The Effect of Dilution Rate upon Protein Content and Cellular Amino Acid Profiles in Chemostat Cultures of Saccharomyces Cerevisiae CABI 039916

2010 ◽  
Vol 6 (4) ◽  
Author(s):  
Beverley Finn ◽  
Linda M Harvey ◽  
Brian McNeil
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Protein Content ◽  
Dilution Rate ◽  
Specific Growth ◽  
Cellular Protein ◽  
Cell Protein ◽  
Protein Pool ◽  
Cellular Protein Content ◽  
Amino Acid Profiles

In the present study we examined the use of a chemostat system to investigate the impact of changes in the specific growth rate of Saccharomyces cerevisiae CABI 039916 on cellular amino acid profiles and total protein content. This experimental system allowed the unambiguous examination of the link between changes in dilution rate and the culture response, which would have been difficult in batch or fed-batch cultures. Alteration of the specific growth rate (via manipulation of the dilution rate) within a carbon and energy-limited chemostat has a significant impact on the physiology of Saccharomyces cerevisiae. Low dilution rates (<0.1h-1) led to predominantly respiratory metabolism and the maximisation of cellular protein content within the cell (58%), by contrast high dilution rates (>0.2h-1) led to respirofermentative metabolism, where the cellular protein content was minimal (~40%). The content of nearly all amino acids in the yeast protein pool fell significantly as dilution rate increased in parallel with the decline in cell protein content. By contrast, the concentration of two related key food/feed amino acids in the cell protein pool—glutamic acid and arginine could be increased within the cellular protein by 5% (increasing the dilution rate from 0.05h-1 to 0.25h-1) and 1.5% respectively (decreasing the dilution rate from 0.05h-1 to 0.2h-1). Despite previous studies showing that metabolic change was associated with major changes in free amino acid levels, the present study indicates that the total cellular yeast protein amino acid composition is largely invariant despite profound metabolic changes, with one or two key exceptions.

ATP and Protein Synthesis Assays to Evaluate the Toxicities of Preservatives in Vitro

1991 ◽  
Vol 19 (1) ◽  
pp. 60-67
Author(s):  
Corrado L. Galli ◽  
Barbara Viviani ◽  
Giampiero Casartelli ◽  
Marina Marinovich
Keyword(s):  
Protein Synthesis ◽  
Protein Content ◽  
Cellular Protein ◽  
Cell Protein ◽  
Leucine Incorporation ◽  
Maximal Effect ◽  
Atp Level ◽  
Cellular Protein Content

Cellular protein content, protein synthesis, ATP level and lactate dehydrogenase (LDH) activity, measured in a murine epidermal cell line (HEL/30), were used as the endpoints for determining the cytotoxicities of 17 antimicrobial chemicals. The relative toxicities of the test compounds were quantified by the determination of the concentrations inducing a 50% inhibition of [3H]-leucine incorporation into proteins (IC50), causing a 50% reduction of ATP level or final cell protein content or producing the maximal effect on LDH leakage (EC50) after 2 hours of treatment. The results indicate a good correlation between both the reduction of ATP level and inhibition of protein synthesis and the minimal inhibitory concentration (MIC) on different microorganisms, suggesting that ATP and protein synthesis assays could be useful as prescreening methods for testing the cytotoxicities of preservatives.

scholarly journals Yeast lines selected for non-quiescence show increased protein content

2019 ◽  
Author(s):  
Monika Opałek ◽  
Agnieszka Marek ◽  
Aleksandra Kałdon ◽  
Barbara Mickowska ◽  
Dominika Wloch-Salamon
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Protein Content ◽  
Total Protein ◽  
Acid Content ◽  
Amino Acid Content ◽  
Dry Mass ◽  
Ancestral Population ◽  
Cell Protein ◽  
Total Protein Content

