scholarly journals Mechanisms of protein degradation in growing and non-growing L-cell cultures

1979 ◽  
Vol 182 (3) ◽  
pp. 847-859 ◽  
Author(s):  
J S Amenta ◽  
M J Sargus
Keyword(s):  
Stationary Phase ◽  
Protein Degradation ◽  
Media Analysis ◽  
Cell Protein ◽  
Minimal Essential Medium ◽  
Buffer Solution ◽  
L Cells ◽  
Effective Inhibition ◽  
Processing Procedure

L-cells prelabelled with [14C]leucine and [3H]thymidine were placed in either fresh growth medium (minimal essential medium with 10% serum) or stepdown medium (minimal essential medium) for 3 days. The 14C/3H ratio remained constant in the growing cultures and decreased in the stationary-phase cultures, indicating no protein turnover in growing cultures and a degradative rate of 0.6%/h in the stationary-phase cultures. Media analysis, however, indicated that 14C-labelled proteins were being degraded at approx. 1.2%/h in growing cultures and 1.7%/h in stationary-phase cultures. Additional studies indicated that a subpopulation of L-cells in the monolayer, comprising approx. 20–30% of the total, were lost in the original processing procedure. Experiments in which recoveries approached 100% by fixation of the monolayer in situ indicated that a protein-degrading subpopulation accounted for all the observed proteolysis in the growing cultures. Proteolysis in these cultures was only partially inhibited with NH4Cl, indicating that only a small part of the protein degradation was occurring in an activated lysosomal-autophagic system. NaF produced a more effective inhibition of proteolysis, but we were not able to distinguish whether this effect was on an ATP-requiring basal-turnover mechanism or a direct effect on unregulated activity of proteinases in the cell hyaloplasm. However, NH4Cl inhibited the proteolysis induced when cells were placed in stepdown medium, suggesting that the induced proteolysis was occurring via the autophagic system. We conclude that L-cells exist in at least two states with respect to protein degradation: (a) a subpopulation that is actively replicating and does not degrade cellular proteins, and (b) a second subpopulation of cells, derived from the preceding one, which degraded most of their labelled proteins, are not capable of further replication, and are not sedimented in an iso-osmotic EDTA buffer solution. In addition, proliferating L-cells, when placed in stepdown medium, begin to degrade cell protein through a mechanism involving autophagolysosomes.

scholarly journals Effect of microtubular or translational inhibitors on general cell protein degradation. Evidence for a dual catabolic pathway

1977 ◽  
Vol 168 (2) ◽  
pp. 223-227 ◽  
Author(s):  
J S Amenta ◽  
M J Sargus ◽  
F M Baccino
Keyword(s):  
Protein Degradation ◽  
Trichloroacetic Acid ◽  
Fold Increase ◽  
Cell Protein ◽  
Inhibitory Effects ◽  
Minimal Essential Medium ◽  
Catabolic Pathway ◽  
Rat Embryo ◽  
Step Down ◽  
Observation Period

Rat embryo fibroblasts were grown in Eagle's minimal essential medium with 10% serum and cell proteins prelabelled with L-[1-(14)C]leucine, followed by a 24h chase. When transferred to medium deprived of serum these cells showed a 2—3-fold increase in the production of trichloroacetic acid-soluble radioactivity during a 4h observation period. The microtubular poisons vinblastine, vincristine and colchicine partially inhibited this induced proteolysis, but had no effect on the proteolytic rate of cells maintained in medium with 10% serum. A similar discriminating effect on induced proteolysis was observed with cycloheximide, puromycin and insulin. The inhibitory effects of cycloheximide and vinblastine were not additive. These data support the hypothesis that, in addition to the basal turnover of cell proteins, a second mechanism of protein degradation involving cytoplasmic autophagy can be activated by nutritional step-down and is selectively inhibited by agents that interfere with microtubular function and protein synthesis.

