The acute phase response alters cationic amino acid transporter expression in growing chickens (Gallus gallus domesticus)

The effect of an acute phase response (APR) on cationic amino acid transporter (CAT1-3) mRNA expression in liver, muscle, bursa and thymus was determined in broiler strain chickens. The APR was initiated by injecting Salmonella typhimurium lipopolysaccharide subcutaneously (LPS; 1 mg/kg bw). In Experiment 1, CAT1-3 mRNA expression was determined at multiple time points following LPS administration. LPS increased bursa and liver total and high affinity CAT mRNA expression (P<0.05) and transiently increased pectoralis total CAT mRNA expression (P<0.05). Total CAT mRNA expression in the thymus decreased 7.7-fold from 0 to 8 h after LPS injection (P<0.05). In Experiment 2, fasted chicks were uninjected or LPS-injected. LPS increased total and high affinity CAT mRNA 2-fold in both the bursa and liver (P<0.05) and did not change thymus total and high affinity CAT mRNA expression (P>0.05). LPS increased liver weight only (P<0.05) and did not alter the plasma lysine and arginine concentration (P>0.05). In Experiments 3 and 4, thymocyte proliferation and total protein content were dependent upon the media lysine concentration (P<0.001). The inability of the thymus to compete for lysine and arginine during the APR may limit the ability of thymocytes to develop during infections.     

Protein metabolism and strength performance after bovine colostrum supplementation

Summary. This study was designed to determine the responses of muscle protein, serum amino acids, and strength performance to bovine colostrum supplementation in physically active men. The rest (R) group (n=6) and the exercise (E) group (n=6) carried out twice a 2-week experiment randomly assigned in a double-blind fashion with either placebo (PLA; consuming daily 20g maltodextrin) or bovine colostrum (COL; consuming daily 20g colostrum supplement) treatment with one month between. On the test day after the treatment period the measurements were carried out in fasting conditions and E carried out a strength training session (STS). The methods involved the infusion of ring-²H₅-phenylalanine, femoral arterial and venous blood sampling, and biopsies from the vastus lateralis muscle. Serum concentration of essential amino acids during recovery was greater (p<0.05) in the COL groups compared with the PLA groups. Both muscle protein synthesis and breakdown increased (p<0.05) with COL. There were no differences in phenylalanine net balance or strength performance between the PLA and COL groups. It was concluded that a 2-week supplementation with bovine colostrum in physically active men increases serum concentration of essential amino acids but has no effect either on strength performance or protein net balance in fasting conditions during recovery after STS.     

The effect of an acute phase response on tissue carotenoid levels of growing chickens (Gallus gallus domesticus)

Plasma, liver and skin carotenoids decrease following infectious disease challenges. Since these challenges often involve substantial host pathology and chronic immune responses, the mechanism underlying altered carotenoid deposition is unclear. Therefore, changes in tissue carotenoid levels were examined during an acute phase response induced by lipopolysaccharide (LPS) or interleukin-1 (IL-1). In two experiments, chicks were hatched from carotenoid-deplete eggs (n=28, n=64, respectively) and fed 0, 8 or 38 mg carotenoids (lutein+canthaxanthin)/kg diet. For chicks fed 38 mg carotenoids, but not those fed 0 or 8 mg, LPS generally reduced plasma lutein, canthaxanthin and total carotenoids (P<0.05), and liver lutein, zeaxanthin, canthaxanthin and total carotenoids (P<0.05). Additionally, LPS reduced thymic total carotenoids (P=0.05) and increased thymocyte lutein (P=0.07), zeaxanthin (P=0.07) and total carotenoids (P=0.07). Finally, LPS increased bursal canthaxanthin (P<0.01), but had no effect on shank carotenoids (P>0.5). In chicks hatched from carotenoid-replete eggs (n=36) and fed dietary lutein (38 mg/kg diet), LPS reduced plasma and liver zeaxanthin and liver total carotenoids (P<0.05); IL-1 reduced plasma and liver lutein, zeaxanthin and total carotenoids (P<0.05). Therefore, an acute phase response plays a role in reduced tissue carotenoids during infectious disease.     