Increasing demand for food production requires improvements as well as openness to new sources of protein. Single Cell Protein derived from microbes has been intensively studied as a supplement for traditional sources of proteins, both for animal feed and direct human consumption. Food grade yeast, including Saccharomyces cerevisiae, has already been successfully used in industry. Here, we describe an artificial selection experiment that resulted in isolating Saccharomyces cerevisiae lines with a heritable phenotype characterized by significantly higher total protein content. Compared to the ancestral population, the average increase in protein content for all the evolved lines containing multiple-evolved clones was 5.4 g per 100g of dry mass (~15% of the total protein content). However, we also obtained specific clones with a total protein content increase of 9.3 g/100g dry mass (increase by 24.6%). Whole genome sequence analysis of mutations acquired by these clones allowed us to hypothesize about the role of the amino acid signaling pathway (SPS) disorders as a genetic base of the increased amino acid content. The proposed method is based on gradient fractionation of starved haploid yeast cells of different density, which reflects their physiological state regarding quiescence. We found a positive correlation in the fraction of non-quiescent cells in the starved populations and their amino acid content. Our method does not require any genetic modification. We believe that it can be successfully applied to other Saccharomyces sp. in order to increase the amino acid content stored in their cells. Beyond its implications for applied science, knowledge of the quiescent state in yeast is of fundamental importance to our understanding of the genetic basis of the G0 state in eukaryotic cells.

scholarly journals Defining the differential sensitivity to norepinephrine and angiotensin II in the ovine uterine vasculature

2012 ◽  
Vol 302 (1) ◽  
pp. R59-R67 ◽  
Author(s):  
Charles R. Rosenfeld ◽  
Kevin DeSpain ◽  
Xiao-tie Liu
Keyword(s):  
Angiotensin Ii ◽  
Protein Content ◽  
Systemic Circulation ◽  
Cellular Protein ◽  
Differential Sensitivity ◽  
Dependent Manner ◽  
Third Generation ◽  
Ang Ii ◽  
Pregnant Sheep ◽  
Cellular Protein Content

The intact ovine uterine vascular bed (UVB) is sensitive to α-agonists and refractory to angiotensin II (ANG II) during pregnancy; the converse occurs in the systemic circulation. The mechanism(s) responsible for these differences in uterine sensitivity are unclear and may reflect predominance of nonconstricting AT2 receptors (AT2R) in uterine vascular smooth muscle (UVSM). The contribution of the placental vasculature also is unclear. Third generation and precaruncular/placental arteries from nonpregnant ( n = 16) and term pregnant ( n = 23) sheep were used to study contraction responses to KCl, norepinephrine (NE), and ANG II (with/without ATR specific inhibitors) and determine UVSM ATR subtype expression and contractile protein content. KCl and NE increased third generation and precaruncular/placental UVSM contractions in a dose- and pregnancy-dependent manner ( P ≤ 0.001). ANG II only elicited modest contractions in third generation pregnant UVSM ( P = 0.04) and none in precaruncular/placental UVSM. Moreover, compared with KCl and NE, ANG II contractions were diminished ≥ 5-fold. Whereas KCl and ANG II contracted third generation>>precaruncular/placental UVSM, NE-induced contractions were similar throughout the UVB. However, each agonist increased third generation contractions ≥ 2-fold at term, paralleling increased actin/myosin and cellular protein content ( P ≤ 0.01). UVSM AT1R and AT2R expression was similar throughout the UVB and unchanged during pregnancy ( P > 0.1). AT1R inhibition blocked ANG II-mediated contractions; AT2R blockade, however, did not enhance contractions. AT2R predominate throughout the UVB of nonpregnant and pregnant sheep, contributing to an inherent refractoriness to ANG II. In contrast, NE elicits enhanced contractility throughout the ovine UVB that exceeds ANG II and increases further at term pregnancy.

