Changes in protein synthesis due to an inflammatory challenge

Rates of protein synthesis in various chick tissues were examined 16 hr after an inflammatory challenge. Protein synthetic rates were calculated from the rate at which [14C]leucine was incorporated into protein and the specific activity of [14C]leucine in the precursor pool. An injection of either Escherichia coli or sheep red blood cells (SRBC) decreased the rate of protein synthesis in the gastrocnemius muscle, and increased the rate in liver, bursa, spleen, and thymus. E. coli, but not SRBC, decreased protein synthesis in the pectoralis muscle. E. coli significantly decreased the aggregation of pectoralis muscle polysomes and increased the aggregation of polysomes in the thymus, bursa, and spleen. E. coli increased the aggregation of free, but not bound, polysomes in liver, suggesting an increase in synthesis of export proteins. SRBC significantly increased polysomal aggregation in bursa and spleen only. A crude preparation of leukocyte endogenous mediator, isolated from peritoneal macrophages, decreased muscle-polysomal aggregation. These studies indicate that tissue-specific changes in protein synthesis occur after a noninfectious inflammatory challenge. These changes may be part of a homeostatic mechanism which supports the immune response.     

Changes in protein degradation in chickens due to an inflammatory challenge

Tissue-specific changes in protein catabolism were examined in chicks 16 hr following an inflammatory challenge. It was determined that tyrosine was not catabolized or converted to phenylalanine in muscle, thymus, bursa, or spleen. Therefore, rates of tyrosine release from protein were used to estimate rates of protein catabolism in these tissues. Arginine was not catabolized to urea by chick liver; consequently, arginine release from liver protein was used to measure protein catabolism in this tissue. An injection of sheep red blood cells (SRBC) or Escherichia coli did not change rates of protein catabolism in liver or bursa as compared to saline-injected controls. SRBC significantly increased protein catabolism in muscle and spleen by 29 and 15%, respectively. E. coli resulted in significant increases in muscle, spleen, and thymus of 43, 30, and 34%, respectively. These changes in protein catabolism, together with known changes in protein synthesis, suggest that an inflammatory response to SRBC and E. coli result in increased protein accretion in the bursa and liver, and net protein loss from muscle.     

Effects of triamcinolone acetonide on plasma amino acids and urinary urea output in rabbits

1. The effect of administering triamcinolone acetonide (10 mg/kg/day), 6 consecutive s.c. injections given daily, on plasma free amino acids and urinary urea output was studied in rabbits. 2. The total free amino acids in plasma decreased significantly from day 2 while ammonia increased significantly only on day 2, glutamine, lysine and branched amino acids increased significantly from day 3 or 5. 3. The output of urinary urea increased significantly from day 3. 4. These findings suggest the inhibition of protein synthesis observed in steroid myopathy may result from a decrease in the amino acid pool in skeletal muscle.     

Uric acid in nucleic and amino acid synthesis in the boll weevil, Anthonomus grandis

When uric acid-2-¹⁴C was injected into the boll weevil, Anthonomus grandis, it was metabolized to RNA, DNA, amino acid, and ¹⁴CO₂ at the end of 2 hr. The free amino acids, lipoamino acids, and protein amino acids were all labelled with the free amino acids showing the highest specific activity. Incorporation in DNA was slight, but it was extensive in RNA; cytidylic acid showed the greatest amount of incorporation.     

Effects of varying doses of methionine sulfoximine on liver glutamine synthetase activity and time courses of blood and urinary nitrogenous compounds in the chicken (Gallus domesticus)

1. The activity of liver glutamine synthetase was inhibited to 7-12% of the control activity by an intracardiac injection with methionine sulfoximine (MSM) at dosages of 20, 50, 75 and 100 mg/kg body wt. 2. Plasma glutamine concentrations in all the MSM treatments decreased sharply, then reached steady-state levels within 0.5-2.5 hr, which were almost proportional to a dose of MSM. 3. Blood ammonia concentration sharply increased to a steady-state level attained at 4.5 hr, which was proportional to a dose of MSM. The excretion rate of urinary ammonia augmented linearly up to the dose dependent maximum rates within 2-5 hr. 4. Plasma uric acid concentration dropped linearly by about 6.4 mg/100 ml at doses of 50, 75 and 100 mg MSM and by 3.7 mg/100 ml at a dose of 20 mg MSM within 2.5 hr, then recovered a little. 5. The decreases in excretion rates of urinary uric acid for the first 4 hr were almost the same at doses of 50 mg and larger, being twice as large as that of the control chicken. 6. Any doses of MSM affected neither the time course of excretion rate of total urinary nitrogen nor its total amounts for 7 hr after MSM treatment.     

