scholarly journals The Sko1p Repressor and Gcn4p Activator Antagonistically Modulate Stress-Regulated Transcription inSaccharomyces cerevisiae

2001 ◽  
Vol 21 (1) ◽  
pp. 16-25 ◽  
Author(s):  
Amparo Pascual-Ahuir ◽  
Ramón Serrano ◽  
Markus Proft
Keyword(s):  
Amino Acid ◽  
Specific Binding ◽  
Ion Homeostasis ◽  
Normal Growth ◽  
Yeast Cells ◽  
Target Sequence ◽  
Acid Uptake ◽  
Amino Acid Availability

ABSTRACT In the transcriptional response of Saccharomyces cerevisiae to stress, both activators and repressors are implicated. Here we demonstrate that the ion homeostasis determinant,HAL1, is regulated by two antagonistically operating bZIP transcription factors, the Sko1p repressor and the Gcn4p activator. A single CRE-like sequence (CRE HAL1 ) at position −222 to −215 with the palindromic core sequence TTACGTAA is essential for stress-induced expression of HAL1. Down-regulation of HAL1 under normal growth conditions requires specific binding of Sko1p to CRE HAL1 and the corepressor gene SSN6. Release from this repression depends on the function of the high-osmolarity glycerol pathway. The Gcn4p transcriptional activator binds in vitro to the same CRE HAL1 and is necessary for up-regulatedHAL1 expression in vivo, indicating a dual control mechanism by a repressor-activator pair occupying the same promoter target sequence. gcn4 mutants display a strong sensitivity to elevated K+ or Na+ concentrations in the growth medium. In addition to reduced HAL1 expression, this sensitivity is explained by the fact that amino acid uptake is drastically impaired by high Na+ and K+concentrations in wild-type yeast cells. The reduced amino acid biosynthesis of gcn4 mutants would result in amino acid deprivation. Together with the induction of HAL1 by amino acid starvation, these results suggest that salt stress and amino acid availability are physiologically interconnected.

Hepatic Amino Acid Uptake Is Decreased in Lactating Rats. In Vivo and In Vitro Studies

1994 ◽  
Vol 124 (11) ◽  
pp. 2163-2171 ◽  
Author(s):  
José García de la Asunción ◽  
Amparo Devesa ◽  
Juan R. Viña ◽  
Teresa Barber
Keyword(s):  
Amino Acid ◽  
Amino Acid Uptake ◽  
In Vitro Studies ◽  
Acid Uptake

scholarly journals A Multiplicity of Coactivators Is Required by Gcn4p at Individual Promoters In Vivo

2003 ◽  
Vol 23 (8) ◽  
pp. 2800-2820 ◽  
Author(s):  
Mark J. Swanson ◽  
Hongfang Qiu ◽  
Laarni Sumibcay ◽  
Anna Krueger ◽  
Soon-ja Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Chromatin Immunoprecipitation ◽  
Target Gene ◽  
Target Genes ◽  
Yeast Cells ◽  
Histone Acetyltransferases ◽  
Vitro Binding ◽  
Paf1 Complex

ABSTRACT Transcriptional activators interact with multisubunit coactivators that modify chromatin structure or recruit the general transcriptional machinery to their target genes. Budding yeast cells respond to amino acid starvation by inducing an activator of amino acid biosynthetic genes, Gcn4p. We conducted a comprehensive analysis of viable mutants affecting known coactivator subunits from the Saccharomyces Genome Deletion Project for defects in activation by Gcn4p in vivo. The results confirm previous findings that Gcn4p requires SAGA, SWI/SNF, and SRB mediator (SRB/MED) and identify key nonessential subunits of these complexes required for activation. Among the numerous histone acetyltransferases examined, only that present in SAGA, Gcn5p, was required by Gcn4p. We also uncovered a dependence on CCR4-NOT, RSC, and the Paf1 complex. In vitro binding experiments suggest that the Gcn4p activation domain interacts specifically with CCR4-NOT and RSC in addition to SAGA, SWI/SNF, and SRB/MED. Chromatin immunoprecipitation experiments show that Mbf1p, SAGA, SWI/SNF, SRB/MED, RSC, CCR4-NOT, and the Paf1 complex all are recruited by Gcn4p to one of its target genes (ARG1) in vivo. We observed considerable differences in coactivator requirements among several Gcn4p-dependent promoters; thus, only a subset of the array of coactivators that can be recruited by Gcn4p is required at a given target gene in vivo.

scholarly journals Effects of p-chlorophenylalanine and α-methylphenylalanine on amino acid uptake and protein synthesis in mouse neuroblastoma cells