Impact of separating amino acids between plasma, extracellular and intracellular compartments on estimating protein synthesis in rodents

Summary. Three models representing different separations of amino acid sources were used to simulate experimental specific radioactivity data and to predict protein fractional synthesis rate (FSR). Data were from a pulse dose of ¹⁴C-U Leu given to a non-growing 20 g mouse and a flooding dose of ³H Phe given to a non-growing 200 g rat. Protein synthesis rates estimated using the combined extracellular and intracellular (Ec + Ic) source pool and extracellular and plasma (Ec + Pls) source pool mouse models were 78 and 120% d⁻¹ in liver, 14 and 16% d⁻¹ in brain and 15 and 14% d⁻¹ in muscle. Predicted protein synthesis rates using the Ec + Ic, Ec + Ic + Tr (combined extracellular, intracellular and aminoacyl tRNA source pool) and Ec + Pls rat models were 57, 3.4 and 57% d⁻¹ in gastrocnemius, 58, 71 and 62% d⁻¹ in gut, 8.3, 8.4 and 7.9% d⁻¹ in heart, 32, 23 and 25% d⁻¹ in kidney, 160, 90 and 80% d⁻¹ in liver, 57, 5.5 and 57% d⁻¹ in soleus and 56, 3.4 and 57% d⁻¹ in tibialis. The Ec + Ic + Tr model underestimated protein synthesis rates in mouse tissues (5.0, 27 and 2.5% d⁻¹ for brain, liver and muscle) and rat muscles (3.4, 5.5 and 3.4% d⁻¹ for gastrocnemius, soleus and tibialis). The Ec + Pls model predicted the mouse pulse dose data best and the Ec + Ic model predicted the rat flooding dose data best. Model predictions of FSR imply that identification and separation of the source specific radioactivity is critical to accurately estimate FSR. Received June 11, 2000 Accepted September 26, 2000     

Effect of alanyl-glutamine on leucine and protein metabolism in irradiated rats

Summary. The mechanism by which glutamine produces a favorable effect in the treatment of sepsis, injury, burns and abdominal irradiation is not completely understood. The main aim of this study was to evaluate the effect of alanyl-glutamine (AlaGln) administration on the metabolism of proteins in irradiated rats. The rats were exposed to whole-body irradiation (8 Gy) and then fed intragastrically with a mixture of glucose and amino acids either with AlaGln or without AlaGln. At 48 hours after irradiation, parameters of whole-body protein metabolism and DNA synthesis in intestinal mucosa were investigated using a primed, continuous infusion of [1-¹⁴C]leucine and [³H]thymidine. In addition, we evaluated the effect of irradiation and AlaGln on gut morphology, blood count and amino acid concentrations in blood plasma and skeletal muscle. Control rats were not irradiated but were given identical treatment. An increase in whole-body leucine oxidation, and insignificant changes in whole-body proteolysis and in protein synthesis were observed after irradiation. In irradiated rats we observed a decrease in muscle glutamine concentration, a decrease in protein synthesis in jejunum, colon and heart, and an increase in synthesis of proteins of blood plasma and spleen. Morphological examination and measurement of DNA synthesis failed to demonstrate any favorable effect of AlaGln supplementation on irradiated gut. However, administration of AlaGln resulted in a decrease in whole-body proteolysis and leucine oxidation which caused an increase in the fraction of leucine incorporated into the pool of body proteins. We conclude that the data obtained demonstrate that irradiation induces metabolic derangement associated with increased oxidation of essential branched-chain amino acids (valine, leucine and isoleucine) and that these disturbances can be ameliorated by administration of AlaGln. Received February 14, 2000 Accepted July 12, 2000     

A comparison of15N proline and13C leucine for monitoring protein biosynthesis in the skin

Summary The tracers L¹⁵N-proline and L(1-¹³C)-leucine were used to explore the synthesis of skin proteins in vivo in rabbits. They orally received a single dose containing an equimolecular mixture of L(1-¹³C)-leucine and L¹⁵N-proline. The changes in the amounts of these tracers in blood and skin were monitored for a total of 8 h. The data showed the appearance of the two tracers in blood within 15 min and their clearance in 8h. They were both rapidly (15 min) incorporated into skin proteins, but more proline was incorporated than leucine. We therefore consider L¹⁵N-proline to be a better tracer than L(1-¹³C)-leucine for studying protein metabolism in the skin.     

