scholarly journals The lipid droplet protein Pgc1 controls the subcellular distribution of phosphatidylglycerol

2019 ◽  
Vol 19 (5) ◽  
Author(s):  
Dominika Kubalová ◽  
Paulína Káňovičová ◽  
Petra Veselá ◽  
Thuraya Awadová ◽  
Vladimíra Džugasová ◽  
...  
Keyword(s):  
Subcellular Distribution ◽  
Fluorescence Recovery After Photobleaching ◽  
In Vitro Assays ◽  
Phospholipase Activity ◽  
Inner Mitochondrial Membrane ◽  
The Individual ◽  
Storage Place ◽  
Lipid Droplet Protein

ABSTRACTThe biosynthesis of yeast phosphatidylglycerol (PG) takes place in the inner mitochondrial membrane. Outside mitochondria, the abundance of PG is low. Here, we present evidence that the subcellular distribution of PG is maintained by the locally controlled enzymatic activity of the PG-specific phospholipase, Pgc1. A fluorescently labeled Pgc1 protein accumulates on the surface of lipid droplets (LD). We show, however, that LD are not only dispensable for Pgc1-mediated PG degradation, but do not even host any phospholipase activity of Pgc1. Our in vitro assays document the capability of LD-accumulated Pgc1 to degrade PG upon entry to the membranes of the endoplasmic reticulum, mitochondria and even of artificial phospholipid vesicles. Fluorescence recovery after photobleaching analysis confirms the continuous exchange of GFP-Pgc1 within the individual LD in situ, suggesting that a steady-state equilibrium exists between LD and membranes to regulate the immediate phospholipase activity of Pgc1. In this model, LD serve as a storage place and shelter Pgc1, preventing its untimely degradation, while both phospholipase activity and degradation of the enzyme occur in the membranes.

scholarly journals The lipid droplet protein Pgc1 controls the subcellular distribution of phosphatidylglycerol

2018 ◽  
Author(s):  
Dominika Kubalová ◽  
Petra Veselá ◽  
Thuraya Awadová ◽  
Günther Daum ◽  
Jan Malínský ◽  
...  
Keyword(s):  
Subcellular Distribution ◽  
Mitochondrial Membrane ◽  
Lipid Droplets ◽  
Phospholipase Activity ◽  
Inner Mitochondrial Membrane ◽  
Present Evidence ◽  
Storage Place ◽  
Lipid Droplet Protein

AbstractThe biosynthesis of yeast phosphatidylglycerol (PG) takes place in the inner mitochondrial membrane. Outside mitochondria, the abundance of PG is low. Here we present evidence that the subcellular distribution of PG is maintained by locally controlled enzymatic activity of the PG-specific phospholipase, Pgc1. We document that the Pgc1 absence leads to spreading of PG over various cellular membranes. Fluorescently labeled Pgc1 protein strongly accumulates at the surface of lipid droplets (LD). We show, however, that LD are not only dispensable for Pgc1-mediated PG degradation, but even host no phospholipase activity of Pgc1. Our in vitro assays document the capability of LD-accumulated Pgc1 to degrade PG upon entry to membranes of the endoplasmic reticulum, mitochondria, and even of artificial phospholipid vesicles. FRAP analysis confirms continuous exchange of GFP-Pgc1 within individual LD in situ, suggesting that a steady-state equilibrium exists between LD and membranes to regulate immediate phospholipase activity of Pgc1. In this model, LD serve as storage place and shelter Pgc1 preventing untimely degradation, while both phospholipase activity and degradation of the enzyme occur in membranes.

