Incorporation Of 14C-phenylalanine Research Articles

Competitive NFAT Bottleneck For Transcriptional Activation of Endogenous Ca2+ ATPASE (SERCA2) in Adrenergic Hypertrophy

A prominent feature of cardiac hypertrophy and failure is reduced SERCA2 expression and deficient Ca2+ signaling. To uncover a causal relationship between hypertrophy and downregulation of SERCA2, we use neonatal rat cardiac myocytes where SERCA2 transcription can be increased by exposure to 10 nM thapsigargin (TG), which allows cytosolic Ca2+ rise and calmodulin activation of calcineurin (CN). This SERCA2 rise is markedly reduced by cyclosporine (CsA), which inhibits CN phosphatase, and increased by KN-93, a calmodulin activated kinase (CAMKII) inhibitor which relieves CAMKII dependent phosphorylation and inhibition of CN. These CsA and KN-93 effects are also produced on TG enhanced luciferase expression, under the control of an NFAT (Nuclear factor of activated T Cells) dependent promoter. We conclude that NFAT dephosphorylation is a limiting factor for SERCA2 transcription. Exposure of myocytes to PE yields adrenergic hypertrophy, with rise of Atrial Natriuretic Factor (ANF) transcript, protein incorporation of 14C-phenylalanine and fluorescent staining of actin, while SERCA2 is downregulated. The adrenergic response can be reproduced by direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate, indicating that inhibition of glycogen synthase kinase (GSK3β) by PKC and consequent reduced NFAT phosphorylation, as well as histone deacetylase (HDAC) phosphorylation by PKC activated MAPK, are involved in the mechanism of adrenergic hypertrophy. The hypertrophy response is markedly reduced by CN inhibition by CsA, indicating that CN dephosphorylation and nuclear import of NFAT play an important role in the development of hypertrophy, and SERCA2 downregulation is produced by competitive utilization of NFAT by the hypertrophy program. Interestingly, CAMKII inhibition limits the development of hypertrophy, emphasizing a rate limiting role of CAMKII dependent phosphorylation and nuclear export of HDAC in the extensive transcriptional hypertrophic program (Supported by 5 R01 HL069830-08).

SB-271258, A major metabolite in the dog of the thiazolidinedione, rosiglitazone, stimulates protein synthesis in re-differentiated rat cardiomyocytes in vitro

In dogs, chronic administration of thiazolidinediones causes cardiac hypertrophy in vivo at high doses. The hypertrophic action of rosiglitazone in dogs might be attributed to production of SB-271258 (a major metabolite in dogs but minor in rats and man), rather than a direct effect on myocardium. The hypothesis that SB-271258 had potential to initiate cardiomyocyte hypertrophy or to modify responses elicited by other hypertrophic stimuli was tested in an in vitro bioassay utilising adult rat ventricular cardiomyocytes. SB-271258 increased protein and incorporation of 14C-phenylalanine, a marker of protein synthesis, in rat cardiomyocytes maintained in serum-free culture (24 h), maximally at 1 μM by 15.0% and 9.1%, respectively. In the presence of serum (10% v/v), SB-271258 elicited a moderate trophic effect: cellular protein and incorporation of 14C-phenylalanine were increased, maximally at 100 nM by 31.7% and 36.3%, respectively, above basal values (18.6% and 13.3% increases above serum response). In the presence of IGF-1 (10 nM) plus SB-271258, protein synthesis was increased, maximally by 45.5% above basal value (increase of 6.9% above IGF-1 alone). In contrast, SB-271258 attenuated the increase (12.0%) in cellular protein elicited by IGF-1. In re-differentiated cardiomyocytes, a model of relevance to established hypertrophy, SB-271258 (1 nM–1 μM) elicited a marked trophic effect per se, as evidenced by the maximum increase (at 100 nM), in protein synthesis of 24.5%. In conclusion, these data imply that cardiac hypertrophy associated with chronic administration of rosiglitazone in dogs in previous in vivo studies might be partly attributable to production of the metabolite SB-271258 since this metabolite was shown to elicit trophic effects directly on rat cardiomyocytes.