Determining synthesis rates of individual proteins in zebrafish (Danio rerio) with low levels of a stable isotope labelled amino acid

The zebrafish is a powerful model organism for the analysis of human cardiovascular development and disease. Understanding these processes at the protein level not only requires changes in protein concentration to be determined but also the rate at which these changes occur on a protein‐by‐protein basis. The ability to measure protein synthesis and degradation rates on a proteome‐wide scale, using stable isotope labelling in conjunction with mass spectrometry is now a well‐established experimental approach. With the advent of more selective and sensitive mass spectrometers, it is possible to accurately measure lower levels of stable isotope incorporation, even when sample is limited. In order to challenge the sensitivity of this approach, we successfully determined the synthesis rates of over 600 proteins from the cardiac muscle of the zebrafish using a diet where either 30% or 50% of the L‐leucine was replaced with a stable isotope labelled analogue ([²H₇]L‐leucine]. It was possible to extract sufficient protein from individual zebrafish hearts to determine the incorporation rate of the label into hundreds of proteins simultaneously, with the two labelling regimens showing a good correlation of synthesis rates.     

Tryptophan Overload in the Pregnant Rat: Effect on Brain Amino Acid Levels and In Vitro Protein Synthesis

The concentration of most amino acids was higher in the brains of 19- and 21-day rat fetuses than in their respective mothers. After an intraperitoneal load of tryptophan to the mother, the intracerebral concentration of several amino acids (including leucine) decreased not only in the mothers, but also in their fetuses. The in vitro incorporation of pHJleucine into proteins in brain postmitochondrial supernatant fractions was enhanced in both the mothers and fetuses after tryptophan administration, but this effect disappeared when protein synthesis was calculated by using specific activities corrected for the amount of unlabeled leucine in the preparation. By this criterion, protein synthesis activity appeared similar in the brains of 19- and 21-day pregnant rats but was higher in their fetuses, especially in the 21-day subjects. Thus, protein synthesis in the brain was not altered by marked changes in the amino acid pool and more profound and prolonged metabolic disturbances must occur to cause permanent damage in the developing brain.     

The relationship between plasma cholesterol,amino acids and acute phase proteins in sepsis

Summary. The purpose of the study was to correlate degree of hypocholesterolemia to changes in plasma levels of amino acids and other metabolic variables in severely injured septic patients. Measurements included plasma cholesterol, full amino-acidograms, acute phase proteins, complementary variables and blood cell counts. The Fischer plasma molar amino acid ratio (leucine+isoleucine+valine)/(phenylalanine+tyrosine) was calculated. Plasma cholesterol for all measurements (n=145) was 3.1±1.1mmol/L and, upon entry in the study, it was correlated inversely with sepsis severity score (p<0.05). Along the clinical course, changes in cholesterol were clearly paralleled by opposite changes in C-reactive protein, which was the best correlate of cholesterol (r²=0.70, p<0.0001). Furthermore cholesterol was inversely related to phenylalanine, fibrinogen, lactate and white blood cell count, and directly to the Fischer molar amino acid ratio, cystathionine, methionine, glycine and transferrin (r² between 0.36 and 0.15, p<0.0001 for all). Within this pattern of correlations, cholesterol was also directly related to alkaline phosphatase, which accounted for the effect of cholestasis, when present. For any given value of the other variables, cholesterol increased significantly with increase in alkaline phosphatase (p<0.0001). C-reactive protein (CRP, mg/dl) and alkaline phosphatase (ALKPH, U/L) together in the same regression explained 79% of the variability of cholesterol (CHOL, mmol/L): CHOL=5.90–0.74[Log_eCRP]+0.004[ALKPH]; multiple r²=0.79, p<0.0001. Inclusion in this regression of other variables did not increase the r². By using only amino acid variables, the best fit was provided by a regression including the Fischer ratio and cystathionine, which explained 55% of the variability of cholesterol (multiple r²=0.55 p<0.0001), and this result was not improved by the inclusion of other amino acids. These data show that severity of hypocholesterolemia in sepsis is quantifiably related to changes in plasma amino acids, and to severity of acute phase response and metabolic decompensation. More study is needed to understand whether hypocholesterolemia in sepsis has only diagnostic or prognostic implications, or that it may also contribute actively to worsening of the disease.     

