Valine Incorporation Research Articles

Isotope-Guided Metabolomics Reveals Divergent Incorporation of Valine into Different Flavor Precursor Classes in Chives.

Plants of the genus Allium such as chives, onions or garlic produce S-alk(en)yl cysteine sulfoxides as flavor precursors. Two major representatives are S-propenyl cysteine sulfoxide (isoalliin) and S-propyl cysteine sulfoxide (propiin), which only differ by a double bond in the C3 side chain. The propenyl group of isoalliin is derived from the amino acid valine, but the source of the propyl group of propiin remains unclear. Here, we present an untargeted metabolomics approach in seedlings of chives (Allium schoenoprasum) to track mass features containing sulfur and/or 13 C from labeling experiments with valine-13 C5 guided by their isotope signatures. Our data show that propiin and related propyl-bearing metabolites incorporate carbon derived from valine-13 C5 , but to a much lesser extent than isoalliin and related propenyl compounds. Our findings provide new insights into the biosynthetic pathways of flavor precursors in Allium species and open new avenues for future untargeted labeling experiments.

Nuclear Magnetic Resonance (NMR)–Based Serum Metabolic Profiles: Impact of Fasting Status

Abstract Introduction Measurement of lipoprotein subclass concentration (-c), particle number (-p), and size (-s) by nuclear magnetic resonance (NMR) has gained traction recently in the clinical laboratory due to associations between smaller lipid particle sizes and atherogenic risk, especially for LDL-p. Since patients may not comply with the clinician’s recommendation of fasting before sample collection, our objective was to evaluate the impact of fasting status on NMR-based lipid and amino acid determinations. Methods Fifty self-reported healthy participants were recruited using an internal review board-approved protocol: males (n = 25; 41.3 ± 10.5 years; BMI 27.7 ± 4.6 kg/m2) and females (n = 25; 35.6 ± 9.1 years; BMI 25.3 ± 5.4 kg/m2). Blood was collected after overnight fast and 4 hours after a high-fat meal. Samples were analyzed using the AXINON lipoFIT by NMR assay (Numares) with an Avance III NMR spectrometer and Ascend 600 MHz magnet (Bruker). This assay included measurements of triglycerides, total cholesterol, IDL-c, and LDL, HDL, VLDL concentrations; particle numbers; and sizes. Additionally, NMR-based glucose, alanine, valine, leucine, and isoleucine concentrations were acquired. Conventional lipid parameters were obtained using cobas 8000 chemistry system (Roche). Fasting and postprandial results were compared for significant differences. Results As expected, mean triglycerides increased after the meal (53%, P < .0001). Increases were also observed for VLDL-c (22%, P < .0001) and large VLDL-p (56%, P < .0001), as well as LDL-s, HDL-s, and VLDL-s (all <3.5%, P < .005). In contrast, LDL-p, small LDL-p, large LDL-p, HDL-p, small HDL-p, glucose, total cholesterol, and LDL, HDL, and IDL cholesterols demonstrated at most an 11% decrease (all P < .002). Valine, leucine, and isoleucine also increased after the meal (31%-46%, P < .0001 for all). Differences in individual lipids or amino acids were not sufficient to distinguish between fasting vs postprandial states, although incorporation of valine into a ratio with VLDL-c and LDL cholesterol (Valine*VLDL-c/LDL cholesterol) provided adequate differentiation (ROC analysis: AUC 0.91 [SD 0.03, P < .0001]). A cutoff of 75 for the ratio had a sensitivity of 88.9% (95% CI, 76.0%-96.3%) and specificity of 85.4% (95% CI, 72.2%-93.9%). Interference with NMR results occurred for three participants’ postprandial samples. These individuals had significantly higher fasting triglycerides, LDL-p, small LDL-p, large VLDL-p, VLDL-s, VLDL-c, IDL-c and lower LDL-s, and HDL-s compared to other participants. Conclusions The clinical impact on results from postprandial samples warrants further evaluation prior to accepting nonfasting samples for NMR analysis. Considering a subpar compliance with fasting, algorithms to differentiate fasting and postprandial specimens may be useful in identifying suboptimal specimens.

Reduced Skeletal Muscle Protein Turnover and Thyroid Hormone Metabolism in Adaptive Thermogenesis That Facilitates Body Fat Recovery During Weight Regain.

