scholarly journals Cysteine Restriction in Murine L929 Fibroblasts as an Alternative Strategy to Methionine Restriction in Cancer Therapy

2021 ◽  
Vol 22 (21) ◽  
pp. 11630
Author(s):  
Werner Schmitz ◽  
Elena Ries ◽  
Corinna Koderer ◽  
Maximilian Friedrich Völter ◽  
Anna Chiara Wünsch ◽  
...  
Keyword(s):  
Amino Acid ◽  
Turnover Rate ◽  
Low Energy ◽  
Single Amino Acid ◽  
Proliferation Inhibition ◽  
Murine Cell Line ◽  
Methionine Restriction ◽  
Cysteine Synthesis ◽  
Amino Acid Analogues

Methionine restriction (MetR) is an efficient method of amino acid restriction (AR) in cells and organisms that induces low energy metabolism (LEM) similar to caloric restriction (CR). The implementation of MetR as a therapy for cancer or other diseases is not simple since the elimination of a single amino acid in the diet is difficult. However, the in vivo turnover rate of cysteine is usually higher than the rate of intake through food. For this reason, every cell can enzymatically synthesize cysteine from methionine, which enables the use of specific enzymatic inhibitors. In this work, we analysed the potential of cysteine restriction (CysR) in the murine cell line L929. This study determined metabolic fingerprints using mass spectrometry (LC/MS). The profiles were compared with profiles created in an earlier work under MetR. The study was supplemented by proliferation studies using D-amino acid analogues and inhibitors of intracellular cysteine synthesis. CysR showed a proliferation inhibition potential comparable to that of MetR. However, the metabolic footprints differed significantly and showed that CysR does not induce classic LEM at the metabolic level. Nevertheless, CysR offers great potential as an alternative for decisive interventions in general and tumour metabolism at the metabolic level.

Site Specific Incorporation of Amino Acid Analogues into Proteins In Vivo

2000 ◽  
Author(s):  
Anne K. Kowal ◽  
Caroline Kohrer ◽  
Uttam L. RajBhandary
Keyword(s):  
Amino Acid ◽  
Site Specific ◽  
Amino Acid Analogues

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

scholarly journals Properties of phosphoenolpyruvate carboxykinase (guanosine triphosphate) synthesized in hepatoma cells in the presence of amino acid analogues

1975 ◽  
Vol 146 (3) ◽  
pp. 585-593 ◽  
Author(s):  
S E Knowles ◽  
J M Gunn ◽  
L Reshef ◽  
R W Hanson ◽  
F J Ballard
Keyword(s):  
Amino Acid ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Hepatoma Cells ◽  
Dibutyryl Cyclic Amp ◽  
Enzyme Degradation ◽  
Amino Acid Analogues ◽  
Enzyme Molecules ◽  
Natural Amino Acids

1. Phosphoenolpyruvate carboxykinase (GTP) was induced by a combination of dibutyryl cyclic AMP, theophyline and dexamethasone in Reuber H35 hepatoma cells under conditions where an amino acid in the medium was replaced by an appropriate analogue. 2. With canavanine replacing arginine or with 5-fluorotryptophan or 6-fluorotryptophan replacing tryptophan the induced enzyme had a lower catalytic activity-relative to antibody reactivity. 3. These aberrant enzyme molecules were heat-labile in vitro. 4. Measurements of enzyme degradation in vivo indicated that the canavanine-containing enzyme and the 6-fluorotryptophan-containing enzyme were degraded more rapidly than the enzyme containing all natural amino acids.

scholarly journals Single amino acid mutations effect Zika virus replication in vitro and virulence in vivo

2020 ◽  
Author(s):  
Nicole M. Collette ◽  
Victoria H.I. Lao ◽  
Dina R. Weilhammer ◽  
Barbara Zingg ◽  
Shoshana D. Cohen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zika Virus ◽  
Model Systems ◽  
Single Amino Acid ◽  
Replication Rate ◽  
Adult Mice ◽  
Naturally Occurring ◽  
Viral Virulence

AbstractThe 2014-2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks.Author summaryTo determine if naturally occurring individual mutations in the Zika virus epidemic genotype effect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

scholarly journals Single Amino Acid Substitutions in the Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Determine Viral Entry and Immunogenicity of a Major Neutralizing Domain

2005 ◽  
Vol 79 (18) ◽  
pp. 11638-11646 ◽  
Author(s):  
Christopher E. Yi ◽  
Lei Ba ◽  
Linqi Zhang ◽  
David D. Ho ◽  
Zhiwei Chen
Keyword(s):  
Amino Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Rational Design ◽  
Antiviral Agents ◽  
Single Amino Acid ◽  
Major Mechanism ◽  
The Difference

