-precipitable protein. Amino acid incorporation into mitochondrial protein was linear with respect to time and was inhibited by chloramphenicol. Radiolabel was selectively enhanced in molecular mass fractions over the same size range as polypeptides known to be encoded by the mitochondrial genome. Protein synthesis was measured in mitochondria isolated from sexually mature animals and from animals subjected to different thermal regimes. The relative ventricular mass of sexually mature male rainbow trout was significantly greater than that of sexually mature females (0. 104+/-0.004 versus 0.087+/-0.002; mean +/- s.e.m.). Mitochondria isolated from the heart of males synthesized protein at a faster rate than mitochondria isolated from the heart of females (0.22+/-0. 02 versus 0.11+/-0.02 pmol phenylalanine mg(-)(1 )protein min(-)(1)). That is, 'male' mitochondria are inherently predisposed to synthesize protein at faster rates. We speculate that the difference may result from higher levels of mitochondrial RNA in males than in females. Mitochondria isolated from the heart of sexually immature rainbow trout acclimated to 13 degrees C synthesized protein at the same rate at 25 degrees C (0.456+/-0.075 pmolphenylalanine mg(-)(1 )protein min(-)(1)) and 15 degrees C (0.455+/-0.027 pmol phenylalanine mg(-)(1 )protein min(-)(1)). However, the rate of protein synthesis was severely impaired at 5 degrees C (0.125+/-0.02 pmol phenylalanine mg(-)(1 )protein min(-)(1)). Since the rate of state 3 respiration by isolated mitochondria decreased in a linear fashion over the temperature range 25 to 5 degrees C, the rate of mitochondrial protein synthesis is not directly coupled to the rate of respiration. Thermal acclimation to 5 degrees C did not result in positive thermal compensation in either the rate of protein synthesis or the rate of oxygen consumption by isolated mitochondria. In a further series of experiments, total protein synthesis and oxygen consumption were measured in isolated myocytes. The rate of oxygen consumption by myocytes remained constant over the temperature range 25 to 5 degrees C. There was no difference in the rate of total cell protein synthesis between 25 degrees C (1.73+/-0. 29 pmol phenylalanine 10(6 )cells(-)(1 )h(-)(1)) and 15 degrees C (2. 12+/-0.19 pmol phenylalanine 10(6 )cells(-)(1 )h(-)(1)), but at 5 degrees C protein synthesis was substantially impaired to approximately one-sixth of the level observed at 15 degrees C. As such, rates of total cell protein synthesis were not directly coupled to rates of respiration and were curtailed at low temperature. In vitro studies show that mitochondria isolated from the heart of sexually mature male rainbow trout are inherently different from mitochondria isolated from the heart of females such that the former are able to synthesize protein at a faster rate. The rate of mitochondrial protein synthesis does not correlate with the greater than twofold changes in rates of oxygen consumption induced by acute changes in assay temperature, suggesting that protein synthesis is not directly coupled to rates of ATP or GTP synthesis.

Overexpression of the sarcolemmal calcium pump in the myocardium of transgenic rats.

