Deprivation Of Essential Amino Acid Research Articles

Essential amino acid starvation induces cell cycle arrest, autophagy, and inhibits osteogenic differentiation in murine osteoblast

This study investigates the effects of essential amino acid (EAA) starvation on murine osteoblasts cells and the underlying mechanisms. We performed and observed the cell proliferation, autophagy, and osteogenic differentiation under deprivation of EAA in vitro. The results showed that EAA starvation resulted in cell cycle arrest via phosphorylation of the MAPK signaling pathway, leading to inhibition of cell proliferation and osteogenic differentiation. Additionally, the LKB1-AMPK signaling pathway was also found to be phosphorylated, inducing autophagy. These findings highlight the significant role of EAA in regulating cellular processes. Furthermore, this study contributes to our understanding of the effects of nutrient deprivation on cellular physiology and may aid in the development of novel therapeutic strategies for diseases associated with amino acid metabolism.

Nutritional and reproductive status affect amino acid appetite in house crickets (Acheta domesticus).

We examined amino acid appetite in the omnivorous house cricket (Acheta domesticus), a common model organism for both research and teaching. Our first experiment addressed the hypothesis that house crickets can discriminate between sucrose and essential amino acids (EAA), and that preference for the latter would be affected by prior feeding experience. To test this hypothesis, we compared feeding responses of juvenile and adult crickets following pre-feeding with sucrose or an essential amino acid mixture, predicting that sucrose-only pre-feeding would enhance subsequent intake of amino acids in a two-choice preference test. Based on previous studies, we also predicted that amino acid consumption would be enhanced in females compared to males, and in mated compared to virgin females. Hence we compared responses in male and female last instar nymphs, adult males, virgin females, mated females, and mated females allowed to lay eggs. The second experiment examined how extended periods of essential amino acid deprivation (48 h to 6 days) affected appetite for these nutrients in adult male and female insects. Finally, we examined growth and survival of juvenile and adult crickets fed a holidic diet lacking all amino acids and protein. Our results demonstrated that house crickets can distinguish EAA from sucrose and that consumption of the former is enhanced following sucrose-only pre-feeding. We also found sex and developmental differences, with juvenile and virgin females showing a greater preference for EAA than juvenile or adult males. Contrary to expectation, mated females preferred sucrose over EAA both prior to and after egg laying. We also found that the crickets of both sexes increased their intake of EAA when exposed to longer periods of deprivation, indicating that they engage in compensatory feeding on these nutrients. Finally, as expected we found that growth was severely limited in juveniles fed a diet lacking all amino acids, but adults and many juveniles survived for 30 days on this diet.

Amino acid sensor GCN2 promotes SARS-CoV-2 receptor ACE2 expression in response to amino acid deprivation

Angiotensin-converting enzyme 2 (ACE2) has been identified as a primary receptor for severe acute respiratory syndrome coronaviruses 2 (SARS-CoV-2). Here, we investigated the expression regulation of ACE2 in enterocytes under amino acid deprivation conditions. In this study, we found that ACE2 expression was upregulated upon all or single essential amino acid deprivation in human colonic epithelial CCD841 cells. Furthermore, we found that knockdown of general control nonderepressible 2 (GCN2) reduced intestinal ACE2 mRNA and protein levels in vitro and in vivo. Consistently, we revealed two GCN2 inhibitors, GCN2iB and GCN2-IN-1, downregulated ACE2 protein expression in CCD841 cells. Moreover, we found that increased ACE2 expression in response to leucine deprivation was GCN2 dependent. Through RNA-sequencing analysis, we identified two transcription factors, MAFB and MAFF, positively regulated ACE2 expression under leucine deprivation in CCD841 cells. These findings demonstrate that amino acid deficiency increases ACE2 expression and thereby likely aggravates intestinal SARS-CoV-2 infection.

Activation of the eIF2α-ATF4 Pathway by Chronic Paracetamol Treatment Is Prevented by Dietary Supplementation with Cysteine.