An assay for the measurement of the protein content of cells immobilized in carrageenan

1984 ◽  
Vol 30 (4) ◽  
pp. 475-481 ◽  
Author(s):  
A. Jones ◽  
T. Razniewska ◽  
B. H. Lesser ◽  
R. Siqueira ◽  
D. Berk ◽  
...  
Keyword(s):  
Protein Content ◽  
Immobilized Cells ◽  
Fine Powder ◽  
Cellular Protein ◽  
Cell Protein ◽  
Specific Productivity ◽  
Immobilized Cell ◽  
Freeze Dried ◽  
Lowry Method ◽  
Reproducible Method

A reliable and reproducible method for the estimation of the protein content of fungal cells immobilized in a carrageenan gel is described. The procedure depends upon the acid lability of the polysaccharide gel at 90 °C and on the acetone solubility of accumulated phenolics. Freeze-dried gel beads (2–3 mm) containing entrapped cells of Penicillium urticae were ground to a fine powder and samples of powder (~20 mg) were sequentially extracted with hot 1 N HCl – 0.9% NaCl and acetone. The precipitated residue contained the cell protein, which was then solubilized with 1 N NaOH at 90 °C and quantitated by the Folin–Lowry method. Interferences from both carrageenan and phenols were thus eliminated. The presence of carrageenan (20–25 mg) did not affect the recovery of varying amounts (0–2500 μg) of bovine serum albumin. The recovery of radiolabeled protein from immobilized cells was parallel to that of Folin–Lowry positive material over a range of 0–60 beads (0–60 mg powder). Cycloheximide (0–100 μg/mL) was shown to progressively inhibit the incorporation of L-[U-14C]leucine so that the radioactivity present in the initial HCl–NaCl extract (i.e., [14C]leucine) increased as that in the final NaOH extract (i.e., 14C-labeled protein) decreased. Using this new assay for cell protein, free and immobilized cell cultures were found to exhibit virtually identical kinetics of glucose utilization, growth, and patulin production. In addition to providing a means of comparing the specific productivity of free versus immobilized cell preparations, this assay accurately measures the incorporation of [14C]leucine into cellular protein and could be used as a measure of cell viability.

Protein prenylation is required for aldosterone-stimulated Na+ transport

1997 ◽  
Vol 272 (6) ◽  
pp. C1928-C1935 ◽  
Author(s):  
B. L. Blazer-Yost ◽  
C. L. Hughes ◽  
P. L. Nolan
Keyword(s):  
Protein Content ◽  
Mevalonate Pathway ◽  
Cellular Protein ◽  
Protein Methylation ◽  
Na Transport ◽  
Protein Prenylation ◽  
Cellular Protein Content ◽  
Farnesylated Protein ◽  
Stimulation Of ◽  
Natriferic Action

Aldosterone stimulation of transcellular Na+ flux in polarized epithelial cells is dependent on at least one transmethylation reaction, but the substrate of this signaling step is unknown. Because it is clear that the majority of cellular protein methylation occurs in conjunction with protein prenylation, we examined the importance of prenylation to aldosterone-stimulated Na+ transport in the A6 cell line. Lovastatin, an inhibitor of the first committed step of the mevalonate pathway, inhibits the natriferic effect of aldosterone but does not inhibit insulin-stimulated Na+ flux. The addition of a farnesyl group does not appear to be involved in aldosterone's action. Neither alpha-hydroxyfarne-sylphosphonic acid, an inhibitor of farnesyl:protein transferase, nor N-acetyl-S-farnesyl-L-cysteine, an inhibitor of farnesylated protein methylation, inhibits the hormone-induced increase in Na+ transport. In contrast, N-acetyl-S-geranyl-geranyl-L-cysteine, an inhibitor of geranylgeranyl protein methylation, completely abolishes the aldosterone-induced increase in Na+ flux with no effect on insulin-mediated Na+ transport or cellular protein content. These data indicate that methylation of a geranylgeranylated protein is involved in aldosterone's natriferic action.

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