The plasma membrane calmodulin-dependent calcium ATPase (PMCA) is a calcium-extruding enzyme controlling Ca2+ homeostasis in nonexcitable cells. However, its function in the myocardium is unclear because of the presence of the Na+/Ca2+ exchanger. We approached the question of the physiological function of the calcium pump using a transgenic "gain of function" model. Transgenic rat lines carrying the human PMCA 4 cDNA under control of the ventricle-specific myosin light chain-2 promoter were established, and expression in the myocardium was ascertained at the mRNA, protein, and functional levels. In vivo hemodynamic measurements in adult homozygous animals showed no differences in baseline and increased cardiac performance recruited by volume overload compared with controls. No differences between transgenic and control cardiomyocytes were found in patch clamp voltage dependence, activation/inactivation behavior of the L-type Ca2+ current, or fast [Ca2+]i transients (assessed by the Fura-2 method). To test whether the PMCA might be involved in processes other than beat-to-beat regulation of contraction/relaxation, we compared growth processes of neonatal transgenic and control cardiomyocytes. A 1.6- and 2.3-fold higher synthesis rate of total protein was seen in cells from transgenic animals compared with controls on incubation with 2% FCS for 24 hours and 36 hours, respectively. An effect of similar magnitude was observed using 20 micromol/L phenylephrine. A 1.4-fold- and 2.0-fold-higher protein synthesis peak was seen in PMCA-overexpressing cardiomyocytes after stimulation with isoproterenol for 12 hours and 24 hours, respectively. Because pivotal parts of the alpha- and beta-adrenergic signal transduction pathways recently have been localized to caveolae, we tested the hypothesis that the PMCA might alter the amplitude of alpha- and beta-adrenergic growth signals by virtue of its localization in caveolae. Biochemical as well as immunocytochemical studies suggested that the PMCA in large part was colocalized with caveolin 3 in caveolae of cardiomyocytes. These results indicate that the sarcolemmal Ca2+-pump has little relevance for beat-to-beat regulation of contraction/relaxation in adult animals but likely plays a role in regulating myocardial growth, possibly through modulation of caveolar signal transduction.

Bioreactor perfusion system for the long-term maintenance of tissue-engineered skeletal muscle organoids.

Three-dimensional skeletal muscle organ-like structures (organoids) formed in tissue culture by fusion of proliferating myoblasts into parallel networks of long, unbranched myofibers provide an in vivo-like model for examining the effects of growth factors, tension, and space flight on muscle cell growth and metabolism. To determine the feasibility of maintaining either avian or mammalian muscle organoids in a commercial perfusion bioreactor system, we measured metabolism, protein turnover. and autocrine/paracrine growth factor release rates. Medium glucose was metabolized at a constant rate in both low-serum- and serum-free media for up to 30 d. Total organoid noncollagenous protein and DNA content decreased approximately 22-28% (P < 0.05) over a 13-d period. Total protein synthesis rates could be determined accurately in the bioreactors for up to 30 h and total protein degradation rates could be measured for up to 3 wk. Special fixation and storage conditions necessary for space flight studies were validated as part of the studies. For example, the anabolic autocrine/paracrine skeletal muscle growth factors prostaglandin F2alpha (PGF2alpha) and insulin-like growth factor-1 (IGF-1) could be measured accurately in collected media fractions, even after storage at 37 degrees C for up to 10 d. In contrast, creatine kinase activity (a marker of cell damage) in collected media fractions was unreliable. These results provide initial benchmarks for long-term ex vivo studies of tissue-engineered skeletal muscle.

O.50 The synthesis rate of total liver protein, but not of albumin, decreases during a surgical trauma

O.50 The synthesis rate of total liver protein, but not of albumin, decreases during a surgical trauma

Suboptimal temperature‐dependent changes in the brain development and activity in Galleria mellonella larvae

The development of Galleria mellonella larvae is strongly affected by suboptimal temperature (18°C). One-day-old last-instar larvae react to 18°C with the arrest of further development for several months described as facultative larval diapause. The aim of this study was to find what type of changes, if any, in the brain correlate with the larval diapause induced by suboptimal temperature. Morphological analysis demonstrated the gradual inhibition of brain development. Paraldehyde-fuchsin (PAF) staining revealed cyclicity in the activity of the medial neurosecretory cells (M-NSC) in the larval brain. SDS-PAGE was used to examine the brain proteins of larvae reared at 30°C and at 18°C. The rate of protein synthesis in the brain of the last instar larvae kept at 18°C, measured as l-[35S]methionine incorporation during 2-h incubation in vitro, was only about 40% of the value characteristic for this tissue during normal development (at 30°C). Despite decrease in the rate of total protein synthesis, suboptimal temperature induced an increase in the level of two major brain proteins: 112 and 84 kDa. In SDS-PAGE analysis, these two proteins appear 21–28 days after transfer to the lower temperature. Whether these proteins are specific for induction of larval diapause of Galleria mellonella remains to be further investigated. Arch. Insect Biochem. Physiol. 38:66–73, 1998. © 1998 Wiley-Liss, Inc.

