Enhancement of resistance to coccidiosis and necrotic enteritis in broiler chickens by dietary muscadine pomace.

Muscadine pomace (MP), a by-product of the production of wine and juice from Vitis rotundifolia, was dried and tested in chickens for effects on primary resistance to coccidiosis, development of protective immunity after vaccination with live coccidia, and resistance to necrotic enteritis (NE) caused by the joint action of Clostridium perfringens and coccidia. To test primary resistance to coccidiosis, 2-wk-old chicks were given 2% or 5% MP in the diet and inoculated with Eimeria acervulina and E. maxima. Birds given MP at either level had significantly (P < 0.05) lower lesion scores at 7 days postinoculation, in comparison with control birds, although weight gains were statistically similar. Broiler chickens were given 2% or 5% MP and grown to 42 days to test the palatability of MP. Birds given 2% MP in feed grew similarly to untreated controls, but birds given 5% had poorer average live weight. This suggested a negative effect on feed intake at the higher level. The effects of dietary 0.5% or 2.0% MP on immune protection were tested after live coccidiosis vaccination in the hatchery. Chicks were removed from each pen at 21 days of age and challenged with E acervulina, E. maxima, and E. tenella. Resistance to infection was improved by MP as suggested by significantly (P < 0.05) lower lesion scores 7 days postchallenge, and improved weight gains in comparison with immunized control birds that did not receive MP. At 42 days of age, birds given MP had higher average live weights than controls, although feed efficiency was not affected. An established model was used to study the effect of MP on NE in broiler chickens. Chicks were inoculated with live coccidia at 14 days of age and dosed orally with live cultures of C perfringens on day 19, day 20, and day 21. Enteritis caused 48% mortality in the first study and 67% mortality in the second study. Dietary MP at 0.5-2.0% significantly (P < 0.05) reduced mortality in both experiments; improved weight gain relative to the unmedicated, infected control; and reduced lesion scores at necropsy. Overall, the results of six experiments suggested that MP given in the diet at 0.5% or higher had a positive effect on primary resistance and development of acquired resistance to two severe intestinal diseases in chickens.

Low dietary protein impairs blood coagulation in BHE/cdb rats.

The influence of dietary protein on blood coagulation tests was evaluated in BHE/cdb rats. Three experiments were conducted in order to compare effects of diets with low (8 g/100 g diet) or high (38 g/100 g diet) protein, to establish values for coagulation tests at intermediate (12-30 g/100 g diet) concentrations of dietary protein, and to compare feeding identical quantities of diets with 8 g protein/100 g diet vs. 18 g protein/100 g diet. After 4 wk of feeding the semipurified diets, bleeding time exceeded 15 min in the groups fed low protein diets, compared to a range of 3-6 min for the groups fed high protein diets. Several in vitro tests of coagulation were abnormal in the rats fed low protein diets. For example, prothrombin time averaged 27 +/- 8 s in rats fed 8 g protein/100 g diet plus beef tallow, but 17 +/- 1 s in rats fed 38 g protein/100 g diet plus tallow. The coagulation deficit in rats fed low protein was not affected by fat source (tallow vs. menhaden oil), but fibrinogen was elevated in rats fed diets with menhaden oil. Conversely, no differences in coagulation tests were observed among rats fed 12-30 g protein/100 g diet. Bleeding times ranged from 7 to 9 min, and prothrombin time was 17-18 s. Significant differences in plasma fibrinogen concentration and prothrombin time were observed in rats fed 8 vs. 18 g protein/100 g diet at a fixed rate of 6 g/100 g body weight. Platelet and blood cell numbers were unaffected by dietary protein. The evidence for multiple deficits in the coagulation system suggests that hepatic function in BHE/cdb rats may become impaired when the rats are fed low protein diets of the composition used here.

Ubiquitin-mediated degradation of tyrosine aminotransferase (TAT) in vitro and in vivo.