Source of amino acids for tRNA acylation in growing chicks

Summary Specific radioactivity in three amino acid compartments was examined in broiler chicks following a flooding dose of leucine or phenylalanine. In general, specific radioactivity of leucine and phenylalanine in deproteinated plasma (SA_e) and tissue (SA_i) compartments, exceeded that in acylated-tRNA (SA_t). In most tissues, SA_e and SA_i rapidly reached a similar peak level by 5 min followed by a slow decline for the next 30 minutes. Many tissues (eg. GI tract, liver, skin, and thigh) failed to maintain equilibrium between SA_e and SA_i over time. More metabolically active tissues, such as GI and liver had the greatest differences between these compartments. The difference between SA_e and SA_i for both leucine and phenylalanine were due to SA_i decreasing faster than SA_e, indicating dilution with unlabelled amino acids from proteolysis. Plasma and tissue specific radioactivity overestimated tRNA specific radioactivity by as much as 5 and 2.8 fold using leucine or 2.7 and 1.4 fold using phenylalanine, respectively. These data suggest that intracellular compartmentation of protein metabolism and the coupling of protein degradation and synthesis occur, in vivo.     

Amino acid studies in transient acute polymorphic psychosis

Summary Data are reviewed on the amino acid metabolism in patients suffering from transient acute polymorphic psychoses according to ICD-10. The psychotic episodes of many of these patients are characterized by distorted sensory perceptions and intense emotional states; plasma amino acid analysis revealed a disturbed serine-glycine metabolism.In remitted patients oral loading with serine induced the characteristic dysperceptions and psychedelic symptoms. Plasma concentrations of serine and methionine were decreased, and the concentration of taurine was increased. Fibroblast experiments suggest that the activities of the serine metabolizing enzymes serine hydroxymethyltransferase and cystathionine-synthase are increased in these patients.The determination of plasma amino acid concentrations proved to be useful in discriminating these patients. The prevalence of this transient psychosis in a psychiatric in-patient population ranged between 1.4 and 3.6%.     

Changes in protein expression in testes of L2 strain Taiwan country chickens in response to acute heat stress

Heat stress causes a decrease of fertility in roosters. Yet, the way acute heat stress affects protein expression remains poorly understood. This study investigated differential protein expression in testes of the L2 strain of Taiwan country chickens following acute heat stress. Twelve 45-week-old roosters were allocated into four groups, including control roosters kept at 25 °C, roosters subjected to 38 °C acute heat stress for 4 hours without recovery, with 2 hours of recovery, and with 6 hours of recovery. Testis samples were collected for morphologic assay and protein analysis. Some of the differentially expressed proteins were validated by Western blot and immunohistochemistry. Abnormal and apoptotic spermatogenic cells were observed at 2 hours of recovery after acute heat stress, especially among the spermatocytes. Two-dimensional difference gel electrophoresis revealed that 119 protein spots were differentially expressed in chicken testes following heat stress, and peptide mass fingerprinting revealed that these spots contained 92 distinct proteins. In the heat-stressed samples, the heat shock proteins, chaperonin containing t-complex, and proteasome subunits were downregulated, and glutathione S-transferase, transgelin, and DJ-1 were upregulated. Our results demonstrate that acute heat stress impairs the processes of translation, protein folding, and protein degradation, and thus results in apoptosis and interferes with spermatogenesis. On the other hand, the increased expression of antioxidant enzymes, including glutathione S-transferase and DJ-1, may attenuate heat-induced damage. These findings may have implications for breeding chickens that can tolerate more extreme conditions.     