Effect of varying amino acid levels on protein metabolism in nephrotic rats during total parenteral nutrition.

In an attempt to determine appropriate diet in nephrotic syndrome, nephrotic rats, induced by puromycin aminonucleoside, were nourished by total parenteral nutrition fluid containing the same energy, but three different levels (1.65, 3.3, and 6.6%) of amino acids for 7 d. The fractional rate of total protein synthesis in the liver was determined by injecting a flooding dose of [3H]phenylalanine. The proportion of newly synthesized proteins retained and exported by the liver was estimated by injecting a tracer dose of [14C]leucine and then measuring the protein radioactivity remaining in the liver and present in the plasma after secretion was completed. Nephrotic animals synthesized more protein than control animals. Although the absolute synthesis rates of total protein in liver were increased with increasing amino acid administration, the absolute rates of synthesis of albumin were higher in the 3.3% group than in the other groups in nephrotic rats. However, kidney protein synthesis in nephrotic rats was higher in the 1.65% group than in the 3.3% group. Interestingly, the 3.3% group revealed the smallest urinary excretion of total protein and albumin. In addition, in the 3.3% group, plasma concentrations of total protein and albumin were higher, and plasma concentrations of total cholesterol and triglyceride were lower than in other groups. It was concluded that the 3.3% group, corresponding to a normal protein diet, has the greatest salutary effect on urinary protein excretion, followed by protein and lipid metabolism, in nephrotic rats. Not only protein intake but also the energy:protein ratio are important for diet therapy in nephrotic animals. The technique of total parenteral nutrition may be useful in defining the factors involved in glomerular permeability or permselectivity and intracellular protein metabolism.

Biochemical and ultrastructural alterations in prothoracic glands of Heliothis virescens (F.) (Lepidoptera: Noctuidae) last instar larvae parasitized by Cardiochiles nigriceps Viereck (Hymenoptera: Braconidae)

The present study describes both the ultrastructural and the biochemical alterations induced by the parasitoid Cardiochiles nigriceps Viereck on the prothoracic glands of the last (fifth) instar larvae of its host Heliothis virescens (F.). Gross morphological degeneration or signs of necrosis of the prothoracic gland cells were not observed. A rough endoplasmic reticulum more developed and electrondense than in non-parasitized controls was the only ultra-structural change occurring in the prothoracic glands of parasitized host last instar larvae. However, although structurally undegraded, 5 days after parasitoid oviposition into newly moulted host last (fifth) instar larvae, the prothoracic gland cells appeared to be functionally dead and became permeable to trypan blue. In contrast, cells of the prothoracic glands explanted on day 4 of the fifth instar, 3 days after parasitism, performed as above, were still alive and did not absorb the trypan blue, as observed in synchronous non-parasitized controls. These glands were used to study in vitro the major biochemical alterations at cellular level induced by the parasitoid. Ecdysteroid production, RNA synthesis, protein synthesis and phosphorylation were studied in response to crude prothoracicotropic hormone (PTTH) extract, forskolin and dibutyryl cAMP (dbcAMP), these latter compounds mimicking the PTTH action at different levels of the signal transduction pathway. The prothoracic glands of H. virescens last instar larvae, parasitized by C. nigriceps, showed a severely depressed biosynthesis of ecdysteroids, which was not resumed with any of the stimulating treatments performed. This severely depressed biosynthetic activity was associated with a remarkable reduction in the rate of total protein synthesis and to a marked underphosphorylation of those proteins normally phosphorylated when healthy glands are challenged with dbcAMP. However, the early steps of the PTTH signal transduction pathway in the host prothoracic glands are apparently not altered by C. nigriceps parasitism. This is supported by the identical effect of PTTH and dbcAMP treatments on RNA synthesis in the prothoracic glands of both parasitized and non-parasitized larvae. In fact, in both cases a reduced level of [ 3H]uridine incorporation into the total RNA synthesized in vitro was observed in stimulated glands. These experimental data would suggest the occurrence of a parasitoid-induced block of protein synthesis translation in host prothoracic glands, partly determined by the marked underphosphorylation of key target regulatory proteins.