Chronic treatment with acetaminophen (APAP) induces cysteine (Cys) and glutathione (GSH) deficiency which leads to adverse metabolic effects including muscle atrophy. Mammalian cells respond to essential amino acid deprivation through the phosphorylation of the eukaryotic translation initiation factor 2α (eIF2α). Phosphorylated eIF2α leads to the recruitment of activating transcription factor 4 (ATF4) to specific CCAAT/enhancer-binding protein-ATF response element (CARE) located in the promoters of target genes. Our purpose was to study the activation of the eIF2α-ATF4 pathway in response to APAP-induced Cys deficiency, as well as the potential contribution of the eIF2α kinase GCN2 and the effect of dietary supplementation with Cys. Our results showed that chronic treatment with APAP activated both GCN2 and PERK eIF2α kinases and downstream target genes in the liver. Activation of the eIF2α-ATF4 pathway in skeletal muscle was accompanied by muscle atrophy even in the absence of GCN2. The dietary supplementation with cysteine reversed APAP-induced decreases in plasma-free Cys, liver GSH, muscle mass, and muscle GSH. Our new findings demonstrate that dietary Cys supplementation also reversed the APAP-induced activation of GCN2 and PERK and downstream ATF4-target genes in the liver.

Activation and execution of the hepatic integrated stress response by dietary essential amino acid deprivation is amino acid specific.

Dietary removal of an essential amino acid (EAA) triggers the integrated stress response (ISR) in liver. Herein, we explored the mechanisms that activate the ISR and execute changes in transcription and translation according to the missing EAA. Wild‐type mice and mice lacking general control nonderepressible 2 (Gcn2) were fed an amino acid complete diet or a diet devoid of either leucine or sulfur amino acids (methionine and cysteine). Serum and liver leucine concentrations were significantly reduced within the first 6 h of feeding a diet lacking leucine, corresponding with modest, GCN2‐dependent increases in Atf4 mRNA translation and induction of selected ISR target genes (Fgf21, Slc7a5, Slc7a11). In contrast, dietary removal of the sulfur amino acids lowered serum methionine, but not intracellular methionine, and yet hepatic mRNA abundance of Atf4, Fgf21, Slc7a5, Slc7a11 substantially increased regardless of GCN2 status. Liver tRNA charging levels did not correlate with intracellular EAA concentrations or GCN2 status and remained similar to mice fed a complete diet. Furthermore, loss of Gcn2 increased the occurrence of ribosome collisions in liver and derepressed mechanistic target of rapamycin complex 1 signal transduction, but these changes did not influence execution of the ISR. We conclude that ISR activation is directed by intracellular EAA concentrations, but ISR execution is not. Furthermore, a diet devoid of sulfur amino acids does not require GCN2 for the ISR to execute changes to the transcriptome.

Amino acid deprivation triggers a novel GCN2-independent response leading to the transcriptional reactivation of non-native DNA sequences.

In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan. However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap. We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation. Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome. Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs. The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated. Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2. Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease.

Amino acid deprivation and central carbon metabolism regulate the production of outer membrane vesicles and tubes by Francisella.

Francisella tularensis is a highly virulent Gram-negative bacterial pathogen that causes the zoonotic disease tularemia. F. novicida, a model tularemia strain, produces spherical outer membrane vesicles (OMV), as well as novel tubular vesicles and extensions of the cell surface. These OMV and tubes (OMV/T) are produced in a regulated manner and contain known virulence factors. Mechanisms by which bacterial vesicles are produced and regulated are not well understood. We performed a genetic screen in F. novicida to decipher the molecular basis for regulated OMV/T formation, and identified both hypo- and hyper-vesiculating mutants. Mutations in fumA and tktA, involved in central carbon metabolism, and in FTN_0908 and FTN_1037, of unknown function, resulted in severe defects in OMV/T production. Cysteine deprivation was identified as the signal that triggers OMV/T formation in F. novicida during growth in rich medium. We also found that fully virulent F. tularensis produces OMV/T in a similarly regulated manner. Further analysis revealed that OMV/T production is responsive to deprivation of essential amino acids in addition to cysteine, and that the hypo-vesiculating mutants are defective in responding to this signal. Thus, amino acid starvation, such as encountered by Francisella during host cell invasion, regulates the production of membrane-derived structures.

Concentration-dependent linkage of dietary methionine restriction to the components of its metabolic phenotype.