ABSTRACT Neutralizing antibodies (NAbs) against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) spike (S) glycoprotein confer protection to animals experimentally infected with the pathogenic virus. We and others previously demonstrated that a major mechanism for neutralizing SARS-CoV was through blocking the interaction between the S glycoprotein and the cellular receptor angiotensin-converting enzyme 2 (ACE2). In this study, we used in vivo electroporation DNA immunization and a pseudovirus-based assay to functionally evaluate immunogenicity and viral entry. We characterized the neutralization and viral entry determinants within the ACE2-binding domain of the S glycoprotein. The deletion of a positively charged region SΔ(422-463) abolished the capacity of the S glycoprotein to induce NAbs in mice vaccinated by in vivo DNA electroporation. Moreover, the SΔ(422-463) pseudovirus was unable to infect HEK293T-ACE2 cells. To determine the specific residues that contribute to related phenotypes, we replaced eight basic amino acids with alanine. We found that a single amino acid substitution (R441A) in the full-length S DNA vaccine failed to induce NAbs and abolished viral entry when pseudoviruses were generated. However, another substitution (R453A) abolished viral entry while retaining the capacity for inducing NAbs. The difference between R441A and R453A suggests that the determinants for immunogenicity and viral entry may not be identical. Our findings provide direct evidence that these basic residues are essential for immunogenicity of the major neutralizing domain and for viral entry. Our data have implications for the rational design of vaccine and antiviral agents as well as for understanding viral tropism.

Recapitulation of the Fetal Pattern of Expression of the Iγ and Vγ-Globin Genes in Cultured Baboon CD34+ Bone Marrow Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1590-1590
Author(s):  
Donald Lavelle ◽  
Kestis Vaitkus ◽  
Mahipal Singh ◽  
Maria Hankewych ◽  
Joseph DeSimone
Keyword(s):  
Bone Marrow ◽  
Amino Acid ◽  
Bone Marrow Cells ◽  
Globin Gene ◽  
Single Amino Acid ◽  
Globin Genes ◽  
Globin Chain ◽  
Hemoglobin Switching ◽  
Globin Chains

Abstract The human Gγ-globin and Aγ-globin genes differ by the presence of a single amino acid, either glycine or alanine, at position 136. The ratio of Gγ/Aγ-globin expression is approximately 7/3 at birth and changes to 2/3 in the adult. The mechanism responsible for this developmental switch is unknown. In the baboon, the duplicated γ-globin genes differ by the presence of a single amino acid at position 75. The Iγ-globin gene contains isoleucine at position 75, while the Vγ-globin gene contains valine at this position. The ratio of expression of the Iγ and Vγ-globin chains also differs in the fetal and adult stages. The Iγ/Vγ ratio is 3/2 in the fetus and 2/3 in the adult. Thus the pattern of expression of the baboon Iγ-globin gene is analogous to the human Gγ-globin gene, and that of the Vγ-globin gene is analogous to the human Aγ-globin gene. During stress erythropoiesis, moderately increased HbF levels are observed (5–10% HbF) and the Iγ/Vγ-globin chains are expressed in the characteristic adult ratio. Decitabine treatment reactivates HbF expression to high levels (50–70% HbF) and Iγ/Vγ ratios of approximately 1:1 have been observed following decitabine treatment. Thus decitabine treatment alters the Iγ/Vγ ratio but does not cause a complete reversion to the fetal pattern of expression. HbF is also reactivated to high levels in cultured baboon BFUe. In this investigation the pattern of expression of the Iγ- and Vγ-globin genes in cultured baboon CD34+ bone marrow (BM) cells was analyzed to determine whether reactivation of HbF in culture was associated with a change in the pattern of expression of the Iγ-and Vγ-globin genes. CD34+ cells were enriched from baboon BM using the 12.8 monoclonal antibody in combination with immunomagnetic microbead columns (Miltenyi) and cultured in Iscove’s media supplemented with 30% fetal bovine serum, stem cell factor (SCF; 200ng/ml), erythropoietin (EPO; 2U/ml), and dexamethasone (Dex; 1μM). The pattern of globin chain expression in d12 cultures, cord blood (CB) of a 58d fetus, and peripheral blood (PB) of adult baboons following phlebotomy and decitabine treatment was compared by HPLC analysis of hemolysates. The baboon 58d CB contained >90% HbF and the ratio of Iγ/Vγ was 1.85. In the adult (phlebotomized) PB the level of HbF was 8.1% and the Iγ/Vγ ratio was 0.75 thus confirming that the ratio of the baboon Iγ and Vγ-globin chains differs in the fetal and adult stages of development in a manner similar to that of the human Gγ and Aγ-globin chains. Following decitabine treatment (PA 7002) an HbF level of 55% was attained with an Iγ/Vγ ratio of 1.1. Hemolysates prepared from d12 cultures of CD34+ baboon (PA 7002) BM cells grown in the presence of SCF, EPO, and Dex contained 57.6% HbF, nearly the same level observed following decitabine treatment in vivo. The Iγ/Vγ ratio was 1.94, markedly different from that observed in this same baboon following decitabine in vivo and, moreover, nearly identical to the fetal ratio. Thus HbF reactivation in cultured adult baboon CD34+ BM cells was associated with a change in the ratio of expression of the two baboon γ-globin genes to that characteristic of the fetal stage. Recapitulation of the fetal pattern of γ-globin chain expression in cultured baboon CD34+ progenitors demonstrates yet another advantage of the baboon model for investigations of hemoglobin switching.

