Novel method for measuring aquatic bacterial productivity using D10-leucine based on protein synthesis rate

2020 ◽  
Vol 85 ◽  
pp. 121-129
Author(s):  
K Tsuchiya ◽  
T Sano ◽  
N Tomioka ◽  
K Kazuhiro ◽  
A Imai ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Bacterial Production ◽  
High Sensitivity ◽  
Radioactive Isotopes ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Liquid Chromatography Mass Spectrometry ◽  
Bacterial Productivity ◽  
Pure Protein ◽  
Novel Method

The most widely used method for measuring bacterial production is tritium-labeled leucine (3H-Leu). Although this method provides methodological simplicity and high sensitivity, the employment of radioactive isotopes is often restricted by regulations, particularly in field settings. In this study, we developed a non-radioactive method for measuring bacterial productivity based on the protein synthesis rate, using deuterium-labeled leucine ((CD3)2CDCD2CD(NH2)COOH; D10-Leu); the proposed method was then compared and verified with the 3H-Leu method. The procedures of the proposed method are (1) incorporation of D10-Leu by bacteria, (2) acid hydrolysis (HCl) to amino acids and (3) quantification of D10-Leu (m/z 142.10) by liquid chromatography mass spectrometry (LC-MS/MS). In the LC-MS/MS analysis, we detected a larger amount of D9-Leu (m/z 141.10) and D8-Leu (m/z 140.10) than that of D10-Leu, suggesting that incorporated D10-Leu was rapidly metabolized such as in deamination and aminotransferase reactions. The incorporation rates of D10-Leu, D10-Leu + D9-Leu (D10+D9-Leu) and D10-Leu + D9-Leu + D8-Leu (D10+D9+D8-Leu) were significantly positively correlated to that of 3H-Leu, confirming the validity of the proposed method. Since D7-Leu (m/z 139.10) could not be detected, the amount of exogenous leucine incorporated into protein can be accurately estimated through D10+D9+D8-Leu measurement. The new compound-based quantification method using stable isotope-labeled leucine can be a powerful tool to estimate pure protein synthesis rate for measuring bacterial production.

scholarly journals Comparison of protein-synthesis rate of alveolar macrophages in vivo and in vitro

1984 ◽  
Vol 217 (3) ◽  
pp. 761-765 ◽  
Author(s):  
M H Oliver ◽  
P J Cole ◽  
G J Laurent
Keyword(s):  
Protein Synthesis ◽  
Alveolar Macrophages ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Novel Method

This paper describes and validates a novel method for measuring rates of protein synthesis of rabbit alveolar macrophages in vivo. A rate of 9.3%/day was obtained, compared with 48.9%/day measured in vitro. This study suggests that the procedures involved in the isolation of alveolar macrophages for study in vitro may themselves activate the cell.

scholarly journals MECHANISMS IN ENDOCRINOLOGY: Exogenous insulin does not increase muscle protein synthesis rate when administered systemically: a systematic review

2015 ◽  
Vol 173 (1) ◽  
pp. R25-R34 ◽  
Author(s):  
Jorn Trommelen ◽  
Bart B L Groen ◽  
Henrike M Hamer ◽  
Lisette C P G M de Groot ◽  
Luc J C van Loon
Keyword(s):  
Systematic Review ◽  
Older Adults ◽  
Protein Synthesis ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Exogenous Insulin ◽  
Insulin Administration ◽  
Muscle Protein Synthesis Rate