The synthesis rates of total liver protein and plasma albumin determined simultaneously in vivo in humans

Although the metabolism of liver-derived plasma proteins such as albumin has been extensively studied, human hepatic protein synthesis as a whole has not been well characterized, because a reproducible model for obtaining human liver tissue has not been available. In this study, the fractional synthesis rates of total liver protein and albumin in vivo were determined simultaneously in nine subjects undergoing elective laparoscopic cholecystectomy. L-[2H5]phenylalanine (45 mg/kg body wt) was administered for 10 minutes intravenously. Blood samples were collected at regular intervals for 90 minutes and a liver biopsy specimen was taken at 35 +/- 7 minutes. The enrichments of plasma free phenylalanine, plasma albumin, and total liver protein were measured with gas chromatography mass spectrometry (GC-MS). The fractional synthesis rate (FSR) of total liver protein was 24.7 +/- 3.1 %/d (mean +/- SD), and that of albumin was 5.9 +/-1.2%/d. The amount of albumin synthesized per day (absolute synthesis rate, ASR) was 109 +/- 21 mg/kg body wt. No correlation between FSR of total liver protein and ASR of albumin was found. It is concluded that the technique of obtaining liver tissue specimens during laparoscopic surgery may serve as a human in vivo model to study total liver protein synthesis. The fractional synthesis rate of total liver proteins (stationary and exported), equals approximately 25% of the liver protein content daily. Within the range of values of this study, the absolute synthesis rate of albumin was not correlated to the fractional synthesis rate of total liver protein.(Hepatology 1997 Jan;25(1):154-8)

The synthesis rates of total liver protein and plasma albumin determined simultaneously in vivo in humans.

Although the metabolism of liver-derived plasma proteins such as albumin has been extensively studied, human hepatic protein synthesis as a whole has not been well characterized, because a reproducible model for obtaining human liver tissue has not been available. In this study, the fractional synthesis rates of total liver protein and albumin in vivo were determined simultaneously in nine subjects undergoing elective laparoscopic cholecystectomy. L-[2H5]phenylalanine (45 mg/kg body wt) was administered for 10 minutes intravenously. Blood samples were collected at regular intervals for 90 minutes and a liver biopsy specimen was taken at 35 +/- 7 minutes. The enrichments of plasma free phenylalanine, plasma albumin, and total liver protein were measured with gas chromatography mass spectrometry (GC-MS). The fractional synthesis rate (FSR) of total liver protein was 24.7 +/- 3.1 %/d (mean +/- SD), and that of albumin was 5.9 +/-1.2%/d. The amount of albumin synthesized per day (absolute synthesis rate, ASR) was 109 +/- 21 mg/kg body wt. No correlation between FSR of total liver protein and ASR of albumin was found. It is concluded that the technique of obtaining liver tissue specimens during laparoscopic surgery may serve as a human in vivo model to study total liver protein synthesis. The fractional synthesis rate of total liver proteins (stationary and exported), equals approximately 25% of the liver protein content daily. Within the range of values of this study, the absolute synthesis rate of albumin was not correlated to the fractional synthesis rate of total liver protein.

Decreased total ventricular and mitochondrial protein synthesis during extended anoxia in turtle heart.