Objective: The recovery of body composition after weight loss is characterized by an accelerated rate of fat recovery (preferential catch-up fat) resulting partly from an adaptive suppression of thermogenesis. Although the skeletal muscle has been implicated as an effector site for such thrifty (energy conservation) metabolism driving catch-up fat, the underlying mechanisms remain to be elucidated. We test here the hypothesis that this thrifty metabolism driving catch-up fat could reside in a reduced rate of protein turnover (an energetically costly “futile” cycle) and in altered local thyroid hormone metabolism in skeletal muscle.Methods: Using a validated rat model of semistarvation-refeeding in which catch-up fat is driven solely by suppressed thermogenesis, we measured after 1 week of refeeding in refed and control animals the following: (i) in-vivo rates of protein synthesis in hindlimb skeletal muscles using the flooding dose technique of 13C-labeled valine incorporation in muscle protein, (ii) ex-vivo muscle assay of net formation of thyroid hormone tri-iodothyronine (T3) from precursor hormone thyroxine (T4), and (iii) protein expression of skeletal muscle deiodinases (type 1, 2, and 3).Results: We show that after 1 week of calorie-controlled refeeding, the fractional protein synthesis rate was lower in skeletal muscles of refed animals than in controls (by 30–35%, p < 0.01) despite no between-group differences in the rate of skeletal muscle growth or whole-body protein deposition—thereby underscoring concomitant reductions in both protein synthesis and protein degradation rates in skeletal muscles of refed animals compared to controls. These differences in skeletal muscle protein turnover during catch-up fat were found to be independent of muscle type and fiber composition, and were associated with a slower net formation of muscle T3 from precursor hormone T4, together with increases in muscle protein expression of deiodinases which convert T4 and T3 to inactive forms.Conclusions: These results suggest that diminished skeletal muscle protein turnover, together with altered local muscle metabolism of thyroid hormones leading to diminished intracellular T3 availability, are features of the thrifty metabolism that drives the rapid restoration of the fat reserves during weight regain after caloric restriction.

Incorporation of methyl-protonated valine and leucine residues into deuterated ocean pout type III antifreeze protein: expression, crystallization and preliminary neutron diffraction studies.

Antifreeze proteins (AFPs) are found in different species from polar, alpine and subarctic regions, where they serve to inhibit ice-crystal growth by adsorption to ice surfaces. Recombinant North Atlantic ocean pout (Macrozoarces americanus) AFP has been used as a model protein to develop protocols for amino-acid-specific hydrogen reverse-labelling of methyl groups in leucine and valine residues using Escherichia coli high-density cell cultures supplemented with the amino-acid precursor alpha-ketoisovalerate. Here, the successful methyl protonation (methyl reverse-labelling) of leucine and valine residues in AFP is reported. Methyl-protonated AFP was expressed in inclusion bodies, refolded in deuterated buffer and purified by cation-exchange chromatography. Crystals were grown in D(2)O buffer by the sitting-drop method. Preliminary neutron Laue diffraction at 293 K using LADI-III at ILL showed in a few 24 h exposures a very low background and clear small spots up to a resolution of 1.80 A from a crystal of dimensions 1.60 x 0.38 x 0.38 mm corresponding to a volume of 0.23 mm(3).

Differential response of protein metabolism in splanchnic organs and muscle to pectin feeding

The aim of the present study was to determine whether the addition of soluble fibre in the diet affected protein metabolism in the intestinal tissues, some visceral organs and in skeletal muscle. A diet supplemented with pectin (80 g/kg) was fed to young growing rats and the effect on organ mass and protein metabolism in liver, spleen, small and large intestines and gastrocnemius muscle was monitored and compared with the control group. Protein synthesis rates were determined by measuring [13C]valine incorporation in tissue protein. In the pectin-fed rats compared with the controls, DM intake and body weight gain were reduced (9 and 20 %, respectively) as well as gastrocnemius muscle, liver and spleen weights (6, 14 and 11 %, respectively), but the intestinal tissues were increased (64 %). In the intestinal tissues all protein metabolism parameters (protein and RNA content, protein synthesis rate and translational efficiency) were increased in the pectin group. In liver the translational efficiency was also increased, whereas its protein and RNA contents were reduced in the pectin group. In gastrocnemius muscle, protein content, fractional and absolute protein synthesis rates and translational efficiency were lower in the pectin group. The stimulation of protein turnover in intestines and liver by soluble fibre such as pectins could be one of the factors that explain the decrease in muscle turnover and whole-body growth rate.