Degradation of a protein via the ubiquitin proteolytic pathway involves two successive steps. Covalent attachment of ubiquitin to the target protein and degradation of the tagged substrate by the 26S proteasome. Most native cellular proteins that are targeted by the ubiquitin system are short-lived transcriptional activators and growth and cell cycle regulators, as well as unstable membrane proteins. In the present study we demonstrate the involvement of the system in the degradation of tyrosine aminotransferase (TAT), a key enzyme in intermediary metabolism. In vitro, we have shown that the native enzyme is conjugated and degraded in a system that requires ATP and ubiquitin. Degradation was monitored by following the decrease of catalytic activity as well as disappearance of the protein molecule. The enzyme could be protected from degradation by association with its specific cofactor, pyridoxal phosphate (PLP). In vivo, we prepared cell extracts from livers of animals in which TAT was induced by starvation and corticosteroid administration. The dramatic increase in the level of the enzyme was accompanied by a concomitant increase in the level of specific TAT-ubiquitin adducts.

Degradation of tyrosine aminotransferase (TAT) via the ubiquitin-proteasome pathway.

Most of the known cellular substrates of the ubiquitin system are short-lived growth regulators and transcriptional activators. Very few enzymes involved in intermediary metabolism have been shown to be targeted by the system. In a reconstituted cell-free system, we show that tyrosine aminotransferase (TAT), a key enzyme involved in amino acid metabolism, is conjugated and degraded in an ATP- and ubiquitin-dependent manner. Degradation of ubiquitin-TAT adducts requires, in addition to the 26S proteasome, a novel, yet unidentified, factor. TAT can be protected from degradation by association with its coenzyme pyridoxal phosphate. To examine the potential role of the ubiquitin system in regulating the stability of the enzyme in vivo, we show that cell extracts derived from livers of animals in which TAT was induced, display a corollary increase in the formation of specific TAT-ubiquitin adducts.

Destabilization of tyrosine aminotransferase by amino acids.

Several L-amino acids (tyrosine, glutamate, methionine, tryptophan, and phenylalanine) and penicillamine destabilized purified tyrosine aminotransferase by removing enzyme-bound pyridoxal 5'-phosphate. The destabilization was measured as a progressive loss of enzyme activity in samples taken at intervals from a primary mixture that was incubated at 37°C. Each destabilizing amino acid either served as a substrate for this enzyme or was a product of transamination. In contrast, L-cysteine destabilized the enzyme only if liver homogenate was added, which generated polysulfide by desulfuration. Cysteine complexed free pyridoxal-5'-phosphate but did not remove it from the enzyme. Other amino acids did not destabilize tyrosine aminotransferase at the concentrations tested.

From genotype to phenotype: computer-based modeling of gene expression with STELLA II.

STELLA II is a microcomputer program that is designed to simulate and animate the dynamics generated by biological systems in a quantitative fashion. The program employs the Macintosh user interface and includes a tool kit for assembling models, plus menus for setting parameters and selecting input or output. Quantitative relationships among components may be expressed with user-defined equations, graphical functions or data from spreadsheets. System dynamics are simulated by running the program after defining initial conditions, and results may be viewed immediately using windows for graphical or tabular plots. The present article describes how STELLA II may be used to simulate gene expression as an adjunct to experimentation or student-directed learning.

Microcomputer-assisted kinetic modeling of mammalian gene expression.

New software for microcomputers enables predictive, quantitative models of genetic systems to be produced that can account for the elements of time, scale, and feedback control of hierarchical systems. The flow of genetic information during protein synthesis can be addressed by treating each intermediate as a kinetic element in a linked series of reactions. When the rate of transcription changes, the time required to achieve a new level of the encoded protein is expected to be a function of the conversion rates or half-lives of all intermediates. Kinetic modeling may be used to make predictions and integrate primary data concerning rates of transcription, nuclear mRNA dynamics, nucleocytoplasmic transport, translational control, and other processes that govern the rate of synthesis for specific proteins.

High casein-lactalbumin diet accelerates blood coagulation in rats.

This study investigates the influence of a high protein intake on normal hemostasis, fluid balance and organ growth. Adult rats were fed semipurified diets that contained either 18 or 56 g/100 g casein-lactalbumin for 2 wk, and the following functions were measured: food and water intake, weight gain, blood pressure, bleeding and clotting time, ADP-stimulated platelet aggregation, thrombin time, prothrombin time and partial thromboplastin time. Although food intake was depressed by the high protein diet, weight gain was not affected by the regimen. Water consumption, 24-h urine excretion and kidney weight were significantly greater in rats fed the high protein diet than in controls. High protein intake resulted in shorter barbiturate-induced sleeping time. Bleeding time and clotting time were significantly lower in rats fed the high protein diet for 7 d. However, heart rate, mean arterial pressure, plasma protein and osmolarity, platelet aggregation, prothrombin time, partial thromboplastin time and thrombin time did not differ significantly. Because a high protein intake caused rapid coagulation of blood in rats without affecting the activity of clotting factors, we suggest that this diet sensitized rats to factors that initiate clotting in vivo.

