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At least 217 records · Page 12Linked to original sources
PubMed1980

Mutagenic activation of N-2-fluorenylacetamide and N-hydroxy-N-2-fluorenylacetamide in subcellular fractions from X/Gf mice.

The Salmonella mutagenesis test system was used to evaluate the in vitro mutagenic potency of N-2-fluorenylacetamide (2-FAA) and N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) mediated by liver and kidney subcellular fractions from X/Gf mice, a strain resistant to 2-FAA carcinogenesis. Pretreatment of the mice with the microsomal inducers 3-methylcholanthrene (MCA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased the number of revertants from both liver and kidney fractions. Mutagenicity of N-OH-2-FAA mediated by liver or kidney microsomes was partially inhibited at 0.001--0.1 microM Paraxon (diethyl-p-nitrophenyl phosphate), an inhibitor of deacetylase enzyme, and the inhibition was complete (98%) in microsomes from control mice (100 microM Paraoxon). Conversely, the liver and kidney microsomal fractions from MCA- and TCDD-treated X/Gf mice were less sensitive to Paraoxon. The inhibition of kidney or liver cytosol-mediated N-OH-2-FAA mutagenicity by Paraoxon was less than that observed with the microsomal fraction (50% inhibition at 1 x 10(-7) and 1 x 10(-5) M Paraoxon, respectively). The mutagenicity of 2-FAA and N-OH-2-FAA mediated by liver or kidney subcellular fractions from X/Gf mice and its response to inducers and inhibitors of mutagenic activation processes appear similar to those observed in species both resistant (cotton rat) and sensitive (Sprague-Dawley rat, NIH Swiss mice) to 2-FAA carcinogenesis.

2-Acetylaminofluorene↗
PubMed1984

PO protein and 2',3'-cyclic-nucleotide 3'-phosphodiesterase activity in the peripheral nerve and subcellular fractions of the Trembler mouse.

To investigate the biochemical abnormalities of the Trembler mouse, the level of the PO protein (as % of total protein) and the activity of CNP was compared in the sciatic nerve and subcellular fractions of normal and mutant littermates. There was a significant decrease in both of these myelin markers in total nerve homgenates of the neurological mutant compared with the control animals. Immunoassay of the PO protein and polyacrylamide gel analysis of proteins indicated an accumulation of a protein with an apparent molecular weight of 67K in mutant nerve extracts. The mutant nerve also had relatively decreased levels of a protein of molecular weight about 41K that cross-reacted with antibody to PO protein. The Trembler mouse exhibited a larger percentage recovery of PO protein and CNP activity in subcellular fractions denser than the myelin sheath. Together these results are consistent with the theories that these denser components represent immature forms of myelin and that the Trembler mutant is characterized by hypomyelination.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1977

Studies on proinsulin and problucagon biosynthesis and conversion at the subcellular level. II. Distribution of radioactive peptide hormones and hormone precursors in subcellular fractions after pulse and pulse-chase incubation of islet tissue.

Anglerfish proinsulin and insulin were selectively labeled with [(14)C]isoleucine, while proglucagon, conversion intermediate(s), and glucagon were selectively labeled with[(3)H]tryptophan. After various periods of continuous or pulse-chase incubation, islet tissue was subjected to subcellular fractionation. Fraction extracts were analyzed by gel filtration for their content of precursor, conversion intermediate(s), and product peptides. Of the seven subcellular fractions prepared after each incubation, only the microsome and secretory granule fractions yielded significant amounts of labeled insulin-related and glucagon-related peptides. After short-pulse incubations, levels of both [(14)C]proinsulin and [(3)H]proglucagon (mol wt approximately 12,000) were highest in the microsome fraction. This fraction is therefore identified as the site of synthesis. With increasing duration of continuous incubation or during chase incubation in the absence of isotopes, proinsulin, proglucagon, and conversion intermediate(s) are transported to secretory granules. Conversion of proinsulin to insulin and proglucagon to a approximately 4,900 mol wt conversion intermediate and 3,500 mol wt glucagon occurs in the secretory granules. Converting activity also was observed in the microsome fraction. The recovery of most of the incorporated radioactivity in microsome and secretory granule fractions indicates that the newly synthesized islet peptides are relegated to a membrane-bound state soon after synthesis at the RER is completed. This finding supports the concept of intracisternal sequestration and intragranular maintenance of peptides synthesized for export from the cell of origin.