In-silico structural, functional and immunogenic characterization of Taenia solium TS14 protein

2017 ◽  
Author(s):  
Chitra Joshi ◽  
Siddharth Gautam
Keyword(s):  
Amino Acid ◽  
In Silico ◽  
Mutational Analysis ◽  
Solvent Accessibility ◽  
Taenia Solium ◽  
Secretory Protein ◽  
Single Amino Acid

TS14, a Cysticercosis cellulosae derived protein, has been exploited for immunodiagnosis of cysticercosis in humans and pigs. However, the information on structure, function, stability and immunogenicity of TS14 derived from different isolates is primarily lacking. The present study deals with in-silico characterization of six TS14 isolates. High thermostability and an isoelectric point of 9.41 were recorded. Based on N-terminal amino acid residues, high resistance to intracellular proteases with extended in-vivo and in-vitro half-lives was predicted. TS14 is foreseen as a secretory protein with a signal peptide and an extracellular localization. Structural analysis of TS14 exhibited the dominance of helices in the secondary structure (92% coverage) with majority of residues showing high and medium solvent accessibility. High lysine content and presence of multiple nucleotide binding sites in TS14 suggests interaction with RNA/DNA and a role in their metabolism. Immunogenic profiling predicted presence of four distinct B-cell epitopes. Mutational analysis based on the single amino acid substitutions among six TS14 isolates demonstrated minor variations in structural stability; however, all the substitutions were well tolerated. Moreover, all the isolates revealed almost identical immunogenic profile with an equivocal potential to elicit the antibody-mediated immune response.

scholarly journals Infection of Human Dendritic Cells by a Sindbis Virus Replicon Vector Is Determined by a Single Amino Acid Substitution in the E2 Glycoprotein

2000 ◽  
Vol 74 (24) ◽  
pp. 11849-11857 ◽  
Author(s):  
Jason P. Gardner ◽  
Ilya Frolov ◽  
Silvia Perri ◽  
Yaying Ji ◽  
Mary Lee MacKichan ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Amino Acid ◽  
Amino Acid Substitution ◽  
Sindbis Virus ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
E2 Glycoprotein ◽  
Human Dendritic Cells ◽  
Antigen Presenting

ABSTRACT The ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55Gag elicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.

Stimulation of human breast cancers by dietary l-arginine

1992 ◽  
Vol 82 (4) ◽  
pp. 413-417 ◽  
Author(s):  
Kenneth G. M. Park ◽  
Steven D. Heys ◽  
Karen Blessing ◽  
Peter Kelly ◽  
Margaret A. McNurlan ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Human Cancer ◽  
Single Amino Acid ◽  
Standard Diet ◽  
Breast Cancers ◽  
Human Breast ◽  
Day Range ◽  
Human Tumours

1. The amino acid L-arginine has been shown to enhance immune mechanisms and inhibit tumour growth in experimental animals, but although many of the immunological effects of arginine have been reproduced in man there have been few studies of its effects on human tumours. In this study the effects of arginine on human breast cancers were determined by measuring tumour protein synthesis and comparing this with immunohistochemical assessments of cell proliferation. 2. Patients with breast cancer were randomized to receive either a standard diet or arginine supplementation. At the time of surgery, the rate of tumour protein synthesis was measured by the incorporation of the stable isotope [1-13C]leucine into tumour protein. Tumours were also assessed histologically and by staining for the presence of the activation antigen Ki67. 3. The median rate of tumour protein synthesis was 10%/ day (range 5.5–15.8%/day) in the control patients and 25.6%/day (range 9-37%/day) in the patients receiving arginine supplements (P < 0.005, Wilcoxon rank sum test). The rates of protein synthesis correlated with Ki67 expression within these tumours (r=0.78, P < 0.001). A double-staining technique confirmed that tumour cells, rather than tumour-infiltrating lymphoreticular cells, expressed Ki67. 4. This study demonstrates that, in contrast to animal studies, L-arginine stimulates human tumours in vivo. This represents the first direct evidence that a single amino acid can modulate the behaviour of a human cancer.

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