ObjectiveRestricting dietary methionine to 0.17% produces a series of physiological responses through coordinated transcriptional effects in liver and adipose tissue. The goal of the present work was to determine the threshold concentrations above and below 0.17% at which the beneficial responses to 0.17% dietary methionine are preserved.MethodsDiets were formulated to restrict methionine to different degrees, followed by evaluation of the transcriptional and physiological responses to the different diets.ResultsRestriction of dietary methionine to 0.25%, but not 0.34%, was partially effective in reproducing the metabolic phenotype produced by restriction of methionine to 0.17%, while restriction of methionine to 0.12% reproduced the responses produced by restriction to 0.17% but failed to support growth and caused excessive weight loss. Restriction beyond 0.12% initiated responses characteristic of essential amino acid deprivation including food aversion and rapid weight loss.ConclusionsRestriction of dietary methionine to levels above 0.25% was without effect while restriction to levels below 0.12% produced responses characteristic of essential amino acid deprivation. In addition, although restriction of dietary methionine to 0.12% does not evoke essential amino acid deprivation responses, it provides insufficient methionine to support growth. The ideal range of dietary methionine restriction is from 0.17% to 0.25%.

Widespread pain and altered renal function in ME/CFS patients

ABSTRACT Background: Widespread pain is noted in many patients with chronic fatigue syndrome (ME/CFS), fibromyalgia and temporomandibular disorders. These conditions usually start as a localized condition and spread to a widespread pain condition with increasing illness duration. Purpose: To aim was to assess the changes in biochemistry associated with pain expression and alterations in renal function. Methods: Forty-seven ME/CFS patients and age/sex-matched controls had a clinical examination, completed questionnaires, standard serum biochemistry, glucose tolerance tests and serum and urine metabolomes in an observational study. Results: Increases in pain distribution were associated with reductions in serum essential amino acids, urea, serum sodium and increases in serum glucose and the 24-hour urine volume; however the biochemistry was different for each pain area. Regression modelling revealed potential acetylation and methylation defects in the pain subjects. Conclusions: These findings confirm and extend our earlier findings. These changes appear consistent with repeated minor inflammatory-mediated alterations in kidney function resulting in essential amino acid deprivation and inhibition of protein synthesis and genetic translation within tissues.

CREB/TRH pathway in the central nervous system regulates energy expenditure in response to deprivation of an essential amino acid.

In the central nervous system (CNS), thyrotropin-releasing hormone (TRH) has an important role in regulating energy balance. We previously showed that dietary deprivation of leucine in mice increases energy expenditure through CNS-dependent regulation. However, the involvement of central TRH in this regulation has not been reported. Male C57J/B6 mice were maintained on a control or leucine-deficient diet for 7 days. Leucine-deprived mice were either third intracerebroventricular (i.c.v.) injected with a TRH antibody followed by intraperitoneal (i.p.) injection of triiodothyronine (T3) or i.c.v. administrated with an adenovirus of shCREB (cAMP-response element binding protein) followed by i.c.v. injection of TRH. Food intake and body weight were monitored daily. Oxygen consumption, physical activity and rectal temperature were assessed after the treatment. After being killed, the hypothalamus and the brown adipose tissue were collected and the expression of related genes and proteins related was analyzed. In other experiments, control or leucine-deficient medium incubated primary cultured neurons were either infected with adenovirus-mediated short hairpin RNA targeting extracellular signal-regulated kinases 1 and 2 (Ad-shERK1/2) or transfected with plasmid-overexpressing protein phosphatase 1 regulatory subunit 3C (PPP1R3C). I.c.v. administration of anti-TRH antibodies significantly reduced leucine deprivation-stimulated energy expenditure. Furthermore, the effects of i.c.v. TRH antibodies were reversed by i.p. injection of T3 during leucine deprivation. Moreover, i.c.v. injection of Ad-shCREB (adenovirus-mediated short hairpin RNA targeting CREB) significantly suppressed leucine deprivation-stimulated energy expenditure via modulation of TRH expression. Lastly, TRH expression was regulated by CREB, which was phosphorylated by ERK1/2 and dephosphorylated by PPP1R3C-containing protein Ser/Thr phosphatase type 1 (PP1) under leucine deprivation in vitro. Our data indicate a novel role for TRH in regulating energy expenditure via T3 during leucine deprivation. Furthermore, our findings reveal that TRH expression is activated by CREB, which is phosphorylated by ERK1/2 and dephosphorylated by PPP1R3C-containing PP1. Collectively, our studies provide novel insights into the regulation of energy homeostasis by the CNS in response to an essential amino-acid deprivation.

