scholarly journals Fractionation of iodinated particles and mitochondria from thyroid by zonal centrifugation and a study of their heterogeneity

1974 ◽  
Vol 138 (2) ◽  
pp. 299-304 ◽  
Author(s):  
Raymond Miquelis ◽  
Claude Simon
Keyword(s):  
Succinate Dehydrogenase ◽  
Radioactive Iodine ◽  
Acid Hydrolases ◽  
Linear Gradient ◽  
Fourth Class ◽  
Secondary Lysosomes ◽  
Gradient Separation ◽  
Zonal Rotor

1. The subcellular particles of horse and rat thyroids were fractionated in a B XIV zonal rotor on a non-linear gradient of Ficoll after labelling with radioactive iodine in vitro (horse) or in vivo (rat). In the horse, the resulting fractions were analysed for radioactive iodine, protein and enzymes representative of certain subcellular particles. In the rat, iodine turnover and thyrotrophin stimulation were studied. 2. The population of iodinated particles could be subdivided into three main classes, characterized by differences in β-galactosidase and acid phosphatase content and position in the gradient. The presence of a fourth class of particles is suggested. 3. It is concluded that iodinated particles isolated from the thyroid are essentially secondary lysosomes. Their heterogeneity is established with respect to their position in the gradient, their content of acid hydrolases and their iodine turnover. 4. The iodine pools of these secondary lysosomes are increased by thyrotrophin without any change in their number. 5. Their functional significance is discussed. 6. The distribution of mitochondria as judged by succinate dehydrogenase was also studied. The succinate dehydrogenase was spread throughout the gradient with a maximum of activity (40%) in the upper layer of the gradient. Separation of mitochondria from lysosomes by this method was not successful.

scholarly journals Bixafen, a succinate dehydrogenase inhibitor fungicide, causes microcephaly and motor neuron axon defects during development

2020 ◽  
Author(s):  
Alexandre Brenet ◽  
Rahma Hassan-Abdi ◽  
Nadia Soussi-Yanicostas
Keyword(s):  
Motor Neuron ◽  
Succinate Dehydrogenase ◽  
Toxicity Testing ◽  
Mitochondrial Respiratory Chain ◽  
Modern Agriculture ◽  
Transgenic Lines ◽  
Vertebrate Model ◽  
The Central Nervous System

AbstractSuccinate dehydrogenase inhibitors (SDHIs), the most widely used fungicides in agriculture today, act by blocking succinate dehydrogenase (SDH), an essential and evolutionarily conserved component of mitochondrial respiratory chain. Recent results showed that several SDHIs used as fungicides not only inhibit the SDH activity of target fungi but also block this activity in human cells in in vitro models, revealing a lack of specificity and thus a possible health risk for exposed organisms, including humans. Despite the frequent detection of SDHIs in the environment and on harvested products and their increasing use in modern agriculture, their potential toxic effects in vivo, especially on neurodevelopment, are still under-evaluated. Here we assessed the neurotoxicity of bixafen, one of the latest-generation SDHIs, which had never been tested during neurodevelopment. For this purpose, we used a well-known vertebrate model for toxicity testing, namely zebrafish transparent embryos, and live imaging using transgenic lines labelling the brain and spinal cord. Here we show that bixafen causes microcephaly and defects on motor neuron axon outgrowth and their branching during development. Our findings show that the central nervous system is highly sensitive to bixafen, thus demonstrating in vivo that bixafen is neurotoxic in vertebrates and causes neurodevelopmental defects. This work adds to our knowledge of the toxic effect of SDHIs on neurodevelopment and may help us take appropriate precautions to ensure protection against the neurotoxicity of these substances.

scholarly journals THE EFFECT OF POLY-L-LYSINE ON THE UPTAKE OF REOVIRUS DOUBLE-STRANDED RNA IN MACROPHAGES IN VITRO

1973 ◽  
Vol 57 (2) ◽  
pp. 484-498 ◽  
Author(s):  
Rolf Seljelid ◽  
Samuel C. Silverstein ◽  
Zanvil A. Cohn
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
Nucleic Acids ◽  
Acid Hydrolases ◽  
Cell Compartment ◽  
Double Stranded Rna ◽  
Secondary Lysosomes ◽  
Toxic Concentrations ◽  
Vacuolar System