Differences in glycogen, lipids, and enzymes in livers from rats flown on COSMOS 2044.

Livers from rats flown aboard COSMOS 2044 were analyzed for protein, carbohydrate (glycogen), and lipids as well as the activities of a number of key enzymes involved in metabolism of these compounds and xenobiotics. The major differences between the flight group and the synchronous control were elevations in microsomal protein, liver glycogen content, tyrosine aminotransferase, and tryptophan oxygenase and reductions in sphingolipids and the rate-limiting enzyme of heme biosynthesis, delta-aminolevulinic acid synthase. These results provide further evidence that spaceflight has pronounced and diverse effects on liver function; however, some of the results with samples from COSMOS 2044 differed notably from those from previous spaceflights. This may be due to conditions of spaceflight and/or the postflight recovery period for COSMOS 2044.

The kinetics of mammalian gene expression.

When rates of transcription from specific genes change, delays of variable length intervene before the corresponding mRNAs and proteins attain new levels. For most mammalian genes, the time required to complete transcription, processing, and transport of mRNA is much shorter than the period needed to achieve a new, steady-state level of protein. Studies of inducible genes have shown that the period required to attain new levels of individual mRNAs and proteins is related to their unique half-lives. The basis for this is a physical principle that predicts rates of accumulation of particles in compartmental systems. The minimum period required to achieve a new level is directly proportional to product half-lives because rates of decay control the ratio between the rate of synthesis and the concentration of gene products at steady state. This kinetic model suggests that sensitivity of gene products to degradation by ribonucleases and proteinases is an important determinant of the time scale of gene expression.

A computer program for modeling the kinetics of gene expression.

Enzyme induction may be modeled on the basis of four, quantifiable processes that control the rates at which specific gene products accumulate and decay. These processes include synthesis of functional mRNA, translation and degradation of mRNA, and degradation of the protein product. We present a simple computer program that permits mathematical simulation of gene expression on the basis of experimentally determined rates of synthesis and degradation. The program was implemented as a spreadsheet using Microsoft Excel for Macintosh and MS-DOS operating systems and also was adapted for HyperCard on the Macintosh. It contains a formula to account for growth of tissue or cell populations. The program predicts amounts of individual mRNAs and proteins (or enzyme activities) in cells as a function of time after a stimulus alters their rates of synthesis or degradation.

Altered carbohydrate, lipid, and xenobiotic metabolism by liver from rats flown on Cosmos 1887.

To determine the possible biochemical effects of prolonged weightlessness on liver function, samples of liver from rats that had flown aboard Cosmos 1887 were analyzed for protein, glycogen, and lipids as well as the activities of a number of key enzymes involved in metabolism of these compounds and xenobiotics. Among the parameters measured, the major differences were elevations in the glycogen content and hydroxymethylglutaryl-CoA (HMG-CoA) reductase activities for the rats flown on Cosmos 1887 and decreases in the amount of microsomal cytochrome P-450 and the activities of aniline hydroxylase and ethylmorphine N-demethylase, cytochrome P-450-dependent enzymes. These results support the earlier finding of differences in these parameters and suggest that altered hepatic function could be important during spaceflight and/or the postflight recovery period.

Stabilization and purification of tyrosine aminotransferase from rat liver.

Purification of unmodified tyrosine aminotransferase from rat liver requires that the activity of cathepsin T be minimized, and that losses of enzyme due to dilution or oxidation by prevented. The enzyme was stabilized by pyridoxal 5'-phosphate, dithiothreitol, and potassium phosphate, but was destabilized by L-tyrosine or L-glutamate. A rapid, efficient method for purification of this enzyme included the following steps: twenty-fold induction with a high-casein diet plus dexamethasone phosphate administered in the drinking water; a heat step (65 degrees C) followed by precipitation from 0.20 M sucrose at pH 5.0; and small-scale chromatography on DEAE-cellulose, hydroxyapatite and CM-Sephadex C50 at pH 6.0. These steps yielded more than 10 mg of native enzyme from 35 rats, with a recovery of 68% of the initial activity.