BackgroundThough it is well appreciated that insulin plays an important role in the regulation of muscle protein metabolism, there is much discrepancy in the literature on the capacity of exogenous insulin administration to increase muscle protein synthesis ratesin vivoin humans.ObjectiveTo assess whether exogenous insulin administration increases muscle protein synthesis rates in young and older adults.DesignA systematic review of clinical trials was performed and the presence or absence of an increase in muscle protein synthesis rate was reported for each individual study arm. In a stepwise manner, multiple models were constructed that excluded study arms based on the following conditions: model 1, concurrent hyperaminoacidemia; model 2, insulin-induced hypoaminoacidemia; model 3, supraphysiological insulin concentrations; and model 4, older, more insulin resistant, subjects.ConclusionsFrom the presented data in the current systematic review, we conclude that: i) exogenous insulin and amino acid administration effectively increase muscle protein synthesis, but this effect is attributed to the hyperaminoacidemia; ii) exogenous insulin administered systemically induces hypoaminoacidemia which obviates any insulin-stimulatory effect on muscle protein synthesis; iii) exogenous insulin resulting in supraphysiological insulin levels exceeding 50 000 pmol/l may effectively augment muscle protein synthesis; iv) exogenous insulin may have a diminished effect on muscle protein synthesis in older adults due to age-related anabolic resistance; and v) exogenous insulin administered systemically does not increase muscle protein synthesis in healthy, young adults.

Relationship between glutamine concentration and protein synthesis in rat skeletal muscle

1988 ◽  
Vol 255 (2) ◽  
pp. E166-E172 ◽  
Author(s):  
M. M. Jepson ◽  
P. C. Bates ◽  
P. Broadbent ◽  
J. M. Pell ◽  
D. J. Millward
Keyword(s):  
Protein Synthesis ◽  
Protein Deficiency ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Glutamine Concentration ◽  
E Coli ◽  
Protein Group ◽  
Highly Correlated ◽  
Rna Concentration

Muscle glutamine concentration ([GLN]) and protein synthesis rate (Ks) have been examined in vivo in well-fed, protein-deficient, starved, and endotoxemic rats. With protein deficiency (8 or 5% casein diet), [GLN] fell from 7.70 to 5.58 and 3.56 mmol/kg in the 8 and 5% diet groups, with Ks falling from 15.42 to 9.1 and 6.84%/day. Three-day starvation reduced [GLN] and Ks to 2.38 mmol/kg and 5.6%/day, respectively. In all these groups food intakes and insulin were generally well maintained (except in the starved group), whereas free 3,5,3'-triiodothyronine (T3) was depressed in the starved and 5% protein group. The E. coli lipopolysaccharide endotoxin (3 mg/kg) reduced [GLN] to 5.85 and 4.72 mmol/kg and Ks to 10.5 and 9.10%/day in two well-fed groups. Insulin levels were increased, and free T3 levels fell. Combined protein deficiency and endotoxemia further reduced [GLN] and Ks to 1.88 mmol/kg and 4.01%/day, respectively, in the 5% protein rats. Changes in both ribosomal activity (KRNA) and concentration (RNA/protein) contributed to the fall in Ks in malnutrition and endotoxemia, although reductions in the RNA concentration were most marked with protein deficiency and reductions in the KRNA dominated the response to the endotoxin. The changes in [GLN] and Ks were highly correlated as were [GLN] and both KRNA and the RNA concentration, and these relationships were unique to glutamine. These relationships could reflect sensitivity of glutamine transport and protein synthesis to the same regulatory influences, and the particular roles of insulin and T3 are discussed, as well as any direct influence of glutamine on protein synthesis.

scholarly journals Dietary γ-Aminobutyric Acid Affects the Brain Protein Synthesis Rate in Ovariectomized Female Rats

2009 ◽  
Vol 55 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Kazuyo TUJIOKA ◽  
Miho OHSUMI ◽  
Kenji HORIE ◽  
Mujo KIM ◽  
Kazutoshi HAYASE ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Female Rats ◽  
Aminobutyric Acid ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Brain Protein ◽  
Brain Protein Synthesis ◽  
The Brain

scholarly journals Dietary Ornithine Affects the Tissue Protein Synthesis Rate in Young Rats