The effect of polycations on cultured mouse peitoneal macrophages has been examined. Polycations, at concentrations greater than 5 µg/ml, are toxic for macrophages) as measured by failure of the cells to exclude vital dyes. At toxic concentrations polycations bind in large amounts to nuclei and endoplasmic reticulum, while at nontoxic levels polycations bind selectively to the cell surface. Nontoxic concentrations of polycations stimulate binding of reovirus double-stranded (ds) RNA to the macrophages by forming polycation-dsRNA complexes either in the medium or at the cell surface. These complexes enter the cell in endocytic vacuoles and are concentrated in secondary lysosomes. Despite exposure to the acid hydrolases within this cell compartment, the dsRNA and the polycation (poly-L-lysine) are conserved in a macromolecular form within the vacuolar system. The mechanism(s) by which the uptake of infectious nucleic acids and the induction of interferon by dsRNA are stimulated by polycations are discussed.

scholarly journals LYSOSOMES IN RAT THORACIC DUCT LYMPHOCYTES FRACTIONATED BY ZONAL CENTRIFUGATION

1973 ◽  
Vol 59 (1) ◽  
pp. 177-184 ◽  
Author(s):  
William E. Bowers
Keyword(s):  
Thoracic Duct ◽  
Light Microscope ◽  
Cathepsin D ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Differential Centrifugation ◽  
Acid Hydrolases ◽  
Fractionation Method

A method of zonal centrifugation was developed which separates rat thoracic duct lymphocytes (TDL) mainly according to size. The validity of the fractionation method was supported by light microscope observations, Coulter Counter sizing, and in vivo and in vitro labeling of lymphocytes. The distributions of lysosomal acid hydrolases in TDL fractionated by zonal centrifugation are similar to the distribution obtained for the cells. This result indicates that the large lymphocyte is not the sole bearer of either lysosomes or the large amount of soluble cathepsin D found in homogenates of TDL. Both reside mainly in small lymphocytes. This point was clearly established by fractionating homogenates of purified small lymphocytes by means of differential centrifugation and isopycnic density gradient centrifugation.

scholarly journals Tumor growth of neurofibromin-deficient cells is driven by decreased respiration and hampered by NAD+ and SIRT3

2021 ◽  
Author(s):  
Ionica Masgras ◽  
Giuseppe Cannino ◽  
Francesco Ciscato ◽  
Carlos Sanchez-Martin ◽  
Marco Pizzi ◽  
...  
Keyword(s):  
Succinate Dehydrogenase ◽  
Complex I ◽  
Nadh Dehydrogenase ◽  
Chain Complex ◽  
Respiratory Chain Complex ◽  
Succinate Dehydrogenase Activity ◽  
Nadh Ratio ◽  
Shed Light

Neurofibromin loss drives neoplastic growth and a rewiring of mitochondrial metabolism. Here, we report that neurofibromin ablation dampens expression and activity of NADH dehydrogenase, the respiratory chain complex I, in an ERK-dependent fashion. This provides cells with resistance to pro-oxidants targeting complex I and decreases both respiration and intracellular NAD+. Expression of the alternative NADH dehydrogenase NDI1 raises NAD+/NADH ratio, enhances the activity of the mitochondrial NAD+-dependent deacetylase SIRT3 and interferes with tumorigenicity in neurofibromin-deficient cells. This anti-neoplastic effect is mimicked both in vitro and in vivo by administration of NAD+ precursors or by rising expression of the NAD+ deacetylase SIRT3, and is synergistic with ablation of the mitochondrial chaperone TRAP1, which augments succinate dehydrogenase activity further contributing to block pro-neoplastic metabolic changes of these cells. These findings shed light on chemotherapeutic resistance and on bioenergetic adaptations of tumors lacking neurofibromin, linking complex I inhibition to mitochondrial NAD+/NADH unbalance and SIRT3 inhibition, as well as to down-regulation of succinate dehydrogenase. This metabolic rewiring could unveil attractive therapeutic targets for neoplasms related to neurofibromin loss.

scholarly journals Sensitivity ofRhizoctonia solanito Succinate Dehydrogenase Inhibitor and Demethylation Inhibitor Fungicides