Animals↗
PubMed1987

Nitroxides as potential contrast enhancing agents for MRI application: influence of structure on the rate of reduction by rat hepatocytes, whole liver homogenate, subcellular fractions, and ascorbate.

With an eye toward the development of nitroxides as potential contrast enhancing agents for MRI applications, we have compared the rates of reduction of 24 nitroxides of diverse structures by rat whole liver homogenate, hepatocytes, subcellular fractions, and ascorbate (10 eq excess). Our results indicate that five-membered ring nitroxides and alpha-carboxy alpha-aryl tert-butyl nitroxides are significantly more resistant toward bioreduction than six-membered ring and heterocyclic-substituted nitroxides in all systems. In the case of six-membered ring nitroxides the presence of either negatively charged or positively charged groups increases the susceptibility toward ascorbate reduction. The presence of carboxylate groups tends to enhance the resistance of all the nitroxides toward reduction by liver homogenate, hepatocytes, and subcellular fractions. trans-Azethoxyl nitroxides exhibited the best overall resistance toward bioreduction. The T1 relaxants of selected nitroxides were measured and found to be similar.

Animals↗
PubMed1987

Mutagenic activation of 2-amino-3-methylimidazo[4,5-f]-quinoline (IQ) and 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline (MeIQ) by subcellular fractions and cells isolated from small intestine, kidney and liver of the rat.

The mutagenic activity of the pyrolysis products 2-amino-3-methyl-imidazo[4,5-f]-quinoline 2-amino-3,4-dimethylimidazo[4,5-f]-quinoline in Salmonella typhimurium TA98 using rat intestinal and renal subcellular fractions as activation systems was approximately 1 and 5 revertants per nmol, respectively. This was 1,000 times less than the activity with a subcellular fraction from rat liver. The mutagenic activity of both compounds was considerably increased using intestinal, renal and hepatic preparations isolated from PCB (Aroclor 1254)-pretreated rats, compared to preparations from control animals. In addition, both compounds displayed a moderate direct-acting mutagenic activity at concentrations above 10(-5) M. Isolated cells from small intestine, kidney and liver incubated in nucleopore chambers were able to convert both compounds into products which mutated bacteria outside the chambers. The concentrations of chemicals required to yield responses of a similar magnitude were approximately 3 orders of magnitude higher in the intestinal and renal systems compared to the hepatic system. The formation of metabolites mutagenic for Salmonella typhimurium by hepatic subcellular and cellular systems was shown to be superior to the respective intestinal and renal systems.

Animals↗
PubMed1988

A Ca2+-stimulated, Mg2+-dependent ATPase activity in subcellular fractions from Schistosoma mansoni.

A Ca2+-stimulated, Mg2+-dependent ATPase activity was found in subcellular fractions from Schistosoma mansoni. Its specific and relative activities were higher in the heterogeneous cuticle fraction and in the microsomal fraction. The K0.5 for ATPase activation by free Ca2+ was 0.2-0.5 microM. This is the first description of an ATPase activity stimulated by Ca2+ in the micromolar range in S. mansoni.

Animals↗
PubMed1983

Activation of free fatty acids in subcellular fractions of human skeletal muscle.

In human pathology little is known about the activating enzymes for fatty acids of different carbon chain length. In order to have a better insight into disorders of lipid metabolism in human skeletal muscle, we studied the distribution of acyl-CoA synthetases in muscular subcellular fractions. We find that in muscle mainly long chain fatty acids are activated to CoA esters. Distribution of palmityl-CoA synthetase in subcellular fractions compared with marker enzymes suggested that this enzymatic activity is located only in the outer mitochondrial membrane, in contrast to human liver, where this enzyme is also located in the microsomes. In human skeletal muscle we also found low butyryl-CoA formation, which was limited to the mitochondrial matrix. This site of activation implies that short chain fatty acids may not depend on carnitine for their oxidation in the mitochondrial matrix, in contrast to long chain fatty acids activated in the outer mitochondrial membrane.