Dietary pectin stimulates protein metabolism in the digestive tract

Objective The aim of this study was to determine if protein metabolism was altered in small and large intestines by feeding pectin, a soluble fiber known to stimulate cecal production of short-chain fatty acids (SCFAs) and to have a trophic effect in these tissues. Methods Twenty-four weanling male Sprague-Dawley rats were fed ad libitum for 14 d with a balanced control diet or an isoproteic, isocaloric pectin (citrus) diet (80 g/kg). SCFA production, intestinal histomorphometry, and protein synthesis were determined in the proximal and distal parts of the small intestine, the cecum, and the colon. Protein synthesis rates were determined by measuring the 13C valine incorporation rate in tissue proteins. Results Pectin feeding slightly decreased food intake and growth rate. It increased the acetate, propionate, and butyrate pools in the cecum. Pectin feeding resulted in heavier intestinal tissues corresponding to higher villus height in the small intestine and crypt depth in the small and large intestines compared with feeding of the control diet. Compared with the control group, the rats fed the pectin diet had significantly higher protein synthesis rates in all the parts of their intestines. Conclusion Supplementation of pectin, as a soluble fiber, in the diets, stimulated SCFA production, had a trophic effect on the different parts of the intestines, and greatly stimulated protein synthesis in those tissues.

Prolactin effect on pre-pubertal Sertoli cell proliferation and metabolism.

Direct effects of PRL on Sertoli cell proliferation were investigated by using Sertoli cell primary cultures isolated from both prepubertal rat and porcine testes. PRL metabolic effects were analyzed in rat Sertoli cell primary cultures. Exposure to physiological doses of PRL resulted in a significant increase (+50-60%) of basal DNA synthesis, as reflected by the pattern of [3H] thymidine incorporation during culture; significant increases in lactate secretion (about 50%), androgen binding protein (ABP) production (about 30%) and basal protein synthesis (25-30%), as reflected in the augmented [14C] valine incorporation, were also evident. Taken together, our present findings, indicating significant effects of PRL on Sertoli cell proliferation and metabolism, demonstrate that Sertoli cells are a potential target for PRL action at testicular level during pre-pubertal development.

L-δ-(α-Aminoadipoyl)-l-cysteine-d-valine Synthetase: Production of Dipeptides Containing Valine Residue at Its C-Terminus

l-δ-(α-Aminoadipoyl)-l-cysteine-d-valine synthetase (ACVS) has been recently studied as a model enzyme for peptide synthetases. It was found that in the absence of α-aminoadipic acid but in the presence of several cysteine analogues it was incorporated into several analogue dipeptides upon incubation of the potential cysteine analogues with ACVS. [14C]Cysteine was incorporated into the[14C]cysteinyl-valine analogue dipeptides. Notably, [14C]valine incorporation in the presence of N-acylated cysteine analogues was observed. The α-aminoadipic acid activation site is influential, inhibitory or promotive, on the production of these putative dipeptide products. The production of dipeptide analogues, containing valine or analogues at the C-terminus, leads to the speculation that the biosynthetic direction of ACV could be from the C-terminus to the N-terminus.

Infliximab for the treatment of abdominal wall fistulas in Crohn’s disease

The effects of some aromatic amines on the protein and ribonucleic acid metabolism in liver and kidney slices from rats and guinea pigs were investigated. 1.1. The incorporation of 14C-valine into protein by rat liver slices was markedly inhibited by 2-aminofluorene (AF) and 2-acetylaminofluorene (AAF), which have a carcinogenic effect on rat liver.2.2. The non-carcinogenic aromatic amines aniline and 1-aminonaphthalene (1-AN) did not inhibit the incorporation of 14C-valine into protein by rat liver slices under similar conditions.3.3. The bladder carcinogen 2-aminonaphthalene (2-AN), which like most other aromatic amines is metabolized by liver, inhibited the incorporation of 14C-valine into protein by rat liver slices at least as much as did AF and AAF.4.4. In slices from guinea pig, an animal which is refractory to the carcinogenic effect of AF and AAF, these compounds inhibited the incorporation of 14C-valine into protein even more than in rat liver slices.5.5. Substitution of fluorine in position 7 of the AF molecule, which increases the carcinogenicity of this compound in liver, rather decreased the inhibitory effect in rat liver slices. A similar picture was observed with guinea pig slices, although the difference in this case was less pronounced.6.6. A certain time of preincubation was needed for the inhibitory effects to become manifest. The incorporation of 14C-valine into protein by rat kidney slices was not inhibited by the above compounds. These facts suggest that the true inhibitors were metabolitic derivatives of the administered substances.7.7. The incorporation of 14C-adenine into ribonucleic acid by rat liver slices was inhibited by AF, FAF and 2-AN under the same conditions as the valine incorporation, although to a somewhat lesser extent.