The role of mRNA and protein stability in gene expression.

How important is the stability of gene products in the process of gene expression? We use a dual-compartment mathematical model to demonstrate the effects that changing the rates of synthesis and degradation of hypothetical mRNAs and proteins would have on the final concentration of protein. The model predicts that the concentration of protein at steady state equals the product of the rate constants for synthesis of mRNA and protein (ks1 and ks2) divided by the product of the rate constants for degradation (kd1 and kd2) and that the rate at which protein concentration changes depends on the rate constants for degradation of both the mRNA and the protein. This permits great flexibility in controlling induction kinetics for particular gene products, since their synthesis, translation, and degradation may be regulated coordinately to permit induction to be stable or transient or to amplify the final yield of protein. We suggest single exons may encode structural features that cause both mRNAs and proteins to be labile, thereby ensuring that modal stabilities of highly regulated macromolecules are similar.

Experimental diabetes increases the formation of sulfane by transsulfuration and inactivation of tyrosine aminotransferase in cytosols from rat liver.

The addition of L-cysteine to hepatic cytosols causes inactivation of tyrosine aminotransferase. We have studied the mechanism of inactivation and the effect of streptozotocin-induced diabetes in the rat on the inactivation of tyrosine aminotransferase in the presence of fractions prepared from livers and kidneys. Diabetes increased the rate at which tyrosine aminotransferase was inactivated after addition of cysteine to hepatic cytosols. The inactivation was due to the production of thiocysteine (which contains sulfane sulfur) from cystine as a result of desulfuration catalyzed by gamma-cystathionase. Diabetes increased the content of cystathionine beta-synthase and gamma-cystathionase in liver. As a result, cytosols from diabetic animals converted homocysteine, cystathionine, cysteine and cystine to sulfane at an elevated rate, with resulting inactivation of tyrosine aminotransferase. In contrast, inactivation in kidney fractions was not affected by diabetes. Incubation with an inhibitor of gamma-cystathionase (propargylglycine) prevented inactivation of tyrosine aminotransferase. These results show that the potential for the formation of sulfane sulfur by the enzymes of the transsulfuration pathway is enhanced by chronic diabetes.

The structure of tyrosine aminotransferase. Evidence for domains involved in catalysis and enzyme turnover.

The primary structure of tyrosine aminotransferase, as deduced from the nucleotide sequence of complementary DNA, was confirmed by fast atom bombardment mass spectrometry of tryptic peptides derived from the purified protein. Limited digestion of the native enzyme with trypsin released an acetylated, amino-terminal peptide; the new amino terminus in the modified enzyme was Val65. Endogenous proteases generated a chromatographically separable form of tyrosine aminotransferase that began at Lys35. Neither trypsin nor the other proteases altered the catalytic activity of tyrosine aminotransferase. Reduction of the holoenzyme with sodium borohydride yielded a major tryptic peptide containing phosphopyridoxamine bound to lysine 280, which probably functions in transamination. The carboxyl terminus of tyrosine aminotransferase contains features that typify proteins with short half-lives; it includes two negatively charged, hydrophilic segments that are enriched for glutamyl residues and are similar to a PEST region in ornithine decarboxylase (Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368). Tyrosine aminotransferase belongs to a superfamily of enzymes which includes aspartate aminotransferase and can be aligned so that many invariant, functional residues coincide. Like the isoenzymes of aspartate aminotransferase, tyrosine aminotransferase may contain two domains, with a central, catalytic core, and a small domain made up of both amino- and carboxyl-terminal components. We speculate that the exposed small domain may confer the unusually rapid degradative rate that characterizes this enzyme.

Persulfide generated from L-cysteine inactivates tyrosine aminotransferase. Requirement for a protein with cysteine oxidase activity and gamma-cystathionase.