Time course of urinary excretion of intraportal ammonia as uric acid and ammonia in chickens fed low- or high-protein diet

15N-ammonia was intraportally infused for 6 hr into chickens fed 5% or 20% protein diet to examine the time course of urinary excretion of intraportal ammonia and dietary effects on it. Urinary ammonia increased linearly for the first hour to the same extent in both dietary groups and thereafter further in the low-protein group. Urinary uric acid derived from the intraportal ammonia adaptively increased and reached a steady state level within 1.5 hr. This level was four times higher in the high-protein group. The infused ammonia was excreted into urine as both ammonia and uric acid, in relatively high proportions in the chickens fed the low-protein diet but was almost all excreted as uric acid in those fed the high-protein diet.     

Changes in ammonia and amino acid levels in the erythrocytes and plasma of carp, Cyprinus carpio, during passage through the gills

Differences in ammonia and free amino acid levels between the dorsal aorta (post-gills) and bulbus arteriosus (pre-gills) were studied in sixteen unanaesthetized carp, Cyprinus carpio , to clarify he roles of erythrocytes in inter-organ transport of these substances. Both erythrocyte and lasma ammonia concentrations decreased significantly during passage through the gills, and the erythrocyte contribution (53%) to the total net decrease of ammonia on a whole blood basis was approximately equal to that by the plasma (47%). The glutamine level in the plasma showed no significant change, but that in the erythrocytes increased slightly. There were losses of some amino acids from the plasma circulating through the gills, while significant increases were noted in the erythrocytes, i.e., changes in the concentrations of most amino acids were in opposite directions in the erythrocytes and plasma across the gills. The results of this study show that not only plasma but also erythrocytes are involved in ammonia and amino acid transportations in carp.     

Stable ammonia-specific NAD synthetase from Bacillus stearothermophilus: purification,characterization, gene cloning,and applications

Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible.     

Metabolism and excretion of nitrogen during metamorphosis and egg production in the sawfly, Neodiprion sertifer

Measurements of whole-body dry matter, total nitrogen, water-soluble protein, amino acids, and uric acid were determined at successive stages during metamorphosis in Neodiprion sertifer. The major change was in the uric acid fraction: in females, it increased up to the non-pharate pupa and then decreased during the subsequent stages of adult development and egg production; in males, it continued to increase during adult development. The decline of uric acid could not be explained by the accumulation of allantoin, allantoic acid, urea, or uric acid riboside. Examination of amino acid levels in the gut revealed an accumulation in the pupa followed by a depletion at the onset of adult development. This was followed by an excretory phase marked by the progressive accumulation of large quantities of uric acid and small quantities of urea, ammonia, and amino acids during the formation of the meconium. Amino acid analysis of the meconium revealed the presence of large proportions of proline, hydroxyproline, and histidine in comparison with the other amino acids.     

The uptake and utilization of Entodinium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa

C oleman , G.S. & H all , F.J. 1984. The uptake and utilization of Entodinium caudatum , bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa. Journal of Applied Bacteriology 56 , 283–294.
Washed suspensions of Entodinium bursa were incubated anaerobically with Entodinium caudatum , ten species of bacteria and a yeast. The rate of uptake and digestion of these micro-organisms was investigated. Protozoa grown in vivo did not engulf Proteus mirabilis or Klebsiella aerogenes but rapidly took up Bacillus mega-terium, Selenomonas ruminantium, Torulopsis glabrata and Streptococcus bouis , although only the last was digested with release of soluble material into the medium. Protozoa grown in vitro engulfed each of the bacteria tested, taking up Megasphaera elsdenii and i>Proteus mirabilis most rapidly. Individual bacterial species and mixed rumen bacteria were engulfed more rapidly (up to 20 times) by protozoa grown in vivo than those grown in vitro , although the latter digested over 80% of the B. megaterium, Escherichia coli and P. mirabilis taken up. Labelled Ent. caudatum was extensively digested after engulfment by Ent. bursa . Some of the digestion products were released into the medium but individual amino acids were transferred as such from Ent. caudatum protein to Ent. bursa protein. Engulfed bacteria and polysaccharide granules were transferred intact from one protozoon to the other. Free amino acids were also taken up intact from the medium into protozoal protein but there was little biosynthesis of amino acids from glucose. When available for engulfment Ent. caudatum was quantitatively a much more valuable source of amino acids for protein synthesis by Ent. bursa than free amino acids or bacteria.     