Evidence for increased synthesis of lipoprotein(a) in the nephrotic syndrome.

In patients with the nephrotic syndrome, markedly increased levels of lipoprotein(a) (Lp(a)) concentration have been frequently reported, and it has been suggested that this may contribute to the increased cardiovascular risk in these patients. The mechanism, however, is not clear. In the present study, in vivo fractional synthesis rate of Lp(a) was measured using incorporation of the stable isotope 13C valine. Under steady-state conditions, fractional synthesis rate equals fractional catabolic rate (FCR). FCR of Lp(a) was estimated in five patients with the nephrotic syndrome and compared with five control subjects. The mean plasma Lp(a) concentration in the patients (1749+/-612 mg/L) was higher than in control subjects (553+/-96 mg/L). Two patients were heterozygous for apolipoprotein(a) (range, 19 to 30 kringle IV domains), whereas all control subjects were each homozygous with regard to apolipoprotein(a) phenotype (range, 18 to 28 kringle IV domains). The FCR of Lp(a) was comparable between control subjects (0.072+/-0.032 pools/d) and patients (0.064+/-0.029 pools/d) despite the wide variance in plasma concentration. This suggests that differences in Lp(a) levels are caused by differences in synthesis rate. Indeed, the absolute synthetic rate of Lp(a) correlated directly with plasma Lp(a) concentration (P < 0.0001) in all subjects. The present results demonstrate that increased synthesis, rather than decreased catabolism, causes elevated plasma Lp(a) concentrations in the nephrotic syndrome.

Increased VLDL in nephrotic patients results from a decreased catabolism while increased LDL results from increased synthesis

Increased VLDL in nephrotic patients results from a decreased catabolism while increased LDL results from increased synthesis

Insulin-like growth factor-I potentiates protein synthesis induced by thyrotropin in FRTL-5 cells: comparison of induction of protein synthesis and DNA synthesis.

In FRTL-5 cells, we and others have shown that TSH and insulin-like growth factor-I (IGF-I) stimulate DNA synthesis synergistically. The present study was undertaken to determine whether interaction between TSH and IGF-I also affects protein synthesis in this cell line, and if so by what mechanism. Quiescent cells were treated with TSH and/or IGF-I and [3H]valine incorporation into the acid-insoluble fraction was measured as a parameter of protein synthesis. Similar to their effects on cell proliferation, TSH or IGF-I alone induced protein synthesis only slightly, but treatment with a combination of TSH and IGF-I (or insulin with about a 100-fold higher concentration than IGF-I) greatly increased protein synthesis. The presence of IGF-I potentiated a TSH-concentration-dependent increase in protein synthesis and in DNA synthesis. In addition, we observed this potentiation when the cells were treated with other cAMP-generating agents and cAMP analogues instead of TSH. We have shown that priming with TSH potentiated DNA synthesis induced by IGF-I, whereas pretreatment with IGF-I enhanced protein synthesis induced by TSH. This observation suggested that protein synthesis and DNA synthesis were potentiated through different mechanisms. From an analysis of cAMP production, it appears that the potentiation of protein synthesis may be explained by an IGF-I-dependent increase in cAMP production induced by TSH at least in part. On the other hand, IGF-I and TSH stimulated (alpha-aminoisobutyric acid (AIB) uptake synergistically, but RNA synthesis induced by IGF-I was depressed by TSH. From these results, we concluded that in FRTL-5 cells, IGF-I potentiated protein synthesis induced by TSH by means of complex mechanisms and the interaction between IGF-I and cAMP-dependent pathways may also have a physiological meaning in regulating protein anabolism.