scholarly journals Effect of Yeast-Fermented Citrus Pulp as a Protein Source on Nutrient Intake, Digestibility, Nitrogen Balance and In Situ Digestion Kinetics in Nili Ravi Buffalo Bulls

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1713
Author(s):  
Muhammad Awais ◽  
Muhammad Sharif ◽  
Khurram Ashfaq ◽  
Amjad Islam Aqib ◽  
Muhammad Saeed ◽  
...  
Keyword(s):  
Nitrogen Balance ◽  
Nutrient Intake ◽  
Dry Matter ◽  
Experimental Period ◽  
Nutrient Digestibility ◽  
Protein Source ◽  
Cell Protein ◽  
Citrus Pulp ◽  
Digestion Kinetics

A study was carried out to evaluate the effect of single cell protein (SCP) supplement as a protein source on nutrient intake, digestibility, nitrogen balance and in situ digestion kinetics in four Nili Ravi buffalo bulls. Four iso-caloric and iso-nitrogenous concentrates containing 3, 6, 9 and 12% of Saccharomyces cerevisiae-fermented citrus pulp were formulated. All animals were fed a ration with a concentrate/forage ratio of 50:50. Diets were provided ad libitum twice a day as a total mixed ration in a 4 × 4 Latin Square Design. Each experimental period lasted 3 weeks while the overall study 12 weeks. The first 2 weeks of each experimental period were used as adaptation period while the third week as collection period. Chemical composition of fermented citrus pulp appeared as an excellent source of protein. No significant difference was observed on dry matter intake, digestibility of nutrients and SCP among all the treatments. Moreover, no significant effect was observed on ruminal pH and ammonia nitrogen at different times. Rate of disappearance and lag time of in situ dry matter digestion kinetics remained nonsignificant regardless of SCP percentage. Based on results of similar nutrients intake, nutrient digestibility, and ruminal parameters it is concluded that SCP could be used in the concentrate diet of ruminant up to 12%. Furthermore, the SCP has the potential of an alternative protein source in animal diet formulation.

The ability of the ficin enzyme technique to predict the in situ degradability of lucerne N

1999 ◽  
Vol 1999 ◽  
pp. 156-156
Author(s):  
G. Gizzi ◽  
E.R. Deaville ◽  
D.I. Givens
Keyword(s):  
Protein Degradation ◽  
Microbial Population ◽  
Reasonable Accuracy ◽  
Complex Process ◽  
Ruminal Protein Degradability ◽  
Protein Degradability

The assessment of protein degradability in the rumen is a complex process. The infinite combination of interaction between the rumen microbial population and the nature of the protein fed to the animal makes the estimation of ruminal protein degradability very arduous. At present the in situ technique is the most popular means of predicting ruminal nitrogen (N) degradation. However this procedure is slow, expensive and relies on the use of numerous surgically prepared animals. A number of studies (Assoumani et al., 1992; Aufrère and Cartailler, 1988) have shown that the use of in vitro methods using proteases can predict with reasonable accuracy the extent of protein degradation. The objective of this experiment was to examine the possibility of replacing the in situ technique with an in vitro procedure based on the use of the ficin protease to predict the extent of N degradation.

scholarly journals Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus α-tocopherol

2016 ◽  
Vol 36 (3) ◽  
pp. 209-215 ◽  
Author(s):  
Carolina R. Jimenez ◽  
Jurandy M. Penitente-Filho ◽  
Ciro A.A. Torres ◽  
Amanda M. Medeiros ◽  
Leandro S. Silva
Keyword(s):  
Liquid Nitrogen ◽  
Trypan Blue ◽  
Ovarian Tissue ◽  
Survival Rates ◽  
Histological Evaluation ◽  
Minimal Essential Medium ◽  
Preantral Follicles ◽  
Vitrification Solution

Abstract: The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles.

scholarly journals Surface Plasmon-Driven Reversible Transformation of DNA-Bound Methylene Blue Detected In Situ by SERS