Coenzyme A Ligases↗
PubMed1993

Topography of apolipoprotein B in subcellular fractions of rabbit liver probed with a panel of monoclonal antibodies.

We have used a panel of anti-rabbit apolipoprotein B monoclonal antibodies in a competitive ELISA to probe the availability of apoB in rough endoplasmic reticulum, smooth endoplasmic reticulum, cis-enriched Golgi, and trans-enriched Golgi fractions from rabbit liver. The ability of each subcellular fraction to inhibit binding of monoclonal antibody to immobilized low density lipoprotein (LDL)-apoB was determined and compared with the expected inhibition based on the apoB content of the fraction. The vesicles remained closed during ELISA, demonstrated by monitoring loss of radiolabeled secretory proteins from the lumen and by measuring leakage of albumin from the vesicles. In control experiments, vesicles were permeabilized using 0.4% taurocholate. All epitopes of apoB were fully expressed in closed trans-Golgi vesicles, indicating that the membrane-bound apoB is at the cytosolic side of this fraction. In the smooth endoplasmic reticulum the epitopes were expressed between 55 and 70%, suggesting that the two pools of apoB may exist in these membranes. These results suggest that newly synthesized apoB has two possible fates. It may be incorporated into the cytosolic side of the endoplasmic reticulum from where it moves to the cytosolic side of the Golgi membrane, or newly synthesized apoB may be translocated to the lumenal surface of the endoplasmic reticulum membrane followed by assembly with lipids for secretion.

Animals↗
PubMed1990

Lung subcellular fractions and surfactant lipid metabolism of rats exposed with DDT or endosulfan intratracheally.

The effect of intratracheally administered DDT (5 mg/100 g body weight) or endosulfan (1 mg/100 g body weight) for three consecutive days has been studied on lipid metabolism of rat lung subcellular fractions. Both the insecticides did not affect the lung weight and the protein contents of microsomes, lamellar bodies and surfactant but significantly increased the phospholipid contents of microsomal and surfactant system. Most of the neutral lipid components of lung subcellular fractions were also increased by DDT or endosulfan treatments, except that of surfactant triglycerides which were decreased by DDT treatment. DDT or endosulfan both increased the incorporation of radioactive [methyl-3H]choline into microsomal phosphatidylcholine (PC) and surfactant dipalmitoylphosphatidylcholine (DPPC) without affecting the incorporation of radioactive [methyl-14C]methionine, showing the increased synthesis of PC via CDPcholine pathway. The results presented in this communication showed that DDT and endosulfan, the two different chloroinsecticides have similar effects on microsomal lipid metabolism but produce different biochemical manifestations on the secretion of surfactant phospholipids.

Animals↗
PubMed1996

Subcellular localization of the Huntington's disease gene product in cell lines by immunofluorescence and biochemical subcellular fractionation.

Huntington's disease is a progressive neurodegenerative disorder, which is caused by expansion of a polymorphic (CAG)n repeat in the coding region of the Huntington's disease gene. The function of huntingtin has not been elucidated so far. Accordingly, detailed subcellular localization studies remain useful. In an immunohistochemical study, we have reported huntingtin to be present in the cytoplasm of cells in the majority of the tissues studied. In addition, we detected a signal in the nucleus of cells in some tissues, including neuronal cells. We have further extended these studies in various mammalian cell lines, using a panel of (affinity-purified) polyclonal huntingtin antibodies in immunofluorescence, confocal laser scanning microscopy and biochemical subcellular fractionation studies. In mouse embryonic fibroblasts, human skin fibroblasts and in mouse neuroblastoma cells huntingtin was present in the cytoplasm. All five antibodies, directed against different parts of huntingtin, also showed a signal in the nucleus. This signal could be competed by the original antigen. The localization of huntingtin in both cytoplasm and nucleus, was confirmed by biochemical subcellular fractionation studies. However, in most other studies, a nuclear location for huntingtin has not been found. Our results suggest, however, that besides its function(s) in the cytoplasm, a nuclear function of huntingtin at some stages of differentiation or in some phases of the cell cycle may not be excluded.