Liver cytosols contain factors that produce an inhibitor of tyrosine aminotransferase and other enzymes when incubated with L-cysteine or L-cystine. Cystine-dependent inactivation was caused by cystathionase and required pyridoxal 5'-phosphate, but a second protein was needed to reconstitute cysteine-dependent inactivation. A cytosolic protein was isolated that oxidized free cysteine and brought about inactivation of tyrosine aminotransferase when coincubated with cystathionase. Hematin also oxidized cysteine, which led to cysteine-dependent inactivation of tyrosine aminotransferase in the presence of cystathionase. The inactivation of tyrosine aminotransferase involved three steps: initial oxidation of cysteine to form cystine; desulfuration of cystine catalyzed by cystathionase to form the persulfide, thiocysteine; and reaction of thiocysteine (or products of its decomposition) with proteins to form protein-bound sulfane. Since dithiothreitol reactivated tyrosine aminotransferase, the sulfane probably inactivated the enzyme by oxidation of thiol groups. The present results do not indicate whether the cysteine oxidase activity is enzymatic nor do they prove which form of polysulfide inactivates tyrosine aminotransferase. Reduced glutathione greatly slowed the rates at which sulfane accumulated and at which tyrosine aminotransferase was inactivated. Incubation of DL-cystathionine with liver cytosols led to formation of cysteine, which was oxidized and cleaved to form persulfide, and caused inactivation of tyrosine aminotransferase. Thus, sulfane sulfur that is generated by an enzyme of the transulfuration pathway inactivates a transaminase by nonselective oxidation of enzyme-bound thiol groups.

A cystine-dependent inactivator of tyrosine aminotransferase co-purifies with gamma-cystathionase (cystine desulfurase).

Tyrosine aminotransferase is stable in homogenates of rat liver, but not when L-cystine or L-cysteine is added, which causes the enzyme to be reversibly inactivated due to oxidation of thiol groups. By monitoring inactivation of the aminotransferase in the presence of L-cystine, a factor responsible for this loss of activity was purified from rat liver. The factor required vitamin B6 and co-purified with gamma-cystathionase during numerous steps. Highly purified inactivating factor contained a protein that was identical in size and isoelectric point to cystathionase but also contained a dissimilar peptide that appeared to be unrelated to cystathionase. Cystathionase and the cystine-dependent inactivator shared several catalytic activities, including the hydrolysis of cystathionine, desulfuration of cystine, and desulfhydration of cysteine. During incubation of L-cysteine with the purified factor, hydrogen sulfide was generated but no inactivation of the aminotransferase occurred, suggesting that cysteine-dependent inactivation requires additional mechanisms. An insoluble inactivator of tyrosine aminotransferase that is produced during the reaction may be elemental sulfur, since colloidal suspensions of sulfur also inhibited the enzyme. Another inhibitor fractionated with high molecular weight substances; this may be protein-bound sulfane.

Hepatic function in rats after spaceflight: effects on lipids, glycogen, and enzymes.

The inclusion of rats aboard Spacelab 3 (SL-3) allowed analyses of liver lipids, glycogen, hepatic enzymes of cholesterol, glycerolipid and sphingolipid biosynthesis, and other enzyme activities. Glycogen content was markedly elevated in livers from the flight animals compared with controls. Cholesterol was 24% (P less than 0.04) lower in livers from the experimental groups, whereas blood cholesterol was 19% higher (P less than 0.05). The activity of 3-hydroxy-3-methylglutaryl-CoA reductase, the rate-limiting enzyme of steroid biosynthesis, was 80% lower (P less than 0.01). Total phospholipids and sphingolipid levels did not differ significantly. The specific activity of fatty acyl-CoA synthetase, which is responsible for activation of fatty acids, was 37% (P less than 0.05) higher in microsomes from the rats on SL-3; however, since these animals had 25% less microsomal protein (P less than 0.02), there was no difference per gram of liver. The initial enzymes of sphingolipid and glycerolipid biosynthesis were assayed; serine palmitoyltransferase was 40% lower (P less than 0.01), and glycerol 3-phosphate acyltransferase did not differ. Hepatic cytochrome P-450 content decreased by 50% after spaceflight. Enzymes that did not differ significantly between the two groups include cytochrome b5, glutathione S-transferase, tyrosine aminotransferase, aspartate aminotransferase, and cystathionase. These findings suggest that spaceflight alters hepatic metabolism of several classes of compounds.