Plasmonics ◽  
2019 ◽  
Vol 15 (2) ◽  
pp. 427-434
Author(s):  
Muhammad R. Shattique ◽  
Maria Stepanova
Keyword(s):  
Methylene Blue ◽  
Surface Plasmon ◽  
Surface Enhanced Raman Spectroscopy ◽  
Laser Exposure ◽  
Chemical Transformations ◽  
Buffer Solution ◽  
Reversible Transformation ◽  
Surface Enhanced Raman ◽  
Methylthioninium Chloride

Abstract We have reported the in situ surface–enhanced Raman spectroscopy (SERS) monitoring of repetitive surface plasmon–mediated chemical transformation cycles in a conjugate nanobiological system. The nanobiological conjugate comprised a gold-coated plasmonic substrate biofunctionalized with thiolated single–stranded DNA carrying a reduction-oxidation indicator methylthioninium chloride, which is also known as methylene blue (MB), in buffer solution at a neutral pH. Exposure to a 523-nm laser excitation produced pronounced SERS bands of oxidized MB. Continued exposure to the laser resulted in disappearance of the SERS bands, which can be interpreted as a reduction of MB. This occurred in the absence of electrochemical stimulation, chemical agents, or catalysts, suggesting a surface plasmon–mediated mechanism of the transformation. The oxidized form of MB was recovered by an addition of fresh buffer solution on the surface of the sample. Continued laser exposure with periodical addition of the buffer resulted in repetitive cycles of changes in the SERS pattern, which were monitored in situ. The chemical transformations of MB were preceded by a buildup of an intermediate SERS pattern, which was attributed to a transient form of MB created by selective surface plasmon-driven excitation.

KGF regulates pulmonary epithelial proliferation and surfactant protein gene expression in adult rat lung

2000 ◽  
Vol 279 (6) ◽  
pp. L1146-L1158 ◽  
Author(s):  
Toshiyuki Yano ◽  
Robert J. Mason ◽  
Tianli Pan ◽  
Robin R. Deterding ◽  
Larry D. Nielsen ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Epithelial Cells ◽  
Surfactant Protein ◽  
Surfactant Proteins ◽  
Clara Cell ◽  
Cell Protein ◽  
Mrna Levels ◽  
Type Ii ◽  
Cell Hyperplasia

Keratinocyte growth factor (KGF, FGF-7) is a potent mitogen for epithelial cells. We instilled recombinant human KGF to determine the effects of KGF on alveolar epithelial cells. Left lungs of adult rats were instilled intrabronchially with KGF (5 mg/kg) or normal saline. KGF instillation resulted in epithelial cell hyperplasia, and the alveolar bromodeoxyuridine (BrdU) labeling index peaked at 35% on day 2 after instillation. The mRNA levels for the surfactant proteins (SPs) SP-A, SP-B, and SP-D were increased in whole lung tissue on days 1 and 2 after KGF treatment and then returned to control levels on days 3–7. SP-C mRNA levels were increased on days 2–5 after KGF instillation. However, all surfactant protein mRNAs were reduced in type II cells isolated from rats instilled with KGF 2 or 3 days before isolation. These observations were confirmed by in situ hybridization. Instillation of KGF also increased the amount of SP-A and SP-D in lavage fluid. Transcripts for CC10, the 10-kDa Clara cell protein, were decreased. KGF increases the mRNA for the surfactant proteins per lung because of type II cell hyperplasia, but the mRNA per cell is slightly diminished as measured in isolated cells or estimated by in situ hybridization.

scholarly journals In Situ Dry Matter and Crude Protein Degradation of Fresh Forages During the Spring Growth

1999 ◽  
Vol 82 (9) ◽  
pp. 1978-1990 ◽  
Author(s):  
J.C. Elizalde ◽  
N.R. Merchen ◽  
D.B. Faulkner
Keyword(s):  
Protein Degradation ◽  
Crude Protein ◽  
Dry Matter
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