scholarly journals Effects of cytochalasin B on membrane-associated microfilaments in a cell-free system.

1981 ◽  
Vol 91 (2) ◽  
pp. 524-530 ◽  
Author(s):  
M J Phillips ◽  
M Oda ◽  
I M Yousef ◽  
K Funatsu
Keyword(s):  
Molecular Weight ◽  
Plasma Membrane ◽  
Rat Liver ◽  
Mode Of Action ◽  
Actin Filaments ◽  
Cytochalasin B ◽  
Bile Canaliculus ◽  
Free System ◽  
A Cell

The mode of action of cytochalasin B was examined in vitro using bile canaliculus-enriched plasma membrane fractions isolated from rat liver. The pericanalicular microfilaments, which are mainly actin filaments and which are normally attached to the canalicular membranes, were dissociated from the membranes by cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin B treatment. A microfilamentous network was found in the supernate of the cytochalasin-treated specimens and a number of polypeptides, of which a polypeptide corresponding in molecular weight to actin was a notable member. These results suggest that actin filaments become detached from the canaliculus membranes by cytochalasin B.

Cell-free translation of mitochondrial nicotinamide nucleotide transhydrogenase

1985 ◽  
Vol 5 (6) ◽  
pp. 483-490 ◽  
Author(s):  
Elisabeth Carlenor ◽  
Vigg Joste ◽  
B. Dean Nelson ◽  
Jan Rydström
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Cytochrome Bc1 Complex ◽  
Free System ◽  
Nicotinamide Nucleotide Transhydrogenase ◽  
Free Translation ◽  
A Cell ◽  
Matrix Fraction

Mammalian nicotinamide nucleotide transhydrogenase is translated as a 5000 daltons larger molecular weight precursor in a cell-free system programmed with rat liver polysomes. The mature rat liver enzyme had the same molecular weight as the purified beef heart enzyme, 115 000 daltons. The precursor was not processed in vitro by liver mitochondria or by a rat liver mitochondrial matrix fraction, nor did it appear to bind to mitochondria. In contrast, pre-FeS protein of the cytochrome bc1 complex was processed in the same samples by both mitochondria and matrix, suggesting an important difference in the processing mechanisms or in the efficiency of processing of the two precursors.

scholarly journals Incorporation of ribonucleic acid in vitro into dense ribonucleoprotein-like materials by isolated rat liver nuclei

1978 ◽  
Vol 174 (2) ◽  
pp. 503-508 ◽  
Author(s):  
I Suzuka
Keyword(s):  
Rat Liver ◽  
Ribonucleic Acid ◽  
Free System ◽  
Isolated Rat Liver ◽  
Cell Free System ◽  
Rat Liver Nuclei ◽  
A Cell ◽  
Liver Nuclei ◽  
Isolated Rat

A cell-free system of isolated rat liver nuclei is described which permits an active incorporation of newly synthesized RNA into ‘dense’ ribonucleoprotein-like materials. The reaction is stimulated with increasing amounts of cytosol protein isolated from rat liver. This indicates that cytosol protein plays an important role in the formation of such material.

scholarly journals Reassembly of Golgi stacks from mitotic Golgi fragments in a cell-free system.

1995 ◽  
Vol 129 (3) ◽  
pp. 605-618 ◽  
Author(s):  
C Rabouille ◽  
T Misteli ◽  
R Watson ◽  
G Warren
Keyword(s):  
Rat Liver ◽  
Biochemical Analysis ◽  
Cytochalasin B ◽  
Free System ◽  
A Cell ◽  
Gamma S ◽  
Total Membrane ◽  
Detailed Picture ◽  
Rat Liver Cytosol ◽  
Electron Lucent