Interleukin-15 decreases proteolysis in skeletal muscle: A direct effect

Incubation of rat isolated skeletal muscles (extensor digitorum longus) in the presence of 100 ng/ml of human recombinant interleukin-15 (IL-15) resulted in a significant decrease in total proteolytic rate, while it had no effect on total protein synthesis as measured by the incorporation of (14)C-phenylalanine into muscle protein. In addition, IL-15 had no effect on either amino acid uptake (as determined by the tissue uptake of labelled [1-(14)C]MeAIB) or alanine utilization by incubated skeletal muscles. Similarly, a single injection of IL-15 (100 microg/kg) in vivo did not result in any changes in amino acid uptake (as measured by the tissue uptake of alpha-[1-(14)C]AIB) or alanine metabolism, with the exception of alanine carbon incorporation into lipids, which was significantly increased in adipose tissue as a result of IL-15 administration. The results suggest that the main mechanism involved in the anabolic effects of IL-15 in skeletal muscle relies on a decrease in the proteolytic rate.

Role of phosphatidylinositol 3-kinase activation in the hypertrophic growth of adult ventricular cardiomyocytes.

The present study investigated whether activation of phosphatidylinositol 3-kinase (PI3-kinase) is involved in the stimulation of hypertrophic growth of adult ventricular cardiomyocytes under alpha- or beta-adrenoceptor stimulation. Adult ventricular rat cardiomyocytes were used either directly after isolation (day 1 culture) or after cultivation for 6 days in presence of 20% fetal calf serum (day 7 culture). PI3-kinase activity was determined in extracts of cardiomyocytes after immunoprecipitation with an antibody against the p85 subunit of PI3-kinase. The influence of PI3-kinase inhibition on myocardial growth was determined using the specific PI3-kinase inhibitors wortmannin and LY294002. In day 1 cultures alpha-adrenoceptor stimulation, but not beta-adrenoceptor stimulation caused activation of PI3-kinase. In response to alpha-adrenoceptor stimulation but not beta-adrenoceptor stimulation an acceleration of protein synthesis (incorporation of 14C-phenylalanine) and an increase in the total masses of cellular protein and RNA was observed. In these cultures inhibition of PI3-kinase attenuated the acceleration of protein synthesis and the increase in cellular masses of protein or RNA in response to alpha-adrenoceptor stimulation. In day 7 cultures alpha- and beta-adrenoceptor stimulation caused activation of PI3-kinase and increased protein synthesis. In these cultures inhibition of PI3-kinase attenuated the growth response to alpha- and beta-adrenoceptor stimulation. PI3-kinase activation via protein kinase C-dependent or cAMP-dependent pathways is required for hypertrophic growth of adult cardiomyocytes.

The anabolic effects of IGF-1 in skeletal muscle after burn injury are not caused by increased cell volume.

In a recent report, insulin-like growth factor 1 (IGF-1) stimulated protein synthesis and inhibited protein breakdown in skeletal muscle after bum injury. The mechanism of the anabolic effects of IGF-1 in skeletal muscle is not known. We tested the hypotheses that IGF-1 stimulates protein synthesis and inhibits protein breakdown in skeletal muscle secondary to cell swelling and that cell swelling in itself induces an anabolic response in muscle tissue. Extensor digitorum longus muscles from control and burned rats were incubated in the absence or presence of 1 microg/mL of IGF-1. Protein synthesis and breakdown rates were determined by measuring incorporation of 14C-phenylalanine into protein and net release of tyrosine, respectively. Cell volume was measured by determining wet and dry weight and by using 3H-mannitol as an extracellular marker. IGF-1 stimulated protein synthesis and inhibited protein breakdown in muscles from nonburned and burned rats without influencing cell volume. Incubating muscles in hypo-osmotic medium increased cell volume by 17% and inhibited protein breakdown by 14% but did not influence protein synthesis. The anabolic effects of IGF-1 in skeletal muscle are not caused by increased cell volume. The results differ from those reported previously in liver cells in which the anabolic effects of IGF-1 were associated with cell swelling. The role of changes in cell volume in the regulation of protein metabolism may be different in skeletal muscle than in other tissues.

Influence of pHi and creatine phosphate on alpha-adrenoceptor-mediated cardiac hypertrophy.