2012 ◽  
Vol 58 (4) ◽  
pp. 297-302 ◽  
Author(s):  
Kazuyo TUJIOKA ◽  
Takashi YAMADA ◽  
Mami AOKI ◽  
Koji MORISHITA ◽  
Kazutoshi HAYASE ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Tissue Protein ◽  
Young Rats ◽  
Tissue Protein Synthesis

scholarly journals Differential regulation of mRNA fate by the human Ccr4-Not complex is driven by coding sequence composition and mRNA localization

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Sarah L. Gillen ◽  
Chiara Giacomelli ◽  
Kelly Hodge ◽  
Sara Zanivan ◽  
Martin Bushell ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Mrna Stability ◽  
Mrna Localization ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Translational Efficiency ◽  
Mrna Levels ◽  
Control Of Gene Expression ◽  
Mrna Fate

Abstract Background Regulation of protein output at the level of translation allows for a rapid adaptation to dynamic changes to the cell’s requirements. This precise control of gene expression is achieved by complex and interlinked biochemical processes that modulate both the protein synthesis rate and stability of each individual mRNA. A major factor coordinating this regulation is the Ccr4-Not complex. Despite playing a role in most stages of the mRNA life cycle, no attempt has been made to take a global integrated view of how the Ccr4-Not complex affects gene expression. Results This study has taken a comprehensive approach to investigate post-transcriptional regulation mediated by the Ccr4-Not complex assessing steady-state mRNA levels, ribosome position, mRNA stability, and protein production transcriptome-wide. Depletion of the scaffold protein CNOT1 results in a global upregulation of mRNA stability and the preferential stabilization of mRNAs enriched for G/C-ending codons. We also uncover that mRNAs targeted to the ER for their translation have reduced translational efficiency when CNOT1 is depleted, specifically downstream of the signal sequence cleavage site. In contrast, translationally upregulated mRNAs are normally localized in p-bodies, contain disorder-promoting amino acids, and encode nuclear localized proteins. Finally, we identify ribosome pause sites that are resolved or induced by the depletion of CNOT1. Conclusions We define the key mRNA features that determine how the human Ccr4-Not complex differentially regulates mRNA fate and protein synthesis through a mechanism linked to codon composition, amino acid usage, and mRNA localization.

Effect of Dietary Restriction on Protein Synthesis and Wound Healing after Surgery in the Rat

1995 ◽  
Vol 89 (4) ◽  
pp. 383-388 ◽  
Author(s):  
Peter W. Emery ◽  
Peter Sanderson
Keyword(s):  
Wound Healing ◽  
Protein Synthesis ◽  
Dietary Restriction ◽  
Muscle Protein ◽  
Muscle Protein Synthesis ◽  
Chronically Ill ◽  
Abdominal Muscle ◽  
Collagen Content ◽  
Synthesis Rate ◽  
Protein Synthesis Rate

1. The healing of an abdominal muscle wound after surgery is associated with a considerable increase in the rate of protein synthesis. We have investigated whether this increase in protein synthesis is affected by chronic undernutrition, and whether this causes a delay in wound healing. 2. A group of rats was fed 58% of the voluntary food intake of a matched control group. After 7 days half the rats in each group underwent abdominal surgery. Forty-eight hours later all the rats were killed and muscle protein synthesis rate was measured by the flooding dose technique. 3. In a second experiment using the same dietary regimen rats were placed in metabolic cages after surgery and killed 7 days later. In addition to measurements of muscle protein synthesis, wound breaking strength was measured with a tensiometer and collagen content was also measured at the wound site. 4. Dietary restriction caused a loss of body weight, a decrease in nitrogen balance and a deficit in muscle protein mass. It also caused a decrease in protein synthesis rate in gastrocnemius muscle and in parts of the abdominal muscle distant from the site of the wound. However, it had no effect on the rate of muscle protein synthesis at the site of the wound either 2 or 7 days after surgery. The tensile strength and the collagen content of the wound were also unaffected by food restriction. 5. It is concluded that the wound healing process is uniquely protected from the effects of moderate undernutrition such as might be experienced by a chronically ill patient.