Plant Disease ◽  
2017 ◽  
Vol 101 (3) ◽  
pp. 487-495 ◽  
Author(s):  
Olutoyosi O. Ajayi-Oyetunde ◽  
Carolyn J. Butts-Wilmsmeyer ◽  
Carl A. Bradley
Keyword(s):  
Succinate Dehydrogenase ◽  
The United States ◽  
Fungicide Sensitivity ◽  
Dmi Fungicides ◽  
Soybean Plants ◽  
Succinate Dehydrogenase Inhibitor ◽  
Collection Date ◽  
Demethylation Inhibitor

Soybean seedling diseases are caused by Rhizoctonia solani and can be managed with seed-applied fungicides that belong to different chemistry classes. To provide a benchmark for assessing a decline in sensitivities to these fungicide classes, R. solani isolates collected prior to 2001 were evaluated for their sensitivities to succinate dehydrogenase inhibitor (SDHI) (penflufen and sedaxane) and demethylation inhibitor (DMI) fungicides (ipconazole and prothioconazole). The effective concentration of each fungicide that reduced mycelial growth by 50% (EC50) was determined in vitro and compared with those of isolates recovered after 2011 from soybean plants with damping off and hypocotyl and root rot symptoms across different soybean-growing regions in the United States and Canada. All isolates, regardless of collection date, were extremely sensitive (EC50< 1 μg/ml) to the SDHI fungicides but were either extremely sensitive or moderately sensitive (1 ≤ EC50≤ 10 μg/ml) to the DMI fungicides. For all four active ingredients, variation in sensitivities was observed within and among the different anastomosis groups composing both isolate groups. Isolates collected after 2011, which also had varying in vitro sensitivities, were further evaluated for in vivo sensitivity to the four fungicides in the greenhouse. In vitro fungicide sensitivity did not always coincide with fungicide efficacy in vivo because all isolates tested, regardless of in vitro sensitivity, were effectively controlled by the application of the seed treatment fungicides in the greenhouse. Overall, our results indicate no shift in sensitivity to the fungicide classes evaluated, although considerable variability in the sensitivities of the two groups of isolates examined was present. Based on this research, continued monitoring of fungicide sensitivities of R. solani populations should occur to determine whether sensitivities become further reduced in the future.

scholarly journals Demonstration of Acid Hydrolase Activity in Primary Cultures of Bovine Brain Microvessel Endothelium

1989 ◽  
Vol 9 (3) ◽  
pp. 280-289 ◽  
Author(s):  
Anna Baranczyk-Kuzma ◽  
Thomas J. Raub ◽  
Kenneth L. Audus
Keyword(s):  
Primary Cultures ◽  
Bovine Brain ◽  
Endocytic Pathway ◽  
Hydrolase Activity ◽  
Acid Hydrolase ◽  
Acid Hydrolases ◽  
Cytochemical Localization ◽  
Brain Microvessel

The existence of lysosomes and acid hydrolase activity was demonstrated in an in vitro blood–brain barrier (BBB) model comprising primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. BMEC lysosomes were observed by the uptake of acridine orange and fluorophore-labeled acetylated low-density lipoprotein by fluorescence microscopy. Cytochemical localization of the acid hydrolase, sulfatase, and acid phosphatase (AcP) activities with light microscopy also revealed hydrolase-positive vacuoles or lysosomes that varied in number from cell to cell. BMEC monolayers were fractionated and biochemical assays of the sulfatase, AcP, and β-galactosidase were performed. Significant activities of the acid hydrolases were found to be associated with lysosome and microsome fractions (69–77%). The majority of β-galactosidase (≈48%) and total sulfatase (≈58%) activity was associated with the lysosome fraction of the BMECs. In contrast, ≈52% of AcP activity was associated with the microsome fraction of the cells. The results of this study are consistent with the demonstration in vivo of acid hydrolases as potential factors in the endocytic pathway for transport of proteins through the BBB and as contributors to the BBB's enzymatic barrier function.

scholarly journals The Dual-Functioning Fumarate Reductase Is the Sole Succinate:Quinone Reductase in Campylobacter jejuni and Is Required for Full Host Colonization