The effects of tobacco mosaic virus synthesis on the free amino acid and amide composition of the host

1. Changes in concentrations of free amino acids and amides have been determined in TMV-infected tobacco leaf discs and in comparable uninfected discs during the time of virus formation. 2. During the period of rapid virus formation the infected discs show a transitory deficiency (as compared to uninfected discs) in glutamine, asparagine, aspartic acid, glutamic acid, serine, and to a lesser extent in valine, threonine, and proline. About 100 hours before this time smaller deficiencies in the concentrations of these components also occur. The latter effect is probably associated with the early synthesis of a non-virus protein in infected tissue. 3. Comparison of the above effects with the known amino acid composition of TMV indicates that it is unlikely that the virus protein is synthesized by condensation of appropriate free amino acids. Rather, the deficiencies observed appear to result from removal of ammonia from the nitrogen pool during synthesis of new proteins in infected tissue. Equilibrium shifts resulting from ammonia withdrawal probably account for the observed deficiencies in amides and free amino acids. TMV protein, therefore, appears to be synthesized de novo, from non-protein nitrogen, probably ammonia. 4. It is suggested that the changes in free amino acid concentrations induced by virus formation may account for some of the symptoms observed in infected plants.     

The uptake and utilization of Entodinium caudatum, bacteria, free amino acids and glucose by the rumen ciliate Entodinium bursa

Washed suspensions of Entodinium bursa were incubated anaerobically with Entodinium caudatum, ten species of bacteria and a yeast. The rate of uptake and digestion of these micro-organisms was investigated. Protozoa grown in vivo did not engulf Proteus mirabilis or Klebsiella aerogenes but rapidly took up Bacillus megaterium. Selenomonas ruminantium, Torulopsis glabrata and Streptococcus bovis, although only the last was digested with release of soluble material into the medium. Protozoa grown in vitro engulfed each of the bacteria tested, taking up Megasphaera elsdenii and Proteus mirabilis most rapidly. Individual bacterial species and mixed rumen bacteria were engulfed more rapidly (up to 20 times) by protozoa grown in vivo than those grown in vitro, although the latter digested over 80% of the B. megaterium, Escherichia coli and P. mirabilis taken up. Labelled Ent. caudatum was extensively digested after engulfment by Ent. bursa. Some of the digestion products were released into the medium but individual amino acids were transferred as such from Ent. caudatum protein to Ent. bursa protein. Engulfed bacteria and polysaccharide granules were transferred intact from one protozoon to the other. Free amino acids were also taken up intact from the medium into protozoal protein but there was little biosynthesis of amino acids from glucose. When available for engulfment Ent. caudatum was quantitatively a much more valuable source of amino acids for protein synthesis by Ent. bursa than free amino acids or bacteria.     

Nitrogen partitioning and blood meal utilization by Aedes aegypti (Diptera Culicidae)

The utilization of the blood meal by mosquitoes was investigated by first feeding females quantities of blood ranging from 1 to 5 mg, and then analyzing the faeces for the various by-products of protein catabolism that were subsequently eliminated. The nitrogeneous waste products in order of importance were uric acid, histidine, ammonia and arginine. Only traces of the other amino acids were excreted.The total amount of each faecal substance varied linearly with the quantity of blood ingested, however their relative proportions did not change. Regardless of blood meal size the quantily of uric acid and ammonia produced indicates that about 80% of the non-histidine and arginine amino acids are deaminated and utilized for metabolic purposes other than egg protein synthesis.Most of the histidine and about one half of the arginine content of the blood were excreted as free amino acids, but the other amino acids were lost in trace amounts.Nineteen per cent of the total ingested amino acids was incorporated into soluble yolk proteins and this proportion was constant even for small blood meals that result in a reduction in the numbers of eggs produced.The comparative aspects of nitrogen partitioning and blood meal utilization by haematophagous insects, as well as the factors that affect blood meal utilization and fecundity in A. aegypti are discussed.     