Stimulation of alpha-adrenoceptors on ventricular cardiomyocytes isolated from adult rat hearts leads to cellular alkalization, increases of creatine phosphate concentration, RNA mass, and protein synthesis. This study investigated whether the increase of creatine phosphate concentrations is causally linked to the hypertrophic response of cardiomyocytes under alpha-adrenoceptor stimulation. Cellular alkalization achieved with phenylephrine (10 microM), an alpha-adrenoceptor agonist, was abolished in the presence of the sodium-proton-exchange (NHE)-inhibitor HOE 694 (1 microM). HOE 694 inhibited also the alpha-adrenoceptor-mediated increase in cellular creatine phosphate and the increase in cellular RNA mass. The phenylephrine-induced stimulation of protein synthesis (determined by incorporation of 14C-phenylalanine) was reduced by one-third when HOE 694 was present. beta-Guanidinopropionic acid was added to cardiomyocytes to reduce cellular creatine phosphate concentrations. In these cultures, alpha-adrenoceptor stimulation activated NHE, but creatine phosphate concentrations were not increased. Protein synthesis was augmented to the same extent as in control cultures, but total RNA mass did not increase. From these results we conclude that alpha-adrenoceptor stimulation causes the increase in protein synthesis via activation of NHE, but independent of the concomitant increase in creatine phosphate contents. The effect of alpha-adrenoceptor stimulation on total RNA mass (translational capacity) is also caused by NHE activation, but depends on the changes in creatine phosphate contents as well.

Metabolic and structural effects of insulin-like growth factor-I and high-protein diet on dystrophic hamster skeletal muscle.

In muscular dystrophy (MD) there is an imbalance between muscle protein synthesis and protein degradation, which results in a net muscle catabolism, along with muscle wasting and weakness. Using a dystrophic hamster model (BIO 53.58), we examined the chronic (8 weeks) effects of two factors that may enhance muscle protein synthesis and inhibit protein degradation, namely, insulin-like growth factor-I (rhIGF-I) and high-protein diet (HPD). Protein synthesis was determined by measuring the incorporation of 14C phenylalanine into perfused leg muscle, while protein degradation was calculated from the release of tyrosine from the same perfused muscle. Urinary 3-methylhistidine excretion was used as an indicator of myofibrillar degradation. Treatment of dystrophic hamsters with rhIGF-I, HPD, or a combination of the two for 8 weeks resulted in significant decreases in total and myofibrillar degradation when compared with untreated dystrophic animals (P < 0.05) but had minimal effects on protein synthesis. Significant morphologic improvements (P < 0.05), including a normalization and greater uniformity of muscle fibers, were also seen in rhIGF-I- and rhIGF-I + HPD-treated animals. rhIGF-I and HPD were effective in reducing the excessive proteolysis seen in dystrophic muscle, and this reduced proteolysis resulted in improvement of muscle morphology.

The effect of photodynamic treatment of yeast with the sensitizer chloroaluminum phthalocyanine on various cellular parameters.

Photodynamic treatment of Kluyveromyces marxianus with chloroaluminum-phthalocyanine resulted in loss of clonogenicity. Several parameters were studied to identify targets that could be related to loss of colony-forming capacity. Inhibition of various plasma membrane-bound processes was observed, such as substrate transport and plasma membrane ATPase activity. Moreover, K+ loss from the cells was observed. Photodynamic treatment also reduced the activity of various enzymes involved in energy metabolism, thereby decreasing the cellular ATP level. It will be discussed however that none of these processes is likely to be related directly to loss of clonogenicity. Treatment with phthalocyanine and light resulted in a strong inhibition of the incorporation of 14C-phenylalanine in trichloracetic acid-precipitable material. The induction of the beta-galactoside utilization system was also strongly inhibited. The latter two processes did not recover during incubation, subsequent to photodynamic treatment. It is concluded that photodynamically induced inhibition of protein synthesis is a critical factor contributing to the loss of clonogenicity.

Is muscle protein turnover regulated by intracellular glutamine during sepsis?