2009 ◽  
Vol 191 (16) ◽  
pp. 5293-5300 ◽  
Author(s):  
Rebecca A. Weingarten ◽  
Michael E. Taveirne ◽  
Jonathan W. Olson
Keyword(s):  
Organic Acids ◽  
Succinate Dehydrogenase ◽  
Campylobacter Jejuni ◽  
Growth Phase ◽  
Tca Cycle ◽  
Fumarate Reductase ◽  
Wild Type ◽  
Succinate Dehydrogenase Activity

ABSTRACT Campylobacter jejuni encodes all the enzymes necessary for a complete oxidative tricarboxylic acid (TCA) cycle. Because of its inability to utilize glucose, C. jejuni relies exclusively on amino acids as the source of reduced carbon, and they are incorporated into central carbon metabolism. The oxidation of succinate to fumarate is a key step in the oxidative TCA cycle. C. jejuni encodes enzymes annotated as a fumarate reductase (Cj0408 to Cj0410) and a succinate dehydrogenase (Cj0437 to Cj0439). Null alleles in the genes encoding each enzyme were constructed. Both enzymes contributed to the total fumarate reductase activity in vitro. The frdA::cat + strain was completely deficient in succinate dehydrogenase activity in vitro and was unable to perform whole-cell succinate-dependent respiration. The sdhA::cat + strain exhibited wild-type levels of succinate dehydrogenase activity both in vivo and in vitro. These data indicate that Frd is the only succinate dehydrogenase in C. jejuni and that the protein annotated as a succinate dehydrogenase has been misannotated. The frdA::cat + strain was also unable to grow with the characteristic wild-type biphasic growth pattern and exhibited only the first growth phase, which is marked by the consumption of aspartate, serine, and associated organic acids. Substrates consumed in the second growth phase (glutamate, proline, and associated organic acids) were not catabolized by the the frdA::cat + strain, indicating that the oxidation of succinate is a crucial step in metabolism of these substrates. Chicken colonization trials confirmed the in vivo importance of succinate oxidation, as the frdA::cat + strain colonized chickens at significantly lower levels than the wild type, while the sdhA::cat + strain colonized chickens at wild-type levels.

Evidence for NAD Nucleosidase in Rabbit-Liver Lysosomes

1975 ◽  
Vol 53 (2) ◽  
pp. 143-148 ◽  
Author(s):  
A. Mellors ◽  
A. K. L. Lun ◽  
O. N. Peled
Keyword(s):  
Plasma Membrane ◽  
Maximum Activity ◽  
Adenyl Cyclase ◽  
Rabbit Liver ◽  
Acid Hydrolases ◽  
Nad Glycohydrolase ◽  
Triton Wr 1339 ◽  
Liver Lysosomes ◽  
Secondary Lysosomes

A method is described for the isolation of secondary lysosomes from homogenates of rabbit liver. The uptake of Triton WR-1339 by rabbit-liver lysosomes when administered by intraperitoneal injection was used to decrease the density of secondary lysosomes. Lysosomal fractions prepared by this method contain an NAD nucleosidase (NAD glycohydrolase, EC 3.2.2.5), an enzyme which has previously been considered to be associated with other subcellular fractions. The enzyme has maximum activity at pH 6 and cleaves both NAD and NADP. It is inhibited by nicotinamide (Ki = 4.5 mM) and by HgCl2. Both nucleosidase and 2′-nucleotidase show in-vitro latency typical of lysosomal acid hydrolases. Rabbit-liver plasma-membrane fractions were isolated which contained most 5′-nucleotidase but relatively little nucleosidase, whereas rabbit liver lysosomes contain both 5′-nucleotidase and nucleosidase enzymes but little adenyl cyclase.