Rat liver Golgi stacks were incubated with mitotic cytosol for 30 min at 37 degrees C to generate mitotic Golgi fragments comprising vesicles, tubules, and cisternal remnants. These were isolated and further incubated with rat liver cytosol for 60 min. The earliest intermediate observed by electron microscopy was a single, curved cisterna with tubular networks fused to the cisternal rims. Elongation of this cisterna was accompanied by stacking and further growth at the cisternal rims. Stacks also fused laterally so that the typical end product was a highly curved stack of 2-3 cisternae mostly enclosing an electron-lucent space. Reassembly occurred in the presence of nocodazole or cytochalasin B but not at 4 degrees C or in the absence of energy supplied in the form of ATP and GTP. Pretreatment of the mitotic fragments and cytosol with N-ethylmaleimide (NEM) also prevented reassembly. GTP gamma S and A1F prevented reassembly when added during fragmentation but not when added to the reassembly mixture. In fact, GTP gamma S stimulated reassembly such that all cisternae were stacked at the end of the incubation and comprised 40% of the total membrane. In contrast, microcystin inhibited stacking so that only single cisternae accumulated. Together these results provide a detailed picture of the reassembly process and open up the study of the architecture of the Golgi apparatus to a combined morphological and biochemical analysis.

scholarly journals Inhibition of the biosynthesis of sterols by phenyl and phenolic compounds in rat liver

1973 ◽  
Vol 134 (3) ◽  
pp. 737-743 ◽  
Author(s):  
S. Ranganathan ◽  
T. Ramasarma
Keyword(s):  
Phenolic Compounds ◽  
Organic Acids ◽  
Aromatic Ring ◽  
Rat Liver ◽  
Carboxyl Group ◽  
Free System ◽  
A Cell ◽  
High Concentrations

The presence of mitochondria increased the incorporation of [2-14C]mevalonate into sterols in a cell-free system from rat liver. Various phenyl and phenolic compounds inhibited the incorporation of mevalonate when added in vitro. p-Hydroxycinnamate, a metabolite of tyrosine, was the most powerful inhibitor among the compounds tested. Catechol, resorcinol and quinol were inhibitory at high concentrations. Organic acids lacking an aromatic ring were not inhibitory. Two hypocholesterolaemic drugs, Clofibrate (α-p-chlorophenoxyisobutyrate) and Clofenapate [α,4-(p-chlorophenyl)phenoxyisobutyrate], which are known to affect some step before the formation of mevalonate in the biosynthesis of cholesterol in vivo, showed inhibition at a step beyond the formation of mevalonate in vitro. The presence of the aromatic ring and the carboxyl group in a molecule appears to be necessary for the inhibition.

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

scholarly journals Trafficking of lipids from the endoplasmic reticulum to the Golgi apparatus in a cell-free system from rat liver

1991 ◽  
Vol 266 (7) ◽  
pp. 4322-4328 ◽  
Author(s):  
P Moreau ◽  
M Rodriguez ◽  
C Cassagne ◽  
D M Morré ◽  
D J Morré
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Free System ◽  
Cell Free System ◽  
A Cell

Brush border myosin-I microinjected into cultured cells is targeted to actin-containing surface structures

1994 ◽  
Vol 107 (6) ◽  
pp. 1623-1631 ◽  
Author(s):  
M. Footer ◽  
A. Bretscher
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Actin Filaments ◽  
Cultured Cells ◽  
Surface Structures ◽  
Specific Expression ◽  
Cultured Fibroblasts ◽  
Myosin I ◽  
Section Electron

The isolated intestinal microvillus cytoskeleton (core) consists of four major proteins: actin, villin, fimbrin and brush border myosin-I. These proteins can assemble in vitro into structures resembling native microvillus cores. Of these components, villin and brush border myosin-I show tissue-specific expression, so they may be involved in the morphogenesis of intestinal microvilli. When introduced into cultured cells that normally lack the protein, villin induces a reorganization of the actin filaments to generate large surface microvilli. Here we examine the consequences of microinjecting brush border myosin-I either alone or together with villin into cultured fibroblasts. Injection of brush border myosin-I has no discernible effect on the overall morphology of the cells, but does become localized to either normal or villin-induced microvilli and other surface structures containing an actin cytoskeleton. Since some endogenous myosin-Is have been found associated with cytoplasmic vesicles, these results show that brush border myosin-I has a domain that specifically targets it to the plasma membrane in both intestinal and cultured cell systems. Ultrastructural examination of microvilli on control cultured cells revealed that they contain a far more highly ordered bundle of microfilaments than had been previously appreciated. The actin filaments in microvilli of villin-injected cells appeared to be more tightly cross-linked when examined by thin-section electron microscopy. In intestinal microvilli, the core bundle is separated from the plasma membrane by about 30 nm due to the presence of brush border myosin-I.(ABSTRACT TRUNCATED AT 250 WORDS)

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