1978 ◽  
Vol 174 (3) ◽  
pp. 931-938 ◽  
Author(s):  
C J Kelly ◽  
T C Johnson
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Animal Models ◽  
Phenylalanine Hydroxylase ◽  
Present Report ◽  
Neuroblastoma Cells ◽  
Acid Uptake

The phenylalanine analogues p-chlorophenylalanine and alpha-methylphenylalanine were used to inhibit phenylalanine hydroxylase in animal models for phenylketonuria. The present report examines the affects of these analogues on the metabolism of neuroblastoma cells. p-Chlorophenylalanine inhibited growth and was toxic to neuroblastoma cells. Although in vivo this analogue increased cell monoribosomes by 42%, it did not significantly affect poly(U)-directed protein synthesis in vitro. P-Chlorophenylalanine did not compete with phenylalanine or tyrosine for aminoacylation of tRNA and was therefore not substituted for those amino acids in nascent polypeptides. The initial cellular uptake of various large neutral amino acids was inhibited by this analogue but did not affect the flux of amino acids already in the cell; this suggested that an alteration of the cell's amino acid pools was not responsible for the cytotoxicity of the analogues. In contrast with p-chlorophenylalanine, alpha-methylphenylalanine did not exert these direct toxic effects because the administration of alpha-methylphenylalanine in vivo did not affect brain polyribosomes and a comparable concentration of this analogue was neither growth inhibitory nor cytotoxic to neuroblastoma cells in culture. The suitability of each analogue as an inhibitor of phenylalanine hydroxylase in animal models for phenylketonuria is discussed.

scholarly journals Stress-activated Protein Kinase-mediated Down-Regulation of the Cell Integrity Pathway Mitogen-activated Protein Kinase Pmk1p by Protein Phosphatases

2007 ◽  
Vol 18 (11) ◽  
pp. 4405-4419 ◽  
Author(s):  
Marisa Madrid ◽  
Andrés Núñez ◽  
Teresa Soto ◽  
Jero Vicente-Soler ◽  
Mariano Gacto ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Ion Homeostasis ◽  
Mitogen Activated Protein Kinase ◽  
Yeast Cells ◽  
Main Role ◽  
Cell Integrity ◽  
Down Regulation ◽  
Mitogen Activated Protein

Fission yeast mitogen-activated protein kinase (MAPK) Pmk1p is involved in morphogenesis, cytokinesis, and ion homeostasis as part of the cell integrity pathway, and it becomes activated under multiple stresses, including hyper- or hypotonic conditions, glucose deprivation, cell wall-damaging compounds, and oxidative stress. The only protein phosphatase known to dephosphorylate and inactivate Pmk1p is Pmp1p. We show here that the stress-activated protein kinase (SAPK) pathway and its main effector, Sty1p MAPK, are essential for proper deactivation of Pmk1p under hypertonic stress in a process regulated by Atf1p transcription factor. We demonstrate that tyrosine phosphatases Pyp1p and Pyp2p, and serine/threonine phosphatase Ptc1p, that negatively regulate Sty1p activity and whose expression is dependent on Sty1p-Atf1p function, are involved in Pmk1p dephosphorylation under osmostress. Pyp1p and Ptc1p, in addition to Pmp1p, also control the basal level of MAPK Pmk1p activity in growing cells and associate with, and dephosphorylate Pmk1p both in vitro and in vivo. Our results with Ptc1p provide the first biochemical evidence for a PP2C-type phosphatase acting on more than one MAPK in yeast cells. Importantly, the SAPK-dependent down-regulation of Pmk1p through Pyp1p, Pyp2p, and Ptc1p was not complete, and Pyp1p and Ptc1p phosphatases are able to negatively regulate MAPK Pmk1p activity by an alternative regulatory mechanism. Our data also indicate that Pmk1p phosphorylation oscillates as a function of the cell cycle, peaking at cell separation during cytokinesis, and that Pmp1p phosphatase plays a main role in regulating this process.

scholarly journals SUN-266 Protein Induced Pancreatic Hormone Secretion Is Modulated by Vagal CaSR

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Mariana Norton ◽  
Simon C Cork ◽  
Aldara Martin Alonso ◽  
Anna G Roberts ◽  
Yateen S Patel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Hormone Secretion ◽  
Sensory Information ◽  
Glucagon Secretion ◽  
Vagal Afferents ◽  
Acid Uptake