The turtle heart provides a model system to study the effects of anoxia on protein synthesis without the potentially confounding factor of contractile failure and decreased ATP levels. Protein synthesis, as measured by 3H-labeled phenylalanine incorporation, was studied under conditions of normoxia and anoxia in isolated perfused turtle [Trachemys (= Pseudemys) scripta elegans] hearts at 15 degrees C. Heart rate, cardiac output, and ventricular pressure development were unaffected by 2 or 3 h of anoxia. Despite the anoxia, energy levels in the heart were presumably still high, since contractility was maintained. RNA content of ventricle decreased after anoxic perfusion. Rates of total protein synthesis rates in ventricle were threefold lower under anoxia than under normoxia. These findings suggest that the total level of RNA is one determinant of protein synthesis. Incorporation of label into protein extracted from mitochondria was also assessed. The ratio of mitochondrial to whole ventricular protein synthesis was significantly lower after anoxia, revealing preferential control mechanisms under anoxia between the synthesis of total cellular protein and protein destined for mitochondria. Isolated mitochondria were still coupled after 2 or 3 h of anoxia. In effect, the mitochondria enter into a state of hypometabolism in terms of rates of ATP synthesis and protein synthesis, but functional integrity is maintained. The decrease in protein synthesis in general and mitochondrial protein synthesis in particular may represent an adaptation to allow the partitioning of the available energy resources toward mechanical function during anoxia.

P.64 The synthesis rates of total liver protein and albumindetermined simultaneously in vivo in man

P.64 The synthesis rates of total liver protein and albumindetermined simultaneously in vivo in man

Comparison of media and sera used for in vitro culture of equine superficial digital flexor tendon explants

Abstract Objective To compare the effects of different commercial nutrient media and sera on protein synthesis and maintenance of cellular density in cultures of the equine superficial digital flexor tendon (SDFT). Animals 8 healthy 2- to 4-year-old horses. Procedure First Dulbecco's modified Eagle's medium, Ham's F12 nutrient mixture, RPMI 1640 medium, minimum essential medium with Earle's salts, minimum essential medium with Hanks' salts, and a Dulbecco's modified Eagle's medium/Ham's F12 nutrient mixture with 10% fetal bovine serum (FBS) were compared. Then FBS, fetal equine serum, and donor horse serum, each at 5, 10, and 15% in RPMI 1640 medium, were compared. Explants were cultured in roller bottles at 37 C and aerated (50% O2/45% N2/5% CO2) daily. Rates of [3H]- proline incorporation were used as a measure of the rates of total protein and collagen synthesis on days 13 and 28. Matrix cellular density of explants at days 14 and 28 was measured by computerized image analysis. Results Equine SDFT explants were cultured in all media for up to 4 weeks. Proline incorporation was greatest in Ham's F12 nutrient mixture and in RPMI 1640 medium, with the concentration of proline in medium correlating to the in vitro response. Total proline incorporation was greater in 15% FBS than in 5 or 10% FBS. Other differences among sera were not detected. Matrix cell density in 15% donor horse serum was equivalent to that in uncultured controls and higher than that in most other sera at week 2. Conclusion The in vitro SDFT culture system described may be used in future studies to enhance knowledge of the biological and biochemical characteristics of intrinsic tendon healing. (Am J Vet Res 1996;57:1118–1123)

N tau-methylhistidine turnover in skeletal muscle cells measured by GC-MS.

A method that employs gas chromatography-mass spectrometry has been developed to measure N tau-methylhistidine (3-methylhistidine; 3-MH) synthesis and release from skeletal muscle myotubes in vitro. It shows excellent linearity (0.9999) over the range studied (0-4 nmol), high recovery (92.6%), and low coefficient of variation (1.6%). 3-MH release from myotubes was essentially linear over a 96-h incubation, whereas the loss of 3-MH from cell protein accelerated with increasing time, an effect due, at lest in part, to decreasing rates of total protein synthesis. When incubated in either glutamine-free or methionine-free medium for 48 h, 3-MH in cell protein and appearing in the medium were greatly reduced compared with the 48-h controls, suggesting that hypertrophy was greatly reduced. Similar but lesser trends were observed with adenosine 3',5' -cyclic monophosphate. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) appeared to both stimulate 3-MII synthesis and inhibit its release during a 48-h incubation. The development of this method facilitates detailed investigation into the mechanisms through which agents such as TPA regulate myofibrillar protein degradation.