Low muscle glutamine levels during sepsis are associated with reduced protein synthesis and elevated protein breakdown, in particular myofibrillar protein breakdown. It is not known if this is a causal or coincidental relationship. We tested the hypothesis that muscle protein turnover rates are directly regulated by glutamine. Paired extensor digitorum longus muscles from nonseptic (sham-operated) and septic rats (16 hours after cecal ligation and puncture) were incubated in the absence or presence of 15 mmol glutamine/L. The effect of glutamine was tested in unsupplemented medium or in medium containing 1 mU/mL of insulin or a mixture of amino acids at normal plasma concentrations. Protein synthesis was measured as incorporation of 14C-phenylalanine into protein; total and myofibrillar protein breakdown was determined by measuring tyrosine and 3-methylhistidine, respectively. Muscles accumulated intracellular glutamine well above normal concentrations in the presence of 15 mmol glutamine/L. In spite of this, protein synthesis was not affected by glutamine, neither when muscles were incubated in unsupplemented medium nor in medium containing insulin or amino acid mixture. Total protein breakdown was not influenced by glutamine when muscles were incubated in unsupplemented medium or with insulin but was reduced by glutamine in the presence of an amino acid mixture. Myofibrillar protein breakdown was unaffected by glutamine in unsupplemented medium and in medium containing insulin but was increased by glutamine in the presence of amino acid mixture. Reduced muscle protein synthesis and increased myofibrillar protein breakdown during sepsis are probably not caused by the low intracellular glutamine levels noticed in this condition.

Effects of a fat emulsion containing medium chain fatty acids and long chain fatty acids on protein and energy metabolism in partially hepatectomized rats

Protein and energy metabolism were examined in 34 partially hepatectomized rats (70%), receiving total parenteral nutrition (TPN). The TPN contained either long chain triglycerides (LCT) or triglycerides comprising both medium- (MCFA) and long chain fatty acids (LCFA) on the same carbon skeleton (MILT, medium- and long chain triglyceride). The rats were divided into 4 groups with and without glucose (G) supplementation: LCT+G, LCT-G, MLT+G, MLT-G. 3 days after surgery protein synthetic rate in skeletal muscle, as evaluated from in vitro incorporation of 14C-phenylalanine into muscle protein, was significantly higher in rats receiving MLT if compared to rats receiving LCT (p < 0.05). Rats receiving MLT lost significantly less weight during the study period when compared to the LCT group (p < 0.005). Increased leucine oxidation was observed in rats receiving TPN without glucose regardless of the type of fat emulsion used (p < 0.05). In conclusion, when given to partially hepatectomized rats TPN containing both MCFA and LCFA exerts a stimulatory effect on muscle protein synthesis and preserves body weight better than an emulsion containing LCT only.

The effect of isolated chloride depletion on growth and protein turnover in young rats.

The effects of feeding a chloride-deficient (CD) diet were examined in young, growing rats. All animals were fed the same sodium-replete, CD diet. The experimental group drank distilled water, while the control group (CS) drank distilled water supplemented with 37 mM sodium chloride. By day 15, the CD rats had negligible concentrations of chloride in their urine and had developed hypochloremic metabolic alkalosis. Both groups had comparable urinary sodium concentrations and creatinine clearances. Food intake (256 vs. 226 g), weight (108.8 vs. 47.0 g) and length (9.6 vs. 7.4 cm) gains were greater in the CS animals and the efficiency of weight gain was lower in the CD rats (25.2 vs. 42.6 g gained/g of food intake). After 15-18 days, blood was drawn for testing, body composition measurements were performed and epitrochlearis muscle protein synthesis and net degradation rates determined. When incubated with or without the addition of insulin (I), epitrochlearis muscle protein synthesis, measured as the incorporation of 14C-phenylalanine, was significantly lower in CD rats [(I+ 45.7 vs. 36.76) and (I-34.72 vs. 26.3) nmol phenylalanine/g wet weight per hour (both P < 0.05)]. Net protein degradation rates were not significantly different between the two groups. Estimated nitrogen balance was significantly diminished in CD compared with CS rats. Gastrocnemius muscle RNA concentrations were also lower in CD rats (1.34 vs. 1.60 mg RNA/g wet weight, P < 0.001), but gastrocnemius protein concentrations were equal.(ABSTRACT TRUNCATED AT 250 WORDS)

Maternal food restriction, brain polysomes and poly-U stimulated protein synthesis in the newborn and 21-day-old rat progeny