Dietary deprivation of each essential amino acid induces differential systemic adaptive responses in mice

Dietary deprivation of essential amino acids (EAAs) in mammals is known to cause reductions in food intake and body weight. The aim of this study was to determine whether and how mice respond to deprivation of individual EAA species. Dietary deprivation of any single EAA (not non-EAA) in mice led to progressive weight loss in the order of Ile(-) > Val(-) > Thr(-) > Leu(-) > Trp(-) > His(-) > Phe(-) > Met(-) > Lys(-) , which correlated with the reduction in food intake. Decreased levels of the deprived EAAs as well as increased levels of all or some of the other amino acids were detected in the serum, although these levels differed among the diets examined. Serum biochemistry identified significant increases in creatine phosphokinase, blood urea nitrogen, alanine aminotransferase, and aspartate aminotransferase, and decreases in glucose and triglycerides; computed tomography revealed a marked reduction in abdominal/femoral fat and muscle depots; histology identified diffuse myofiber atrophy in the rectus femoris muscle, all in that approximate order. In contrast, amino acid response, autophagy, and ubiquitination marker genes as well as amino acid transporter genes were induced in both deprived EAA-specific and tissue-specific manners. Dietary deprivation of individual EAAs induced systemic adaptive responses that differed in magnitude and molecular machinery.

Passport control for foreign integrated DNAs

The endless battle between mammalian host cells and microbes has evolved mechanisms to shut down the expression of exogenous transcriptional units integrated into the genome with the goal of limiting their spreading. Recently, we observed that deprivation of essential amino acids leads to a selective, reversible upregulation of expression of exogenous transgenes, either carried by integrated plasmids or retroviral vectors, but not of their endogenous counterparts. This effect was dependent on epigenetic modifications and was mediated by the downregulation of the class II histone deacetylase-4 (HDAC4). Indeed, HDAC4 expression inversely correlated with that of the transgene and its inhibition or downregulation enhanced transgene expression. Could this be true also for “naturally” integrated proviruses? We investigated this question in the case of HIV-1, the etiological agent of AIDS and we observed that both amino acid starvation and HDAC4 inhibition triggered HIV-1 reactivation in chronically infected ACH-2 T lymphocytic cells (HDAC4+), but not in similarly infected U1 promonocytic cells (HDAC4-negative). Thus, an HDAC4-dependent pathway may contribute to unleash virus expression by latently infected cells, which represent nowadays a major obstacle to HIV eradication. We discuss here the implications and open questions of these novel findings, as well as their serendipitous prelude.

Effects of essential amino acids or glutamine deprivation on intestinal permeability and protein synthesis in HCT-8 cells: involvement of GCN2 and mTOR pathways

GCN2 and mTOR pathways are involved in the regulation of protein metabolism in response to amino acid availability in different tissues. However, regulation at intestinal level is poorly documented. The aim of the study was to evaluate the effects of a deprivation of essential amino acids (EAA) or glutamine (Gln) on these pathways in intestinal epithelial cells. Intestinal epithelial cell, HCT-8, were incubated during 6h with 1/DMEM culture medium containing EAA, non EAA and Gln, 2/with saline as positive control of nutritional deprivation, 3/DMEM without EAA, 4/DMEM without Gln or 5/DMEM without Gln and supplemented with a glutamine synthase inhibitor (MSO, 4mM). Intestinal permeability was evaluated by the measure of transepithelial electric resistance (TEER). Using [L-(2)H(3)]-leucine incorporation, fractional synthesis rate (FSR) was calculated from the assessed enrichment in proteins and free amino acid pool by GCMS. Expression of eiF2α (phosphorylated or not), used as marker of GCN2 pathway, and of 4E-BP1 (phosphorylated or not), used as a marker of mTOR pathway, was evaluated by immunoblot. Results were compared by ANOVA. Six-hours EAA deprivation did not significantly affect TEER and FSR but decreased p-4E-BP1 and increased p-eiF2α. In contrast, Gln deprivation decreased FSR and p-4E-BP1. MSO induced a marked decrease of TEER and FSR and an increase of p-eiF2α, whereas mTOR pathway remained activated. These results suggest that both mTOR and GCN2 pathways can mediate the limiting effects of Gln deprivation on protein synthesis according to its severity.

Gene expression and integrated stress response in HepG2/C3A cells cultured in amino acid deficient medium.