Lysine metabolism in higher plants

Summary. The essential amino acid lysine is synthesised in higher plants via a pathway starting with aspartate, that also leads to the formation of threonine, methionine and isoleucine. Enzyme kinetic studies and the analysis of mutants and transgenic plants that overaccumulate lysine, have indicated that the major site of the regulation of lysine synthesis is at the enzyme dihydrodipicolinate synthase. Despite this tight regulation, there is strong evidence that lysine is also subject to catabolism in plants, specifically in the seed. The two enzymes involved in lysine breakdown, lysine 2-oxoglutarate reductase (also known as lysine α-ketoglutarate reductase) and saccharopine dehydrogenase exist as a single bifunctional protein, with the former activity being regulated by lysine availability, calcium and phosphorylation/dephosphorylation. Received December 21, 1999 Accepted February 7, 2000     

CO_2对离体欧芹叶片中蛋白质代谢的影响

以离体欧芹叶片为实验系统 ,研究了CO2 对叶片中蛋白质降解的影响。对离体欧芹叶片中游离氨基酸含量及可溶蛋白含量的分析结果表明 :CO2 具有抑制离体欧芹叶片中蛋白质降解的作用 ,这为进一步了解CO2 延缓叶片衰老的作用机理很有意义。     

A food web analysis of the juvenile blue crab, Callinectes sapidus, using stable isotopes in whole animals and individual amino acids

The stable isotope compositions (C and N) of plants and animals of a marsh dominated by Spartina alterniflora in the Delaware Estuary were determined. The study focused on the juvenile stage of the Atlantic blue crab, Callinectes sapidus, and the importance of marsh-derived diets in supporting growth during this stage. Laboratory growth experiments and field data indicated that early juvenile blue crabs living in the Delaware Bay habitat fed primarily on zooplankton, while marsh-dwelling crabs, which were enriched in ¹³C relative to bay juveniles, utilized marsh-derived carbon for growth. In laboratory experiments, the degree to which juvenile blue crabs isotopically fractionated dietary nitrogen, as well as the growth rate, depended on the protein quality of the diet. The range of δ¹³C of amino acids in laboratory-reared crabs and their diets was almost 20‰, similar to the isotopic range of amino acids of other organisms. In laboratory studies, the δ¹³C of nonessential and essential amino acids in the diet were compared to those in juvenile crabs. Isotopic fractionation at the molecular level depended on diet quality and the crabs' physiological requirements. Comparison of whole-animal isotope data with individual amino acid C isotope measurements of wild juvenile blue crabs from the bay and marsh suggested a different source of total dietary carbon, yet a shared protein component, such as zooplankton. Received: 1 July 1998 / Accepted: 15 March 1999     

13C-Leucine-tracer-technique for in-vivo measurement of amino acids' metabolism by human colon carcinomas

Summary Own investigations on in-vivo tumor metabolism of the malignant human colon tumor showed a significant uptake of branched chain amino acids by the tumor itself. To study the quantitative tumor protein metabolism (compartment tumor) the¹³C-leucine-tracer-technique was modified.Beside the common¹³C-leucine-breath-test we measured also the AV-differences of¹³C-leucine,¹³C-ketoisocaproate and¹³CO₂. The Tumorblood flow was measured by venous-outflow-technique as well as the tumor mass.In this way it is possible to get quantitative results in substrate exchange of branched chain amino acids in malignant human colon tumors.     

Protein turnover in exponential and stationary phase Vibrio cells

Vibrio strain 14 supports phage alpha 3a growth in standing stationary phase cells but not in shaking (aerated) stationary phase cells. In exponential cells, protein was turned over at 1.8% h-1, and the rate was increased by starvation or inhibition of protein synthesis. In shaking stationary phase cells the rate of protein turnover was low (1.0% h-1) for proteins synthesised during growth but high (20% h-1) for recently synthesised proteins. In contrast recently synthesised proteins in standing stationary phase cells were stable over 60 min and proteins synthesised during growth were turned over at 2.9% h-1. ppGpp and pppGpp were detected in exponential cells, but were not detected in stationary phase cells.