Chronic Low Protein Intake Reduces Tissue Protein Synthesis in a Pig Model of Protein Malnutrition

To determine the effect of severe chronic protein deficiency on protein synthesis in different tissues and total protein in plasma, and on plasma biochemical constituents involved in amino acid metabolism, we fed diets containing either 20 or 3% protein to two groups of four age-matched piglets. After consuming the diets for 8 wk, the pigs received a primed-constant infusion of 2H3-leucine for 8 h to measure the fractional synthesis rates (FSR) of tissue protein and total protein in plasma. Plasma urea and amino acid concentrations, particularly indispensable amino acids, were significantly lower in protein-deficient pigs. Fractional protein synthesis rates were lower in skin by 66% (P < 0.01), in jejunal mucosa by 50% (P < 0.05), in masseter muscle by 40% (P < 0.05), and in liver by 25% (P < 0.02); the fractional synthesis rate of the longissimus muscle was not different than controls. Although plasma protein concentration was significantly (P < 0.01) lower in protein-deficient pigs, the fractional synthesis rate of the total intravascular plasma protein pool was not different. We conclude that adaptation to a low protein diet involves a reduction in the rate of protein synthesis in most body tissues, with the most marked changes occurring in skin and intestine, two tissues which frequently exhibit severe functional impairment in protein malnutrition.

Muscle and Liver Protein Synthesis Adapt Efficiently to Food Deprivation and Refeeding in 12-Month-Old Rats

Our aim was to analyze mechanisms involved in the adaptation of protein metabolism to food deprivation and refeeding in adult rats. Twelve-month-old rats, which had been food-deprived for 113 h and refed for 6 h, were injected subcutaneously with a flooding dose of valine (with 50% [113C]-L-valine) to measure in vivo protein synthesis in tibialis anterior, soleus and liver. Protein and RNA contents were also measured. In both muscles, protein mass was maintained during food deprivation. Due to a drop in protein synthetic capacity (Cs), total and myofibrillar protein synthesis rates were reduced in food-deprived rats and were not stimulated by a 6-h refeeding. In contrast, protein levels were maintained lower than RNA levels in liver during food deprivation, and Cs was higher than in fed rats. Protein synthesis rates and ribosomal efficiency were reduced in food-deprived rats. Due to maintenance of protein synthetic capacity, there was a rapid stimulation of liver protein synthesis with refeeding, which induced a significant rise in protein mass (also related to an inhibition of protein degradation). In conclusion, coordinated responses of liver and muscles allowed a sparing of muscle proteins during food deprivation and a rapid recovery of liver proteins during refeeding. Control of ribosome quantity could play a critical role in these adaptations in tissue protein synthesis in adult rats.

Physiological responses of Pseudomonas putida KT2442 to phosphate starvation.

The physiological responses of Pseudomonas putida KT2442 to phosphate starvation were examined with respect to cell morphology, qualitative demonstration of the accumulation of the intracellular storage component poly-3-hydroxyalkanoate (PHA), cellular ATP and ribosome content, and the rate of total protein synthesis. Upon prolonged incubation under phosphate-limiting conditions, the number of viable cells decreased by two to three orders of magnitude during the first 3 weeks. However, after this decline, viability of the cultures remained remarkably constant for many weeks. The cells remained rod-shaped under phosphate starvation conditions with a tendency to swell in parallel with the accumulation of PHA. Protein synthesis and ribosome concentration were gradually reduced, and ATP levels dropped to very low values after the onset of starvation; later, however, there was a return to near-normal ATP concentrations. Evidence was obtained that the strong selective pressure imposed by phosphate deprivation forces the selection of mutants with a competitive advantage. These mutants are able to grow, possibly utilizing nutrients derived from dead cells, and eventually take over the cultures. One frequently encountered mutant formed smaller colonies on rich solidified medium and displayed an altered cell morphology. This mutant was isolated and further characterized. By employing a bioluminescence-based marker system, we demonstrated that this mutant is able to replace wild-type cells in mixed culture experiments. Thus, long-term phosphate-deprived cultures represent dynamic regimes that can undergo population shifts.