The Use of the Epithelial Kidney OK Cell Line for Studying the Nephrotoxicity of Anticancer Platinum Coordination Complexes

1993 ◽  
Vol 21 (1) ◽  
pp. 30-37
Author(s):  
Hervé Toutain ◽  
Françoise Courjault
Keyword(s):  
Protein Synthesis ◽  
Glucose Uptake ◽  
Succinate Dehydrogenase ◽  
Platinum Complexes ◽  
Marker Enzyme ◽  
Ok Cells ◽  
Dna And Protein Synthesis ◽  
Release Method

Nephrotoxicity is one of the most important dose-limiting side-effects of cis-diaminedichloroplatinum (II) (cDDP) in humans. Quiescent OK cells grown in hormonally-defined, serum-free medium in the total absence of antibiotics were used to study the in vitro nephrotoxicity of three platinum complexes which produce different renal toxicity in vivo: cDDP, its stereoisomer trans-diaminedichloroplatinum (II) (tDDP), and cis-diamine-1,1-cyclobutane-dicarboxylateplatinUm (II) (CBDCA). The uptake and cytotoxicity of these compounds at concentrations of 3-l,600μM and their impact on DNA and protein synthesis, glucose uptake, Na+-K+-ATPase and succinate dehydrogenase activities, as well as intracellular total glutathione level, were measured. The results showed that the cytotoxicity ranking of these three compounds, assessed by the LDH release method, was not in agreement with their in vivo nephrotoxic potentials (tDDP > cDDP > CBDCA00 in OK cells versus cDDP > CBDCA > tDDP in vivo). cDDP and tDDP showed similar uptakes at all the concentrations studied, which demonstrated that their cytotoxic potential was not directly related to intracellular levels of platinum. At non-cytotoxic concentrations, both cDDP and CBDCA decreased DNA and protein synthesis and, to a lesser extent, Na+-K+-ATPase activity, whereas no effect on glucose uptake and succinate dehydrogenase activity (a mitochondrial marker enzyme) was observed. Nevertheless, 5–10 times greater concentrations of CBDCA were required to induce effects similar to those induced by cDDP. Our results did not show a rapid and early depletion of intracellular glutathione after exposure to cDDP or CBDCA. tDDP exhibited the characteristics of a non-specific cytotoxic chemical which was unable to markedly inhibit the biochemical and functional parameters studied at non-cytotoxic concentrations. These results underline the key role of the inhibition of synthetic activities in the pathogenesis of cDDP-induced and CBDCA-induced nephrotoxicity in OK cells.

IODINATED PARTICLES IN THE RAT THYROID

1975 ◽  
Vol 79 (3) ◽  
pp. 459-473 ◽  
Author(s):  
J. Dang ◽  
R. Miquelis ◽  
P. Bastiani ◽  
C. Simon
Keyword(s):  
Acid Phosphatase ◽  
Thyroid Gland ◽  
Phosphatase Activity ◽  
Acid Phosphatase Activity ◽  
High Turnover ◽  
Secondary Lysosomes ◽  
Rat Thyroid ◽  
Tsh Stimulation

ABSTRACT In a previous study (Simon et al. 1971) a procedure for the preparation and separation of iodinated particles was described in the rat. The present paper deals with further investigations on the nature of these particles. Acid phosphatase and iodine are conjointly sedimentable and display a latency that is unmasked on dilution in a hypo-osmotic medium and under acidification to pH 5.0. These properties together with the sensitivity to Triton X-100 are best accounted for by assuming that iodinated particles of the thyroid gland are lysosomes. Part of the particulate iodine is soluble in n-butanol (BEI fraction). The existence of this BEI fraction demonstrates that hydrolysis of thyroglobulin occurs within the particles which thus exhibit an acid protease activity. Both the sedimentable iodine pool and acid phosphatase are increased under TSH stimulation and decreased after thyroxine treatment. In addition, the general activity of the iodinated particles is dependent on the daily iodine intake as shown by the variation of their iodine pool, acid phosphatase activity and BEI fraction with the iodine diet. It is concluded that iodinated particles of the thyroid gland are secondary lysosomes which participate in iodine secretion under TSH control. By in vitro treatment with destabilizing media or after in vivo treatment with thyroxine, iodinated particles exhibit a parallel loss of iodine and acid phosphatase. After a short-term TSH treatment in vivo, their iodine pool is more increased than their acid phosphatase activity. It is concluded that, at least in the normal rat thyroid, iodinated particles are essentially secondary lysosomes; true colloid droplets actually accumulate only after sufficient TSH stimulation. After ultracentrifugation, 3 main subpopulations are separated for which iodine and acid phosphatase patterns are superimposed. In addition, they all exhibit properties characteristic of secondary lysosomes. Finally, the presence of a fourth sedimentable iodinated fraction with a high turnover rate is postulated.

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