Abstract The existence of a vago-vagal entero-pancreatic pathway, where sensory information from the gut can signal via vagal afferents to the brain to mediate changes in pancreatic function, has been recognised for over a century, and investigated extensively with regards to pancreatic exocrine secretions. However, the role of such pathways in pancreatic endocrine secretions has received less attention. The secretion of insulin and glucagon in response to protein and amino acids is conserved across species. This effect is thought to promote amino acid uptake into tissues without concomitant hypoglycaemia. We found that the essential amino acid L-Phenylalanine potently stimulates glucagon secretion, even when administered directly into the gut at small doses unlikely to significantly raise systematic levels. Administration of L-Phenylalanine also increased neuronal activation in the rat and mouse dorsal vagal complex, the central nervous system region directly innervated by vagal afferents. L-Phenylalanine modulates the activity of the calcium sensing receptor (CaSR), a nutrient sensor more commonly known for its role in calcium homeostasis, but which is thought to also act as a sensor of aromatic amino acids. Interestingly, the CaSR is one of the few nutrient sensors expressed in vagal afferents and in vitro calcium imaging revealed CaSR synthetic agonists activate subpopulations of vagal afferents. The role of CaSR in vivo was investigated further by selectively knocking down the CaSR in vagal afferents. Briefly, CaSR floxed mice were bilaterally injected directly into the nodose ganglion, where the cell bodies of vagal afferents are located, with a cre expressing adeno-associated virus. CaSR knockdown did not interfere with normal food intake, nor the vagal-dependent anorectic effects of cholecystokinin, or of L-Phenylalanine. However, it did blunt protein-induced glucagon secretion, suggesting involvement of the CaSR in the vagus nerve in protein sensing and glucose homeostasis. Future studies are required to determine the importance of vagal CaSR in protein induced pancreatic endocrine secretions, and the possibility of exploiting this circuit to develop new anti-diabetic therapies.

scholarly journals Relation between in vitro and in vivo assessment of amino acid availability

1989 ◽  
Vol 29 (4) ◽  
pp. 495-507 ◽  
Author(s):  
I. Galibois ◽  
L. Savoie ◽  
C. Simoes Nunes ◽  
A. Rérat
Keyword(s):  
Amino Acid ◽  
Amino Acid Availability ◽  
In Vivo Assessment

scholarly journals Mutational analysis of the Saccharomyces cerevisiae ABD1 gene: cap methyltransferase activity is essential for cell growth.

1996 ◽  
Vol 16 (2) ◽  
pp. 475-480 ◽  
Author(s):  
X Mao ◽  
B Schwer ◽  
S Shuman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Yeast Cells ◽  
Terminal Deletion ◽  
Methyltransferase Activity

RNA (guanine-7-)-methyltransferase is the enzyme responsible for methylating the 5' cap structure of eukaryotic mRNA. The Saccharomyces cerevisiae enzyme is a 436-amino-acid protein encoded by the essential ABD1 gene. In this study, deletion and point mutations in ABD1 were tested for the ability to support growth of an abd1 null strain. Elimination of 109 amino acids from the N terminus had no effect on cell viability, whereas a more extensive N-terminal deletion of 155 residues was lethal, as was a C-terminal deletion of 55 amino acids. Alanine substitution mutations were introduced at eight conserved residues within a 206-amino-acid region of similarity between ABD1 and the methyltransferase domain of the vaccinia virus capping enzyme. ABD1 alleles H253A (encoding a substitution of alanine for histidine at position 253), T282A, E287A, E361A, and Y362A were viable, whereas G174A, D178A, and Y254A were either lethal or severely defective for growth. Alanine-substituted and amino-truncated ABD1 proteins were expressed in bacteria, purified, and tested for cap methyltransferase activity in vitro. Mutations that were viable in yeast cells had either no effect or only a moderate effect on the specific methyltransferase activity of the mutated ABD1 protein, whereas mutations that were deleterious in vivo yielded proteins that were catalytically defective in vitro. These findings substantiate for the first time the long-held presumption that cap methylation is an essential function in eukaryotic cells.