Studies on enzymes of nitrogen metabolism, and nitrogen end products in the developing chick (Gallus domesticus) embryo.

1. A total of 450 fertilized eggs were used to study the concentrations of uric acid, urea and ammonia in allantoic and amniotic fluids, and some enzymes of nitrogen metabolism in the liver and kidney during the development of the chick embryo from the 5th to 21st day of incubation. 2. Concentrations of the compounds studied were higher in allantoic fluid. The molar concentration of allantoic uric acid increased steadily with time. The pattern of urea and ammonia in both allantoic and amniotic fluids were the same. 3. Arginase (E.C.3.5.3.1) activity in both embryonic kidney and definitive kidney was higher than that in the liver. The specific activity of arginase (mumole urea formed/hr per g wet wt kidney) dropped during development. 4. Little arginine synthetase activity (argininosuccinate synthetase, E.C.6.3.4.5; and argininosuccinate lyase, E.C.4.3.2.1) was found in kidney, but none in the liver. 5. The complete urea cycle function was absent in both the liver and the kidney of the chick embryo.     

Do mammals,birds, reptiles and fish have similar nitrogen conserving systems?

Comparative physiological studies are a powerful tool for revealing common animal adaptations. Amino acid catabolism produces ammonia which is detoxified through the synthesis of urea (mammals, some fish), uric acid (birds), or urea and uric acid (reptiles). In mammalian herbivores and omnivores, urea nitrogen is salvaged by a series of steps involving urea transfer into the intestine, microbial mediated urea hydrolysis with synthesis of amino acids utilizing the liberated ammonia and transfer of the amino acids back to the host. A similar series of steps occur in omnivorous/granivorous and herbivorous birds, although in this case urine, containing uric acid, is refluxed directly into the intestine where microbes degrade the uric acid and utilize the liberated ammonia for amino acid synthesis. These amino acids are transferred back to the host. In reptiles and ureotelic fish not all of these steps have been experimentally confirmed. Reptiles like birds, reflux urine into the intestine where it is exposed to the microflora. However, the capacity of these microbes to breakdown the uric acid and urea and utilize ammonia for amino acid synthesis has not been documented. Ureotelic fish transfer urea into the intestine where urease (presumably of bacterial origin) hydrolyzes the urea. However, the amino acid synthesizing capacity of the intestinal microflora has not been studied. The series of steps, as outlined, would define the prevailing nitrogen conservation system for herbivores and omnivores at least. However, it would appear that some animals, in particular the fruit-eating bat and perhaps the fruit-eating bird, may have evolved alternative, as yet uncharacterized, adaptations to a very limited nitrogen intake.     

13N isotope studies on the pathway of ammonia assimilation in Bacillus megaterium and Escherichia coli.

The pathway of ammonia incorporation into amino acids was studied by use of 13N-ammonium ions in Bacillus megaterium and Escherichia coli that had been grown aerobically on a minimal salts medium containing NH4Cl as the source of nitrogen. Anion- and cation-exchange high-pressure-liquid chromatography was used to separate amino acids relevant to the several possible pathways for ammonia assimilation in bacteria. At an initial concentration of added NH4+ of 1 microM, the glutamine synthetase-glutamate synthase pathway represented the major pathway in both bacteria on the basis of the effects of inhibitors of that pathway (L-methionine-DL-sulfoximine and azaserine) and of transamination (aminooxy-acetate) and the observation that the specific activity of glutamine was greater initially than that of any other amino acid likely to be the first product of an assimilation pathway. The study provides (i) a new analytical method for 13N-tracer investigation of amino acids, (ii) confirmation of conclusions from enzymological studies on the pathway of ammonia assimilation in B. megaterium and E. coli, and (iii) proof that alanine dehydrogenase and aspartate ammonia lyase (aspartase) are not important pathways in B. megaterium at low NH4+ concentrations.     