The Effect of Different Bovine Viral Diarrhea Virus Genotypes and Biotypes on the Metabolic Activity and Activation Status of Bovine Peripheral Blood Mononuclear Cells

The effects of cytopathic (cp) and non-cytopathic (ncp) bovine viral diarrhea virus (BVDV) on the cellular metabolic activity and activation status of bovine peripheral blood mononuclear cells (PBMC) were investigated. Cellular DNA and protein synthesis was determined by [3H]thymidine and [3H]valine incorporation, respectively, in phytohemagglutinin (PHA)-stimulated PBMC. All cp strains and most ncp BVDV strains significantly inhibited DNA synthesis in PHA-stimulated PBMC; however, only cp BVDV strains inhibited protein synthesis. A plaque assay and immunofluorescence test confirmed productive BVDV infection of PBMC. In addition, viral RNA synthesis was demonstrated in BVDV-infected PBMC by RT-PCR. The interleukin-2 receptor (IL-2R) was used as a marker for the activation status of BVDV-infected PBMC. The expression of IL-2R was preserved in virus-infected cells, even though DNA and protein synthesis was suppressed. These findings suggest a novel mechanism of virus-induced immune suppression in which BVDV inhibits basic metabolic activities of bovine PBMC. The activation signals, however, are maintained.

Physiological changes in extracellular sodium directly control human proximal tubule growth and transport.

In order to examine the nature and potential mechanisms of action of extracellular sodium on human proximal tubule growth and transport, quiescent primary cultures of human proximal tubule cells (PTC) were incubated for 24 h in serum-free, growth-factor-free culture media containing low (130 mmol/l), control (140 mmol/l) or high (150 mmol/l) Na+. Compared to control conditions, cells exposed to a high Na+ concentration demonstrated stimulated thymidine incorporation (121.8 +/- 7.6%, P < 0.05) and increased cellular protein content (139.7 +/- 9.9%, P < 0.05); the latter arising from suppressed protein degradation ([3H]valine release 72.3 +/- 2.5%, P < 0.01) and unchanged protein synthesis ([3H]valine incorporation 98.5 +/- 2.6%, P > 0.1). Substitution of choline chloride for NaCl did not replicate these effects. Conversely, cells incubated in low-Na+ media showed reduced thymidine incorporation (77.2 +/- 4.4%, P < 0. 05), reduced protein synthesis (60.6 +/- 4.3%, P < 0.01), reduced protein degradation (79.5 +/- 1.8%, P < 0.01) and an unaltered protein content (102.4 +/- 8.8%). A role for apical Na+/H+ exchange (NHE) activity in mediating Na+-dependent alterations in PTC growth was suggested by the findings of increased apical, ethylisopropylamiloride- (EIPA)-sensitive 22Na+ uptake in the presence of a high Na+ concentration (159 +/- 19% of control, P < 0. 05) and concentration-dependent inhibition of cellular growth by EIPA at levels corresponding to those producing inhibition of apical NHE. Conditioned media from low Na+, control or high Na+ PTC contained comparable amounts of platelet-derived growth factor-AB (1. 19 +/- 0.23, 1.14 +/- 0.22 and 1.28 +/- 0.20 ng/mg protein, P > 0.1) and transforming growth factor-beta1 (1.76 +/- 0.32, 1.73 +/- 0.33 and 1.45 +/- 0.28 ng/mg protein, P > 0.1), and did not exhibit autocrine growth factor activity on separate PTC following adjustment of Na+ concentrations to 140 mmol/l by dialysis. Similarly, low-Na+, control or high-Na+ media did not modify the mitogenic responsiveness of PTC to insulin-like growth factor-I (IGF-I) or alter the affinity or number of PTC IGF-I binding sites. The results confirm that physiological increases in extracellular Na+ concentration directly stimulate human proximal tubule growth and Na+ transport. Such stimulation does not appear to be mediated by altered PTC secretion of, or responsiveness to, cytokines known to affect tubule growth and transport.

The rate of valine incorporation into proteins with correction for valine recycling, measured in two brain tumor models and the cortex.