scholarly journals Phosphorylation of the α subunit of initiation factor 2 correlates with the inhibition of translation following transient cerebral ischaemia in the rat

1994 ◽  
Vol 302 (2) ◽  
pp. 335-338 ◽  
Author(s):  
J Burda ◽  
M E Martín ◽  
A García ◽  
A Alcázar ◽  
J L Fando ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Cerebral Ischaemia ◽  
Ternary Complex ◽  
Initiation Factor ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Vessel Occlusion ◽  
Α Subunit ◽  
Free System ◽  
Initiation Factor 2

Rats were subjected to the standard four-vessel occlusion model of cerebral transient ischaemia (vertebral and carotid arteries) for 15 and 30 min. After a 30 min recirculation period, protein synthesis rate, initiation factor 2 (eIF-2) and guanine nucleotide exchange factor (GEF) activities, and the level of phosphorylation of the alpha subunit of eIF-2 (eIF-2 alpha) were determined in the neocortex region of the brain from sham-operated controls and ischaemic animals. Following reversible cerebral ischaemia, the protein synthesis rate, as measured in a cell-free system, was significantly inhibited (70%) in the ischaemic animals. eIF-2 activity, as measured by its ability to form a ternary complex, also decrease parallel to the decrease in protein synthesis. As eIF-2 activity was assayed in the presence of Mg2+ and GTP-regenerating capacity, the decrease in ternary-complex formation indicated the possible impairment of GEF activity. Since phosphorylated eIF-2 [eIF-2(alpha P)] is a powerful inhibitor of GEF, the levels of phosphorylated eIF-2 alpha were determined, and an increase from 7% phosphorylation in sham control rats to 20% phosphorylation in 15 min and 29% phosphorylation in 30 min in ischaemic rats was observed, providing evidence for a tight correlation of phosphorylation of eIF-2 alpha and inhibition of protein synthesis. Moreover, GEF activity measured in the GDP-exchange assay was in fact inhibited in the ischaemic animals, proving that protein synthesis is impaired by the presence of eIF-2(alpha P), which blocks eIF-2 recycling.

Regulation of hepatic protein synthesis in chronic inflammation and sepsis

1992 ◽  
Vol 262 (2) ◽  
pp. C445-C452 ◽  
Author(s):  
T. C. Vary ◽  
S. R. Kimball
Keyword(s):  
Protein Synthesis ◽  
Sterile Inflammation ◽  
Chain Elongation ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Translational Efficiency ◽  
Liver Protein ◽  
Ribosomal Subunits ◽  
Hepatic Protein Synthesis

The regulation of protein synthesis was determined in livers from control, sterile inflammatory, and septic animals. Total liver protein was increased in both sterile inflammation and sepsis. The rate of protein synthesis in vivo was measured by the incorporation of [3H]phenylalanine into liver proteins in a chronic (5 day) intra-abdominal abscess model. Both sterile inflammation and sepsis increased total hepatic protein synthesis approximately twofold. Perfused liver studies demonstrated that the increased protein synthesis rate in vivo resulted from a stimulation in the synthesis of both secreted and nonsecreted proteins. The total hepatic RNA content was increased 40% only in sterile inflammation, whereas the translational efficiency was increased twofold only in sepsis. The increase in translational efficiency was accompanied by decreases in the amount of free 40S and 60S ribosomal subunits in sepsis. Rates of peptide-chain elongation in vivo were increased 40% in both sterile inflammation and sepsis. These results demonstrate that sepsis induces changes in the regulation of hepatic protein synthesis that are independent of the general inflammatory response. In sterile inflammation, the increase in protein synthesis occurs by a combination of increased capacity and translational efficiency, while in sepsis, the mechanism responsible for accelerated protein synthesis is an increased translational efficiency.

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