Amino Acid Uptake and Metabolism by Embryos of the Blenny Zoarces Viviparus

1992 ◽  
Vol 171 (1) ◽  
pp. 315-328 ◽  
Author(s):  
BODIL KORSGAARD
Keyword(s):  
Amino Acid ◽  
High Performance ◽  
Acid Uptake ◽  
Late Development ◽  
Zoarces Viviparus ◽  
Uptake Rates ◽  
Net Uptake ◽  
Uptake And Metabolism

The ability of blenny Zoarces viviparus (L.) embryos in early and late development to assimilate and metabolize ambient L-alanine was investigated in vitro and in vivo by means of autoradiographic and radiochemical methods. Autoradiograms showed that after 24 h of exposure to L-[14C]alanine, label was distributed in the tissues of the embryos. Uptake rates for 14C-labelled L-alanine in vitro were estimated by measuring the disappearance of radioactivity from the medium. Net uptake rates were measured by high performance liquid chromatography of samples taken simultaneously from the medium. Uptake rates, based on uptake of the tracer (0.60 μmol g−1 h−1), were similar to net uptake rates (0.54 μmol g−1 h−1) in embryos in late development in the in vitro incubations in 60 μmoll−1 alanine. In vivio, the injected tracer was completely cleared from the ovarian fluid over 24h. After intraovarian and in vitro incubation of the embryos with labelled alanine, there was evidence for both catabolic and anabolic metabolism of the amino acid by production of 14CO2 and by incorporation of radioactivity into molecules insoluble in trichloro-acetic acid, respectively. The data provide evidence for uptake and metabolism of amino acids by embryos in vitro and in vivo. An increase in the capacity for uptake and metabolism of L-alanine may occur during development of the embryos in the ovary.

scholarly journals Amino acid availability in poultry—in vitro and in vivo measurements

1999 ◽  
Vol 50 (5) ◽  
pp. 889 ◽  
Author(s):  
V. Ravindran ◽  
Wayne L. Bryden
Keyword(s):  
Amino Acid ◽  
Protein Quality ◽  
Poultry Feed ◽  
In Vitro Assays ◽  
Total Amino Acid ◽  
Amino Acid Availability ◽  
Amino Acid Contents ◽  
In Vivo Growth

Methodology to evaluate the protein quality or amino acid availability in feed ingredients for poultry using in vitro (enzymic, chemical, or microbiological assays), indirect in vivo (plasma amino acid assays), or direct in vivo (growth or digestibility assays) measurements has been reviewed. The specific applications and limitations of these methods are examined. In vitro assays are useful in providing information on heat damage in selected protein sources under defined conditions, and on relative ranking of different samples, but they cannot form the basis of practical feed formulations. While growth assays remain the only direct means of confirming nutritional relevance of values obtained by other procedures, in vivo digestibility assays appear to be most useful, at present, to estimate amino acid availability. Amino acid digestibility assays in poultry should be based on the analysis of digesta from the terminal ileum rather than excreta, because of the variable and modifying effects of hindgut microflora. Techniques used to estimate endogenous amino acid losses in poultry are discussed. The needs for correction of endogenous losses in amino acid digestibility calculations and the relative merits of apparent and true digestible amino acid systems are still being debated. It is, however, clear that both digestible amino systems are superior to the total amino acid system currently employed to formulate practical diets. Digestible amino acid values are likely to form the basis of poultry feed formulations in the future. In particular, there is an urgent need for more precise information on the variation in digestible amino acid contents of locally grown ingredients and on the factors causing this variation (e.g. variety, location, season, agronomic practices, processing, etc.).

Correlation between in vitro and in vivo Data of Radiolabeled Peptide for Tumor Targeting

2019 ◽  
Vol 19 (12) ◽  
pp. 950-960
Author(s):  
Soghra Farzipour ◽  
Seyed Jalal Hosseinimehr
Keyword(s):  
Tumor Targeting ◽  
Single Photon ◽  
Specific Binding ◽  
Specific Interaction ◽  
Photon Emission ◽  
Emission Computed Tomography ◽  
Mixed Effect ◽  
Radiolabeled Peptides

Tumor-targeting peptides have been generally developed for the overexpression of tumor specific receptors in cancer cells. The use of specific radiolabeled peptide allows tumor visualization by single photon emission computed tomography (SPECT) and positron emission tomography (PET) tools. The high affinity and specific binding of radiolabeled peptide are focusing on tumoral receptors. The character of the peptide itself, in particular, its complex molecular structure and behaviors influence on its specific interaction with receptors which are overexpressed in tumor. This review summarizes various strategies which are applied for the expansion of radiolabeled peptides for tumor targeting based on in vitro and in vivo specific tumor data and then their data were compared to find any correlation between these experiments. With a careful look at previous studies, it can be found that in vitro unblock-block ratio was unable to correlate the tumor to muscle ratio and the success of radiolabeled peptide for in vivo tumor targeting. The introduction of modifiers’ approaches, nature of peptides, and type of chelators and co-ligands have mixed effect on the in vitro and in vivo specificity of radiolabeled peptides.

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