The tissue dilution factor (lambda) for the incorporation of valine into proteins in the rat cortex and in two different tumors, AA ascites and C6 glioma, was determined from measurements of specific activities in the tissue acid-soluble and aminoacyl-tRNA pools and in the plasma. A constant plasma specific activity was achieved by a constant infusion rate of [3H] valine. The data showed that the lambda for valine was the same in the cortex as in the tumors, and the recycling was approximately 36%. There was no difference in the lambda calculated on the basis of the specific activities in the tissue acid-soluble or aminoacyl-tRNA pools. The average dilution factor was found to be 0.64+/-0.05. The rate of valine incorporation into proteins was on average 3.2+/-0.4 and 4.9+/-0.4 nmol/g/min in the cortex for the groups of rats used in the AA ascites and C6 glioma experiments, respectively. In the AA ascites tumor the rate was approximately 41 and 29 nmol/g/min 4 and 7 days after tumor implantation, respectively, whereas in the C6 glioma the rate was approximately 41 and 72 nmol/g/min 6 and 13 days after inoculation, respectively. The tumors had, in comparison with the cortex, a significantly greater volume of distribution of valine. The amounts of valyl-tRNA were significantly greater in the tumors as compared with the normal cortex, with the exception of the glioma 6 days after implantation where the concentration was the same as in the cortex.

Leucine Affects the Metabolism of Valine by Isolated Perfused Rat Hearts: Relation to Branched-Chain Amino Acid Antagonism

This study was conducted to determine the effects of different concentrations of leucine on the transport, transamination and oxidation of valine and on incorporation of valine into heart proteins in the isolated perfused rat heart. Valine metabolism was studied in rat hearts perfused with medium containing glucose and graded levels of L-leucine. In transport studies L-phenylalanine was also tested. Uptake of L-[1-14C]valine (0.2 mmol/L) was significantly reduced (∼50%) by inclusion of 0.2 mmol/L phenylalanine or leucine, and by ∼70% by inclusion of 1.0 mmol/L phenylalanine or leucine in the perfusate. Transamination of valine decreased by 37 and 48%, and oxidation of valine by 53 and 71%, respectively, when 0.2 or 1.0 mmol/L leucine was included in the perfusate. Tissue concentrations of valine decreased by 43, 48 and 62% in the presence of 0.2, 0.5 and 1.0 mmol/L leucine, respectively; tissue concentrations of leucine, glutamate and alanine increased ∼11-fold, 1.2-fold and 0.5-fold, respectively, when 1.0 mmol/L leucine was present in the perfusate. Addition of 0.2–1.0 mmol/L leucine did not affect incorporation of valine into heart proteins. We conclude that 1) competition among large neutral amino acids for transport into heart occurs at physiological concentrations of these amino acids in plasma; 2) inhibition of valine uptake by leucine can limit the rate of valine catabolism in heart; and 3) depletion of tissue valine concentration by an excess of leucine did not affect the rate of protein synthesis.

Role of amino acid‐induced changes in ion fluxes in the regulation of hepatic protein synthesis

Alanine is a powerful stimulator of hepatic protein synthesis whose mechanism of action has not yet been ascertained. The present work aimed to elucidate whether rate changes in ion fluxes accompanying the transport of this amino acid could play a role in the stimulation of protein synthesis. In perfused livers, the utilization of alanine produced a net uptake of K+ of 1.5 mumol/min/liver, a progressively increasing efflux of Ca2+ to reach a maximum of 0.9 mumol/min/liver, and alkalization of the extracellular medium. Inhibition of Na+/K+ exchange by ouabain reversed only the uptake of K+, indicating that this is the main way for the efflux of Na+ cotransported with alanine. In isolated hepatocytes, the uptake of alanine increased the intracellular content of K+ and the cell volume. The following observations suggest that these changes, and not an increased intracellular concentration of Na+, are associated with the stimulation of protein synthesis: 1) Ouabain inhibited the alanine stimulation of L-[3H]-valine incorporation into protein without altering the basal rate of protein labeling; 2) ouabain had no effects on alanine uptake indicating that Na+ influx is not involved in the alanine stimulation of protein synthesis; 3) disruption of Na+ gradient across the plasma membrane by specific ionophores, monensin and gramicidin D, inhibited both basal and alanine-stimulated protein synthesis, but substitution of extracellular Na+ by K+ did not prevent the stimulatory action of alanine. The observation that hypotonic buffer enhanced protein synthesis to the same degree than alanine in liver cells indicates that alanine-induced cell swelling could be sufficient to stimulate protein synthesis.