Effect of maternal dietary restriction during the period of gestation (EI) and during the period of growth, gestation and lactation (EII) on (i) the polysomal profiles (ii) distribution of 14C-phenylalanine and 3H-orotate into membrane bound and free polysomes and ribosomes and (iii) ribosomal response to the addition of poly-U in the incorporation of 14C-phenylalanine was investigated in the brain of the neonatal (N) and 21-day-old (T) progeny. Polysomal profile showed variable degrees of reductions in the membrane bound ribosomes, free polysomes and monoribosomes in the brain of N and T progeny of EI and EII groups compared with WF group. Distribution of radioactive phenylalanine and orotate also showed variable responses in the membrane bound ribosomes free polysomes and the monoribosomes of the brain of N and T progeny of EI and EII groups. EII N progeny generally had a lower distribution and EII group T progeny had a higher distribution than EI group in each component of the polysomal profile. Ribosomes from the brain of N and T progeny of EI and EII groups responded less actively to the addition of poly-U than WF group. Synthesis of protein/synthesis of polyphenylalanine ratio (which roughly represents polysomes/monosomes ratio) demonstrated variable responses in the brain of N and T progeny of EI and EII groups compared with WF group. In T progeny when WF polysomes were incubated with EI or EII group pH-5 enzymes, the incorporation increased significantly and was higher in EII group than EI group. Incubation of EI polysomes with EII pH-5 enzyme demonstrated a significant increase in the incorporation. These results clearly demonstrate that maternal food restriction during various physiological periods had a profound effect on brain polysomes and consequently the protein synthesizing machinery in the N and T progeny.

In vivo administration of interleukin-1α induces muscle proteolysis in normal and adrenalectomized rats

The effect on muscle protein turnover of recombinant interleukin-1α (rlL-1α), 300 μg/kg body weight (BW) administered intraperitoneally (IP) in three divided doses over 18 hours, was studied in rats. Protein synthesis rate was determined by measuring incorporation of 14C-phenylalanine into protein, and total and myofibrillar protein breakdown rates were determined by measuring release of tyrosine and 3-methylhistidine, respectively, in incubated extensor digitorum longus muscles. To assess the role of glucocorticoids in rlL-1α-related metabolic alterations, plasma levels of corticosterone following rlL-1α injection and the effect of rlL-1α on muscle protein breakdown in adrenalectomized and sham-adrenalectomized rats were determined. Total and myofibrillar protein breakdown rates were increased by 45% and 167%, respectively, following treatment of normal rats with rlL-1α; muscle protein synthesis was not altered by the cytokine. Plasma corticosterone levels were markedly elevated following rlL-1α injection, with a maximal level occurring at 30 minutes. However, administration of rlL-1α resulted in increased total and myofibrillar protein breakdown rates in both adrenalectomized and shamadrenalectomized rats. The results suggest that increased muscle proteolysis following administration of rlL-1α is independent of glucocorticoids.

Protein and RNA biosynthesis in various cellular fractions of the brain of undernourished rats

Body and brain weight; DNA, RNA, and protein content; total free nucleotides; amino acids and their specific activities; as well as the in vitro incorporation of 14C-phenylalanine into protein and of 2H-orotate into RNA of homogenates and various cellular brain fractions were measured in well-fed (WF) and undernourished rats during gestation (EI) and during growth, gestation, and lactation (EII). Body weight was significantly decreased in both EI and EII while brain weight and DNA content per organ were lower in EII than in WF and EI rats. The brain weight:DNA ratio was increased significantly in EII animals compared with the WF group. RNA content did not show any significant alterations in homogenates, nuclear, microsomal and pH-5 enzyme fractions but was elevated in mitochondrial and soluble fractions of the brain of EI rats compared with the WF group. Brain RNA content in EII animals was decreased in homogenates and all cellular fractions with the exception of the nuclear fraction in which it did not change significantly. The RNA:DNA ratio tended to increase (non-significantly) in the EI and EII groups as compared with the WF controls. Protein content was decreased in homogenates and all cellular fractions of EII rats while it was reduced only in the mitochondrial and pH-5 enzyme fractions of the EI group. The RNA:protein ratio fell in the microsomal fraction and rose in the soluble and pH-5 enzyme fractions of EI and EII animals. The protein:DNA ratio increased significantly in homogenates and the nuclear fraction of the EII Group: 14C-phenylalanine/mg protein incorporation was augmented in homogenates, mitochondrial, and soluble fractions, was diminished in the microsomal fraction, and did not change in the nuclear fraction of the brain of EI and EII rats. 3H-Orotate/mg RNA incorporation was decreased in the nuclear and microsomal fractions and was increased in the soluble and pH-5 enzyme fractions of the brain of EI and EII groups. Incorporation was also elevated in brain homogenates of EII rats and was reduced in the EI group. It was increased in the mitochondrial fraction of EI animals but did not show any change in the EII group. From these results, it can be concluded that dietary stress during gestation alone (EI) and during growth, gestation, and lactation (EII) modulates the metabolism of nucleic acids and proteins in the whole brain and in various cellular brain fractions.