In situ and in vitro techniques for estimating degradation parameters and digestibility of diets based on maize or sorghum

2020 ◽  
Vol 158 (1-2) ◽  
pp. 150-158 ◽  
Author(s):  
B. C. Silva ◽  
M. V. C. Pacheco ◽  
L. A. Godoi ◽  
F. A. S. Silva ◽  
D. Zanetti ◽  
...  
Keyword(s):  
Dry Matter ◽  
Latin Square ◽  
In Vitro Techniques ◽  
Sorghum Grains ◽  
Sorghum Silage ◽  
The Individual ◽  
Individual Grains

AbstractAn experiment was conducted to evaluate: (1) the effects of ensiling maize or sorghum grains after reconstitution on readily soluble fraction (a), potentially degradable fraction in the rumen (b) and rate constant for degradation of b (c) of dry matter (DM), organic matter (OM) and starch (STA); and (2) an appropriate incubation time for in situ or in vitro procedures to estimate in vivo digestibility. Four rumen-cannulated Nellore bulls (body weight = 262 ± 19.6 kg) distributed in a 4 × 4 Latin square were used. Diets were based on dry ground maize (DGM); or dry ground sorghum (DGS); or reconstituted ground maize silage; or reconstituted ground sorghum silage. In vitro and in situ incubations of the individual grains and diets were simultaneously performed with in vivo digestibility. In general, reconstituted grains and diets based on reconstituted grains presented greater (P < 0.05) fraction a and lower (P < 0.05) fraction b of DM, OM and STA compared to dry grains and diets based on dry grain. However, the magnitude of response of the reconstitution and ensiling process on DM and OM degradability parameter was greater for maize than that for sorghum. Moreover, no differences (P > 0.05) were observed between DGM- and DGS-based diets for c estimates. The results suggest that the reconstitution process promotes grains protein matrix breakdown increasing STA availability. The incubation times required for in vivo digestibility estimations of DM, OM and STA are 24 h for in situ and 36 h for in vitro procedures.

SUBCELLULAR DISTRIBUTION AND FATE OF RADIOACTIVITY IN OVARIES OF PSEUDO-PREGNANT RATS AFTER IN VIVO ADMINISTRATION OF [131I]HCG

1973 ◽  
Vol 74 (2) ◽  
pp. 361-370 ◽  
Author(s):  
Wilhelm Braendle ◽  
Meinert Breckwoldt ◽  
Dieter Graesslin ◽  
Hans-Christoph Weise
Keyword(s):  
Binding Sites ◽  
Subcellular Distribution ◽  
Nuclear Fraction ◽  
Pregnant Rats ◽  
Subcellular Compartments ◽  
The Individual ◽  
Lh Rh ◽  
Higher Doses

ABSTRACT After in vivo application of [131I]HCG to pseudo-pregnant rats most of the radioactivity is found in the ovaries. Ovarian homogenate contains per mg protein 50 times as much radioactivity as the liver and 7 times as much as the kidney. The relatively high amount of radioactivity in the kidney possibly reflects a rapid excretion of metabolized or damaged hormone. The subcellular distribution of radioactivity after in vivo application of the labelled hormone indicates a biologically occurring process which cannot be studied by in vitro experiments. Various binding components are shown to exist in the individual subcellular compartments of ovaries which diverge in their binding affinity and capacity for HCG. Binding sites in the nuclear fraction are already saturated when injecting 10 μg HCG together with the label. The uptake of radioactivity in the cytosol, however, is only inhibited when using higher doses of HCG. 1 μg LH-RH provokes maximum release of endogenous LH, inhibits radioactivity uptake by the corpuscular subcellular compartments and does not affect the binding components in the cytosol. It may be concluded that the hormone itself penetrates the cell membrane to reach its target site within the cell or that the recovered radioactivity in the different fractions is due to plasma membrane contamination which may represent the actual hormone binding sites.

In vitro degradability of feed proteins in the rumen: use of non-rumen proteases

2005 ◽  
Vol 56 (8) ◽  
pp. 797 ◽  
Author(s):  
M. Aslam Mirza ◽  
E. L. Miller
Keyword(s):  
Correlation Coefficient ◽  
Soybean Meal ◽  
Streptomyces Griseus ◽  
Correlation Coefficients ◽  
In Vitro Assays ◽  
Vitro Assay ◽  
Bacterial Protease ◽  
Ruminal Protein Degradability