The integrated stress response (ISR), a defense mechanism cells employ when under stress (e.g., amino acid deprivation), causes suppression of global protein synthesis along with the paradoxical increased expression of a host of proteins that are useful in combating various stresses. Genes that were similarly differentially expressed under conditions of either leucine- or cysteine-depletion were identified. Many of the genes known to contain an amino acid response element and to be induced in response to eIF2α phosphorylation and ATF4 heterodimer binding (ATF3, C/EBPβ, SLC7A1, SLC7A11, and TRIB3), as well as others shown to be induced downstream of eIF2α phosphorylation (C/EBPγ, CARS, SARS, CLCN3, CBX4, and PPP1R15A) were among the upregulated genes. Evidence for the induction of the ISR in these cells also included the increased phosphorylation of eIF2α and increased protein abundance of ATF4, ATF3, and ASNS in cysteine- and leucine-depleted cells. Based on genes highly differentially expressed in both leucine- and cysteine-deficient cells, a list of 67 downregulated and 53 upregulated genes is suggested as likely targets of essential amino acid deprivation in mammalian cells.

Metabolic Regulation of Manganese Superoxide Dismutase Expression via Essential Amino Acid Deprivation

Organisms respond to available nutrient levels by rapidly adjusting metabolic flux, in part through changes in gene expression. A consequence of adaptations in metabolic rate is the production of mitochondria-derived reactive oxygen species. Therefore, we hypothesized that nutrient sensing could regulate the synthesis of the primary defense of the cell against superoxide radicals, manganese superoxide dismutase. Our data establish a novel nutrient-sensing pathway for manganese superoxide dismutase expression mediated through essential amino acid depletion concurrent with an increase in cellular viability. Most relevantly, our results are divergent from current mechanisms governing amino acid-dependent gene regulation. This pathway requires the presence of glutamine, signaling via the tricarboxylic acid cycle/electron transport chain, an intact mitochondrial membrane potential, and the activity of both the MEK/ERK and mammalian target of rapamycin kinases. Our results provide evidence for convergence of metabolic cues with nutrient control of antioxidant gene regulation, revealing a potential signaling strategy that impacts free radical-mediated mutations with implications in cancer and aging.

Regulation of Global and Specific mRNA Translation by Amino Acids

A continuous supply of a complete complement of essential amino acids is a prerequisite for maintenance of optimal rates of protein synthesis in both liver and skeletal muscle. Deprivation of even a single essential amino acid causes a decrease in the synthesis of essentially all cellular proteins through an inhibition of the initiation phase of mRNA translation. However, the synthesis of all proteins is not repressed equally. Specific subsets of proteins, in particular those encoded by mRNAs containing a 5'-terminal oligopyrimidine (TOP) motif, are affected to a much greater extent than most proteins. The specific decrease in TOP mRNA translation is a result of an inhibition of the ribosomal protein S6 kinase, S6K1, and a concomitant decline in S6 phosphorylation. Interestingly, many TOP mRNAs encode proteins involved in mRNA translation, such as elongation factors eEF1A and eEF2, as well as the ribosomal proteins. Thus, deprivation of essential amino acids not only directly and rapidly represses global mRNA translation, but also potentially results in a reduction in the capacity to synthesize protein.

Essential Amino Acid Deprivation Induces Monocytic Differentiation of the Human HL-60 Myeloid Leukemia Cell Line

In this study we examine the effects of amino acid deprivation on the growth and differentiation of the human HL-60 myeloid leukemia cell line. The HL-60 cell line was chosen for study because of its ability to differentiate along either a granulocytic or monocytic pathway under appropriate culture conditions. Differentiation was determined by changes in cell morphology, nonspecific esterase (NSE) content, hydrogen peroxide (H2O2) production, and expression of the cell surface differentiation antigens LeuM3 (CD14) and OKM1 (CD11). Using a model system in which HL-60 cells were cultured in medium that selectively lacked one amino acid (AA), it was seen that deprivation of HL-60 cells for essential (but not nonessential) AAs results in decreased cell growth and viability and in differentiation of 30% to 60% of the surviving population of cells specifically along the monocytic pathway. This differentiation is irreversible as well as time- and dose-dependent. Culture of HL-60 cells in essential AA-deficient medium potentiated the differentiative effects of recombinant human interferon-gamma (IFN-gamma), recombinant human tumor necrosis factor (TNF), and dihydroxyvitamin D3 (D3), all of which have previously been shown to induce monocytic differentiation of HL-60 cells. Differentiated cells had decreased DNA and RNA synthesis, but protein synthesis was unchanged compared with control cells. The protein synthesis inhibitor cycloheximide prevented differentiation, indicating the necessity of protein synthesis in this process. Cell cycle analysis revealed that an increased proportion of cells cultured in AA-deficient medium was arrested in G0-G1 (80% and 50% for AA-deficient and control cells, respectively). These results suggest that alterations of AA metabolism and subsequent perturbations in DNA and RNA synthesis may be important in initiating differentiation or in augmenting cytokine-induced differentiation of HL-60 cells into more mature, nonreplicating, monocyte-like cells.