In vivo incorporation of 14C-phenylalanine into ascidian tunichrome.

Ascidia ceratodes exposed to 14C-phenylalanine in the surrounding seawater incorporates the radiolabel into newly biosynthesized tunichrome molecules. Radioactivity can be detected in tunichrome extracted from circulating blood cells within one day following initial exposure to the radiolabel; weak activity (less than or equal to 4 microCi/mol tunichrome = 22 nmol phenylalanine/mol tunichrome) is detected in 1 to 10 days; significantly higher amounts of radiolabel (57 microCi/mol tunichrome = 318 nmol phenylalanine/mol tunichrome) appear 20 days after seawater exposure. Therefore, phenylalanine can function as a precursor in the biosynthesis of tunichrome.

Influence of sepsis in rats on muscle protein turnover in vivo and in tissue incubated under different in vitro conditions

We studied the influence of sepsis on muscle protein synthesis and degradation in vivo and in muscles, incubated flaccid or at resting length. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham-operated. A flooding dose of 14C-phenylalanine was used to determine muscle protein synthesis rate in vivo, and protein breakdown was calculated from the difference between protein synthesis and growth rates. Protein synthesis rate in vitro was assessed by determining incorporation of 14C-phenylalanine into protein in incubated extensor digitorum longus (EDL) and soleus (SOL) muscles. Total and myofibrillar protein breakdown rates were determined from release into incubation medium of tyrosine and 3-methylhistidine (3-MH), respectively. Muscle protein synthesis rate in vivo was reduced by 35%, similar to the reduction observed in muscles incubated flaccid or at resting length. The calculated protein breakdown rate in vivo was increased by 31% in septic rats. In incubated muscles, the increase in total protein breakdown (ie, tyrosine release) during sepsis was almost identical in muscles incubated flaccid or at resting length, ie, 83% to 88% in EDL and 47% to 49% in SOL. Myofibrillar protein degradation in vitro (ie, 3-MH release) was increased approximately 10-fold in EDL muscles incubated flaccid or at resting length, but was not significantly affected by sepsis in SOL. Results suggest that sepsis-induced changes in protein synthesis observed in muscles incubated either flaccid or at resting length reflect changes in vivo. Changes in protein breakdown were qualitatively similar in vivo and in vitro, but results in incubated muscles may overestimate the increase in muscle proteolysis caused by sepsis.

Effect of catabolic hormone infusion on protein turnover and amino acid uptake in skeletal muscle

Increased plasma levels of the catabolic hormones glucagon, epinephrine, and cortisol have been implicated in mediating various metabolic alterations in trauma and sepsis. Their role in altered protein turnover and amino acid transport in skeletal muscle during sepsis, however, is not known. In the current study, rats were infused with a mixture of the catabolic hormones for 16 hours. Control animals were infused with vehicle solution. Protein synthesis and degradation rates were measured in incubated, intact soleus muscles as incorporation of 14C-phenylalanine into protein and release of tyrosine into incubation medium, respectively. Muscle amino acid uptake was determined by measuring the intracellular to extracellular ratio of [3H]-alpha-aminoisobutyric acid after incubation for 2 hours. Infusion of catabolic hormones for 16 hours resulted in elevated plasma glucose and lactate levels, reduced plasma concentrations of most amino acids, and accelerated muscle protein breakdown, similar to previous findings in septic rats. Protein synthesis rates and amino acid uptake in incubated muscles were not significantly different in control and hormone-infused rats. The current study suggests that increased muscle proteolysis in sepsis and severe injury may be mediated in part by catabolic hormones. In contrast, reduced muscle protein synthesis and amino acid uptake are probably signaled by other substances or mechanisms.