Animals↗
PubMed1981

Properties of LH/hCG receptors in porcine corpus luteum homogenates and subcellular fractions, and factors influencing the recovery of membrane-bound hormone.

An evaluation of several techniques commonly used to separate bound from free hormone was made for the binding of radiolabelled human chorionic gonadotropin to mid-luteal pig corpus luteum homogenates and subcellular fractions. Compared to millipore filtration with 0.22 or 0.45-mu HAWP filters, centrifugation of homogenates at 2 500 x gav or 1 000 x gav for 15 min recovered only 50 and 33% respectively of the total bound hormone present. This discrepancy was accentuated further when hormone binding to post-nuclear subcellular fractions was studied. Centrifugation of a nuclear fraction at 2 500 x gav for 15 min recovered 70% of the membrane-bound receptors present (as measured by filtration), whereas centrifugation of mitochondrial, lysosomal and microsomal fractions under these conditions recovered only 46, 16 and 4% respectively. Divalent metal ions influenced hormone binding in 2 ways: (i) Specific bonding of hCG was greatest in the absence of metal ions and in the presence of low levels of chelating agents. Increasing concentrations of magnesium and calcium ions appeared to interfere with the interaction of hormone and receptor. (ii) Divalent metal ions reduced the recovery of particle-bound hormone by centrifugation, at both high and low speeds. The results illustrate the pitfalls of using low-speed centrifugation to recover membrane-bound hormone-receptor complexes when small membrane vesicles are present.

Animals↗
PubMedDate not supplied

[Activity of DNAase I inhibitor in subcellular fractions of rat tissues].

The inhibitory activity in the mitochondrial and postmitochondrial fractions of the liver, spleen and thymus of albino rats was studied with respect to pancreatic DNase I. It is shown to be due to the presence of thermolabile substances of protein nature (natural inhibitors of DNase I) in the subcellular fractions of the organs under study. A higher inhibitory activity is detected in the postmitochondrial fraction of all the tissues under investigation. The lowest inhibitory activity is found in the mitochondrial and postmitochondrial fractions of the liver. The data of a simultaneous study of changes in the activity of tissue DNase and inhibitory activity in the subcellular fractions give reasons to suppose the presence of the nuclease-inhibitory complex in the tissues.

Animals↗
PubMed1985

Antigenic and immunogenic characteristics of subcellular fractions and whole cells of a rough E. coli 0111 (J5) mutant.

Little information is available on the antigenic and immunogenic properties of an E. coli rough mutant: J5 derived from E. coli 0111:B4, and the relationship of J5 to other cross-reacting antigens of Enterobacteriaceae. Subcellular fractions of J5 and various antigen preparations were tested against antisera to Enterobacterial Common Antigen (ECA-Kunin) and to the Re mutant of S. minnesota (R595). ECA-Kunin was demonstrated in all subcellular fractions of the J5 mutant by means of indirect hemagglutination and by hemagglutination inhibition tests. This common antigen was separable from J5 LPS by ethanol fractionation and by phenol-chloroform-petroleum ether extraction. Treatment with alkali destroyed the hemagglutinating reactivity of ECA-Kunin and revealed the complex nature of J5 surface antigens; an alkalinized 20p30 fraction (containing cell wall components) contained both specific J5 antigen and an antigen which cross-reacted with S. minnesota and S. typhimurium. This antigen was shown by absorption studies to be a new common antigen other than Re LPS or ECA-Kunin. Studies of J5 LPS by ELISA demonstrated that there was a shared cross-reactive immunodeterminant between the glycolipids of J5 and Re. Accordingly, heat-killed J5 preparations were complex vaccines which were able to elicit an antibody response with at least two specificities: ECA-Kunin and the specific J5 antigen.

Antibodies, Bacterial↗
PubMed1988

Protein synthesis rates in rat brain regions and subcellular fractions during aging.