Various feed proteins were incubated independently with bacterial protease from Streptomyces griseus (SGP), papain (Corica papaya), and ficin (Ficus glabrata) in a simple laboratory assay to predict ruminal protein degradability. The estimates obtained from in vitro assays were compared with those obtained from an in situ analysis using synthetic fibre bags. The rate and extent of degradation in vitro using proteases from non-rumen sources differed among substrates used. A high correlation coefficient (r2 = 0.99) was observed between N-degradability from the in vitro method using SGP and in situ estimates when soybean meal was the substrate. Soybean meal nitrogen (N) was almost completely hydrolysed (0.99) in vitro. The correlation coefficients were low and variable with assays using other enzymes. The correlation coefficient was also high (r2 = 0.77–0.84) with in vitro methods using either SGP, papain, or ficin when incubated with fish meal. The N disappearance from barley in vitro was slow to moderate. The ‘b’ estimate of barley obtained with the in vitro assay was significantly (P < 0.01) lower than that observed in situ. Slower proteolysis observed in barley may possibly be linked to poor accessibility of structural proteins rather than the degradability of N per se. None of the enzymes could rank barley in the same order as the in situ method.

scholarly journals The Microtubule Plus-End Proteins EB1 and Dynactin Have Differential Effects on Microtubule Polymerization

2003 ◽  
Vol 14 (4) ◽  
pp. 1405-1417 ◽  
Author(s):  
Lee A. Ligon ◽  
Spencer S. Shelly ◽  
Mariko Tokito ◽  
Erika L.F. Holzbaur
Keyword(s):  
Cultured Cells ◽  
Cell Types ◽  
Microtubule Dynamics ◽  
In Vitro Assays ◽  
Nucleation Effect ◽  
Microtubule Polymerization ◽  
The Individual ◽  
Different Cell Types ◽  
Dynactin Complex

Several microtubule-binding proteins including EB1, dynactin, APC, and CLIP-170 localize to the plus-ends of growing microtubules. Although these proteins can bind to microtubules independently, evidence for interactions among them has led to the hypothesis of a plus-end complex. Here we clarify the interaction between EB1 and dynactin and show that EB1 binds directly to the N-terminus of the p150Glued subunit. One function of a plus-end complex may be to regulate microtubule dynamics. Overexpression of either EB1 or p150Glued in cultured cells bundles microtubules, suggesting that each may enhance microtubule stability. The morphology of these bundles, however, differs dramatically, indicating that EB1 and dynactin may act in different ways. Disruption of the dynactin complex augments the bundling effect of EB1, suggesting that dynactin may regulate the effect of EB1 on microtubules. In vitro assays were performed to elucidate the effects of EB1 and p150Glued on microtubule polymerization, and they show that p150Gluedhas a potent microtubule nucleation effect, whereas EB1 has a potent elongation effect. Overall microtubule dynamics may result from a balance between the individual effects of plus-end proteins. Differences in the expression and regulation of plus-end proteins in different cell types may underlie previously noted differences in microtubule dynamics.

scholarly journals Regional Evaluation of Fungal Pathogen Incidence in Colombian Cocoa Crops

Agriculture ◽  
2019 ◽  
Vol 9 (3) ◽  
pp. 44
Author(s):  
Raquel Villamizar-Gallardo ◽  
Johann Osma ◽  
Oscar Ortíz-Rodriguez
Keyword(s):  
Fungal Pathogens ◽  
Morphological Variability ◽  
Phytopathogenic Fungi ◽  
Phytophthora Palmivora ◽  
In Vitro Assays ◽  
Post Conflict ◽  
Solid Media ◽  
Black Pod