Indirect inhibition of myocyte RNA and protein synthesis by interleukin-1

Soluble mediators of the inflammatory response may directly influence myocardial function and metabolism in the absence of myocardial cell necrosis. Previous reported experimental data have demonstrated that the monokine interleukin-1 (IL-1) can produce myocardial depression and may influence muscle protein metabolism. To further investigate this hypothesis, IL-1 was added to neonatal rat cardiac muscle cell (MC) cultures with and without additional rat cardiac non-muscle cells (NMC). Incorporation of 3H-uridine or 14C-phenylalanine into acid-insoluble material was utilized as a measure of RNA or protein synthesis. IL-1 in concentrations of up to 500 units/ml had no effect on MC RNA or protein synthesis. When NMC were added to the MC culture, IL-1 exhibited a concentration-dependent inhibition of both RNA and protein synthesis, with effects apparent at concentrations as low as 5 units/ml. Supernatants from IL-1-treated NMC cultures exerted a dose dependent reduction on the incorporation of radiolabeled precursor into MC cultures, suggesting production of a soluble substance mediating the IL-1 effect. Supernatants from IL-1 treated rat skin fibroblasts or rat skeletal muscle myoblasts increased MC radiolabeled precursor incorporation slightly, in contrast to the decrease seen with NMC supernatant. Furthermore, IL-1 treated NMC supernatant had no inhibitory effect on skeletal myoblasts. We conclude that IL-1 decreases protein and RNA synthesis in MC cultures through a second mediator elaborated from the NMC population.

Flavour formation in callus cultures of guava (Pridium guajava) fruit

AbstractExplants from guava Pridium guajava L fruit, when cultured on Murashige and Skoog media containing different combinations of IAA, NAA, 2,4‐D and kinetin, yielded unorganised callus cultures. Light (&gt;2000 lux) and kinetin (&gt;5 mg litre−1) promoted chlorophyllous callus. However, none of the tested variations yielded organised cultures. Increase in kinetin from 0.2 to 0.8 mg litre−1 resulted in a slight increase in the growth of the callus.There was perceptible guava fruit flavour in all three passages of callus cultures, all of which exhibited a slow growth rate. GC‐MS analysis of the volatile flavour fraction of the callus tissue revealed (Z)hex‐3‐en‐1‐ol, 3‐phenylpropyl acetate, (E)hex‐3‐en‐1‐ol, hexan‐1‐ol, (Z)hex‐3‐ynyl acetate, (E)hex‐2‐enal and cinnamyl acetate as prominent peaks. Incorporation of 14C glucose, 14C phenylalanine and 14C acetate from the growth medium into the volatile fraction of the callus cultures was 0.07, 0.09 and 0.14% respectively during a 4‐day incubation.

Altered growth and protein turnover in rats fed sodium-deficient diets.

The effects of feeding a sodium-deficient (NaD) diet were examined in young, growing rats. All animals were fed the same diet and drank distilled water. In the control group the water contained 37 mM sodium chloride. After 2-3 wk body composition measurements were performed and epitrochlearis muscle protein synthesis and degradation rates determined. The experimental group gained only 45% of the wt and 70% of the length seen in the control group. The difference in wt gain could not be accounted for by differences in extracellular volume which averaged only 4% of body wt. Although total food intake was equivalent in both groups, urinary ammonia plus urea nitrogen excretion was higher in the experimental animals. Protein synthesis, measured as the incorporation of 14C-phenylalanine into muscle protein was significantly lower in NaD rats (56.58 versus 68.79) and (65.26 versus 83.88 nmol phenylalanine/h/g wet wt (both p less than 0.01) when incubated with or without the addition of insulin (1 mU/mL). Net protein degradation rates were unchanged. Gastrocnemius muscle RNA concentrations were also lower in NaD rats (1.09 versus 1.52 mg/g wet wt, p less than 0.001). There were no changes noted in the concentration of protein within either gastrocnemius or epitrochlearis muscles. These results suggest that in rats, the growth failure seen in sodium deficiency: 1) affects both length and wt gain; 2) is not associated with decreased nutrient intake; 3) is due to decreased rates of muscle protein synthesis without affecting net protein degradation rates; and 4) is associated with diminished muscle tissue RNA concentrations.