真养产碱杆菌112R4的二羧酸单酰胺酰胺水解酶

在一株具有环酰亚胺转化活性的真养产碱杆菌112R4中发现了一种特异性的二羧酸单酰胺酰胺水解酶（半酰胺酶）,它催化环酰亚胺代谢的第二步反应,将二羧酸单酰胺水解为二羧酸和氨。该酶的底物仅限于此代谢途径的第一个酶——酰亚胺酶的产物二羧酸单酰胺,而对其它的酰胺类化合物没有明显水解活性。真养产碱杆菌112R4中的半酰胺酶和酰亚胺酶在表达上具有相关性,环酰亚胺（如琥珀酰亚胺）和二羧酸单酰胺（如琥珀酰胺酸）对它们有正调控作用,游离氨离子显示出负调控作用,琥珀酸则在酶合成和活性两方面均表现出影响作用。对重组大肠杆菌中表达的半酰胺酶粗酶的部分性质进行了研究。钴离子对半酰胺酶的活性表现出促进作用,比活力提高到3.37倍,表明半酰胺酶可能是一种金属结合酶。     

Changes in blood metabolite concentrations in response to repeated capture,anaesthesia and blood sampling in the golden perch,Macquaria ambigua

• 1.1. The major metabolic changes associated with repeated capture, aquarium transfer, anaesthesia and blood sampling were investigated in an Australian freshwater fish, the golden perch (Macquaria ambigua),
• 2.2. A compounded stress response was seen after repetition of the procedure, in which the plasma glucose rose within 3 hr and amino acid concentrations rose and the serum free fatty acids concentration fell after 24 hr.
• 3.3. Alanine was identified as an important circulating energy store in the stress response of golden perch.
• 4.4. No change was noted in the serum protein, plasma lactate or β-hydroxybutyrate concentrations, indicating that tissue damage and hypoxia were absent, and that degradation of free fatty acids did not produce metabolites excess to the requirements of gluconeogenesis and the tricarboxylic acid cycle.

     

刀豆氨酸对亚洲玉米螟的生理生化效应

本文报道亚洲玉米螟Ostrinia furnacalis五龄幼虫注射0.25-1.0毫克/克刀豆氨酸48小时后有关生理生化的变化,其中血淋巴蛋白质电泳区带数目减少,浓度降低,而游离氨基酸总量明显增加,其中脯氨酸和谷氨酸的变化最为明显。虫体酸性磷酸酯酶的活性显著升高,而精氨酸酶的活性却有所降低。刀豆氨酸可引起虫体内尿素含昆下降,而对尿酸含量影响不大。经刀豆氨酸处理后发育成长的成虫其雌虫卵巢和雄虫附腺的蛋白质合成均显著下降,并对上述结果做了简要的讨论。     

Amino acid sequence of Escherichia coli glutamine synthetase deduced from the DNA nucleotide sequence

Glutamine synthetase is encoded by the glnA gene of Escherichia coli and catalyzes the formation of glutamine from ATP, glutamate, and ammonia. A 1922-base pair fragment from a cDNA containing the glnA structural gene for E. coli glutamine synthetase has been sequenced. An open reading frame of 1404 base pairs encodes a protein of 468 amino acid residues with a calculated molecular weight of 51,814. With few exceptions, the amino acid sequence deduced from the DNA sequence agreed very well with the amino acid sequences of several peptides reported previously. The secondary structure predicted for the E. coli enzyme has approximately 36% of the residues in alpha-helices which is in agreement with calculations of approximately 39% based on optical rotatory dispersion data. Comparison of the amino acid sequences of glutamine synthetase from E. coli (468 amino acids) and Anabaena (473 amino acids) (Turner, N. E., Robinson, S. T., and Haselkorn, R. (1983) Nature 306, 337-342) indicates that 260 amino acids are identical and 80 are of the same type (polar or nonpolar) when aligned for maximum homology. Several homologous regions of these two enzymes exist, including the sites of adenylylation and oxidative modification, but the regulation of each enzyme is different.