The production of cocoa (Theobroma cacao L.) in Colombia has a significant environmental and socioeconomic importance as a promissory crop in the post-conflict process. The department of Norte de Santander has cocoa crops that are dramatically affected by fungal pathogens causing important losses during harvest and post-harvest. Therefore, the current study focused on the determination of the incidence of diseases caused by phytopathogenic fungi in cocoa crops, and the identification of primary phytopathogenic fungi found in biological material from different farms of the region. The study was conducted in four municipalities of the department, by sampling fruits infected with frosty pod rot (FPR) and black pod rot (BPR) that presented in situ incidence ranging from 0.37 to 21.58% and from 1.75 to 35.59%, respectively. The studied hybrid materials, together with clone TSH 65, were found to be the most susceptible, while the remaining clones were more tolerant, especially CCN 51, IMC 67, and ICS95. Fifteen strains were isolated using in vitro assays and then morphologically characterized both in solid media and by microscopy. Nine of them corresponded to the pathogen Moniliophthora roreri, and other six to Phytophthora palmivora. The isolated agents showed in vitro morphological variability, as well as the ability to adapt to different environments when growing in situ.

scholarly journals Synthesis and Biological Screening of New Cyano-Substituted Pyrrole Fused (Iso)Quinoline Derivatives

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 2066
Author(s):  
Maria Cristina Al-Matarneh ◽  
Roxana-Maria Amărandi ◽  
Ionel I. Mangalagiu ◽  
Ramona Danac
Keyword(s):  
Cell Lines ◽  
Cancer Cell ◽  
Human Cancer ◽  
Cancer Cell Lines ◽  
In Vitro Assays ◽  
Biological Screening ◽  
Human Cancer Cell Lines ◽  
Breast And Prostate Cancer

Several new cyano-substituted derivatives with pyrrolo[1,2-a]quinoline and pyrrolo[2,1-a]isoquinoline scaffolds were synthesized by the [3 + 2] cycloaddition of (iso)quinolinium ylides to fumaronitrile. The cycloimmonium ylides reacted in situ as 1,3-dipoles with fumaronitrile to selectively form distinct final compounds, depending on the structure of the (iso)quinolinium salt. Eleven compounds were evaluated for their anticancer activity against a panel of 60 human cancer cell lines. The most potent compound 9a showed a broad spectrum of antiproliferative activity against cancer cell lines representing leukemia, melanoma and cancer of lung, colon, central nervous system, ovary, kidney, breast and prostate cancer. In vitro assays and molecular docking revealed tubulin interaction properties of compound 9a.

scholarly journals Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity

2011 ◽  
Vol 2011 ◽  
pp. 1-18 ◽  
Author(s):  
Rachel S. Lee ◽  
Colin M. House ◽  
Briony E. Cristiano ◽  
Ross D. Hannan ◽  
Richard B. Pearson ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Specific Activity ◽  
Dominant Role ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Disease States ◽  
The Individual

The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ.

In situ determination of [Ca2+]i threshold for translocation of the alpha-protein kinase C isoform

1994 ◽  
Vol 266 (6) ◽  
pp. C1544-C1551 ◽  
Author(s):  
R. A. Khalil ◽  
C. Lajoie ◽  
K. G. Morgan
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Second Messengers ◽  
In Vitro Assays ◽  
Intact Cells ◽  
Kinase C ◽  
Biochemical Assays

Because of inherent difficulties in maintaining physiological conditions in biochemical assays, the intracellular free Ca2+ concentration ([Ca2+]i) required for activation of protein kinase C (PKC) in intact cells remains unclear. In the present study, [Ca2+]i was measured in freshly isolated vascular smooth muscle cells loaded with fura 2 while, in parallel, the distribution of the Ca(2+)-dependent alpha-PKC isoform was monitored using digital imaging microscopy. The [Ca2+]i alpha-PKC translocation threshold was determined by changing extracellular free Ca2+ concentration in steps while monitoring [Ca2+]i. In the absence of agonists, increasing [Ca2+]i caused < 25% of maximal translocation. In the presence of phenylephrine, maximum translocation occurred at [Ca2+]i > or = 198 nM. Phenylephrine augmented translocation of alpha-PKC primarily by increasing the slope of the [Ca2+]i-PKC translocation relationship. These results indicate that the [Ca2+]i threshold of alpha-PKC translocation in situ is less than that reported in most in vitro assays and are consistent with an effect of agonist-induced generation of other second messengers that cause cooperative interactions leading to translocation.

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