Influence of valine flooding on channeling of valine into tissue pools and on protein synthesis.

Rates of valine incorporation into protein were measured under control and valine-"flooding" conditions and included correction for the degree of recycling of unlabeled valine derived from the steady-state breakdown of tissue protein into the precursor pool (tRNA bound). The correction factor lambda, which is the ratio of the steady-state specific activity of valine in the tissue tRNA-bound pool to that in the arterial plasma, was determined for each of the tissues. In controls, values of lambda ranged from 0.31 in adrenals to 0.54 in heart; in flooded animals, values were higher, but only in liver was the value of lambda close to 1.0. In control and flooded rats, rates of protein synthesis were highest in liver and adrenals and lowest in skeletal muscle, with intermediate values in brain and heart. Flooding resulted in increased rates of protein synthesis in liver and decreased rates in adrenals. Rates of protein synthesis in brain, heart, and skeletal muscle were not statistically significantly affected by flooding.

Parallel formation and synergism of hydrolytic enzymes and peptaibol antibiotics, molecular mechanisms involved in the antagonistic action of Trichoderma harzianum against phytopathogenic fungi

Chitinase, beta-1,3-glucanase, and protease activities were formed when Trichoderma harzianum mycelia, grown on glucose as the sole carbon source, were transferred to fresh medium containing cell walls of Botrytis cinerea. Chitobiohydrolase, endochitinase, and beta-1,3-glucanase activities were immunologically detected in culture supernatants by Western blotting (immunoblotting), and the first two were quantified by enzyme-linked immunosorbent assay. Under the same conditions, exogenously added [U-14C]valine was incorporated in acetone-soluble compounds with an apparent M(r) of < 2,000. These compounds comigrated with the peptaibols trichorzianines A1 and B1 in thin-layer chromatography and released [U-14C]valine after incubation in 6N HCl. Incorporation of radioactive valine into this material was stimulated by the exogenous supply of alpha-aminoisobutyric acid, a rare amino acid which is a major constituent of peptaibols. The obtained culture supernatants inhibited spore germination as well as hyphal elongation of B. cinerea. Culture supernatants from mycelia placed in fresh medium without cell walls of B. cinerea did not show hydrolase activities, incorporation of [U-14C]valine into peptaibol-like compounds, and inhibition of fungal growth. Purified trichorzianines A1 and B1 as well as purified chitobiohydrolase, endochitinase, or beta-1,3-glucanase inhibited spore germination and hyphal elongation, but at concentrations higher than those observed in the culture supernatants. However, when the enzymes and the peptaibols were tested together, an antifungal synergistic interaction was observed and the 50% effective dose values obtained were in the range of those determined in the culture supernatants. Therefore, the parallel formation and synergism of hydrolytic enzymes and antibiotics may have an important role in the antagonistic action of T. harzianum against fungal phytopathogens.

Renal tubular cell protein breakdown in uninephrectomized and ammonium chloride-loaded rats.

Kidney enlargement after unilateral nephrectomy or the induction of a systemic acidosis with ammonium chloride is associated with an increase in kidney protein content. This reflects an imbalance between protein breakdown and protein synthesis. Because it has been shown in diabetic nephromegaly that depressed protein breakdown contributes to the increase in kidney protein content, this study examined whether altered protein breakdown is common to all forms of renal hypertrophy. Accordingly, protein turnover was measured in isolated proximal tubules from kidney in rats undergoing renal enlargement after uninephrectomy or chronic ammonium chloride-induced acidosis. In both conditions, kidney protein content and protein synthesis ([14C]valine incorporation) increased significantly. Fractional protein degradation was depressed in renal tubules isolated from the acidotic rats and was accompanied by a decrease in proximal tubule cathepsin B and combined B and L activities. These changes are comparable to earlier observations with the diabetic kidney. In contrast, after unilateral nephrectomy, protein breakdown is not reduced, and it can reasonably be concluded that, in this condition, protein gain reflects increased protein synthesis alone. It was concluded that the pattern of protein turnover leading to protein accretion in renal hypertrophy varies according to the initial stimulus for renal growth.