Essential amino acid deprivation induces monocytic differentiation of the human HL-60 myeloid leukemia cell line

In this study we examine the effects of amino acid deprivation on the growth and differentiation of the human HL-60 myeloid leukemia cell line. The HL-60 cell line was chosen for study because of its ability to differentiate along either a granulocytic or monocytic pathway under appropriate culture conditions. Differentiation was determined by changes in cell morphology, nonspecific esterase (NSE) content, hydrogen peroxide (H2O2) production, and expression of the cell surface differentiation antigens LeuM3 (CD14) and OKM1 (CD11). Using a model system in which HL-60 cells were cultured in medium that selectively lacked one amino acid (AA), it was seen that deprivation of HL-60 cells for essential (but not nonessential) AAs results in decreased cell growth and viability and in differentiation of 30% to 60% of the surviving population of cells specifically along the monocytic pathway. This differentiation is irreversible as well as time- and dose- dependent. Culture of HL-60 cells in essential AA-deficient medium potentiated the differentiative effects of recombinant human interferon- gamma (IFN-gamma), recombinant human tumor necrosis factor (TNF), and dihydroxyvitamin D3 (D3), all of which have previously been shown to induce monocytic differentiation of HL-60 cells. Differentiated cells had decreased DNA and RNA synthesis, but protein synthesis was unchanged compared with control cells. The protein synthesis inhibitor cycloheximide prevented differentiation, indicating the necessity of protein synthesis in this process. Cell cycle analysis revealed that an increased proportion of cells cultured in AA-deficient medium was arrested in G0-G1 (80% and 50% for AA-deficient and control cells, respectively). These results suggest that alterations of AA metabolism and subsequent perturbations in DNA and RNA synthesis may be important in initiating differentiation or in augmenting cytokine-induced differentiation of HL-60 cells into more mature, nonreplicating, monocyte-like cells.

The Effect of Single Essential Amino Acid Deprivation on Chick Growth and Nitrogen and Energy Balances at Ad Libitum- and Equalized-Food Intakes

Diets completely devoid of single essential amino acids were fed to chicks from Day 14 to Day 24 after hatching to investigate the effect on chick growth and nitrogen and energy balances under ad libitum- and equalized-feeding conditions. The effects were different depending upon the amino acid omitted from the diet and were significantly associated with voluntary food intake. Among the essential amino acids, lysine and histidine had the least effect and methionine plus cystine had the severest effect. With equalized-food intake, body weight loss and the reduction in nitrogen balance were diminished by about 44 and 29%, respectively, and the differences in energy utilization disappeared. The possible factors that may explain the nutritional specificity of amino acids are discussed.

The effect of essential amino acid deprivation on poly(A) metabolism in Neurospora crassa

Poly(A) messenger RNA was isolated from a methionine auxotroph of Neurospora crassa grown to exponential and early stationary phases, and from mycelia deprived of the essential amino acid. The polyadenylate regions were isolated from the mRNA preparations by enzymatic digestion and affinity chromatography, and their lengths determined by electrophoresis on 10% acrylamide 90% formamide gels. There was no significant difference in the lengths of the poly(A) regions between exponential and early stationary phase cultures. However, the poly(A) regions of mRNA isolated from cells undergoing amino acid deprivation were larger and more heterogeneous than the poly(A) regions isolated from cells grown in amino acid-supplemented medium. Whole cell extracts prepared from both amino acid-supplemented and amino acid-deprived cultures were assayed for their ability to synthesize and degrade poly(A). No significant difference between the two extracts was detected in either poly(A) polymerase or poly(A)-degrading activities. It was concluded that continuing translational activity is required for the shortening of poly(A) tracts in N. crassa.