In vivo protein synthesis rates in various brain regions (cerebral cortex, cerebellum, hippocampus, hypothalamus, and striatum) of 4-, 12-, and 24-month-old rats were examined after injection of a flooding dose of labeled valine. The incorporation of labeled valine into proteins of mitochondrial, microsomal, and cytosolic fractions from cerebral cortex and cerebellum was also measured. At all ages examined, the incorporation rate was 0.5% per hour in cerebral cortex, cerebellum, hippocampus, and hypothalamus and 0.4% per hour in striatum. Of the subcellular fractions examined, the microsomal proteins were synthesized at the highest rate, followed by cytosolic and mitochondrial proteins. The results obtained indicate that the average synthesis rate of proteins in the various brain regions and subcellular fractions examined is fairly constant and is not significantly altered in the 4 to 24-month period of life of rats.

Aging↗
PubMed1995

Effects of metformin treatment on glucose transporter proteins in subcellular fractions of skeletal muscle in (fa/fa) Zucker rats.

1. The present study was designed to clarify the cellular mechanism through which the antihyperglycaemic drug, metformin, exerts its effects. For this purpose the contents of glucose transporter protein isoforms GLUT1 and GLUT4 were measured in plasma membrane and intracellular membrane fractions of skeletal muscle obtained from genetically obese, insulin-resistant Zucker rats. 2. Hindlimb muscles were dissected from metformin-treated (300 mg kg-1 day-1, p.o., for 12 days) and control rats in basal treatment state, and after acute stimulation with insulin (22 u kg-1, i.p.). Since metformin treatment reduces food intake, we also used a pair-fed control group to investigate the effects of altered insulinaemia per se. Glucose transporter levels were analysed by Western blot and slot blot-techniques. In addition, 2-deoxy-[14C]-glucose uptake in isolated muscle strips was evaluated. 3. No changes were noted in the contents of GLUT1 proteins in any of the subcellular fractions after metformin treatment. The contents of GLUT4 in subcellular fractions were not altered in the basal treatment state. After acute insulin exposure the content of GLUT4 in the intracellular membrane fraction declined significantly in the metformin-treated group, while no significant effect was seen in the plasma membrane fraction. In agreement with these results, metformin treatment did not alter 2-deoxyglucose uptake into isolated muscle strips. 4. In conclusion, the present study does not support the concept that metformin would enhance translocation of glucose transporter proteins from the intracellular compartment to the plasma membrane in skeletal muscle in vivo.

Animals↗
PubMed1977

Studies on the effects of heterologous antisera against subcellular thymocyte fraction.

Subcellular fractions of mice thymocytes were used for sensitization of rabbits. The antisera were examined for their immunosuppressive potency in vivo by allogeneic murine tumor metastases system and on skingraft survival and in vitro by leukocyte agglutination tests. The results indicated that the most potent immunosuppressive antisera was that against the second fraction (Fr. 2) of the detergent soluble endoplasmic reticulum fraction from thymocytes.

Agglutination Tests↗
PubMed1975

Renal subcellular fractions producing angionecrosis and increased vascular permeability.

In order to define the subcellular localization and characters of substances in the rat kidney which increase vascular permeability and produce angionecrosis, the following investigations have been undertaken: (1) subcellular fractionation by use of differential centrifugation and osmotic shock treatment with enzyme profile determination; (2) chromatographic separation of lysosomal contents with concanavalin A affinity column. Lysomal contents contained substances that induced an increase of vascular permeability of the rabbit skin and angionecrosis in the pancreas of the bilaterally nephrectomized rats and the spontaneously hypertensive rats. Lysosomal contents treated at 60 degrees for 30 min showed no renin activity and yet produced angionecrosis. Non-affinity fraction from concanavalin A column chromatography showed no renin activity but produced angionecrosis and an increase of vascular permeability of rabbit skin.

Acid Phosphatase↗
PubMed1986

Subcellular fractionation and distribution of cholinergic binding sites in fetal human brain.

Conventional subcellular fractionation techniques have been applied to human fetal brain (13-15 weeks gestation) and the fractions have been characterized by assaying for marker enzymes, cholinergic binding sites and electron microscopy. Fractionation of the homogenate resulted in a nuclear pellet (P1), a crude mitochondrial pellet (P2) and a supernatant (S2). Further resolution of the P2 fraction by density gradient centrifugation resulted in two bands at the gradient interfaces and a pellet. The P2 and subsequently the P2B fraction contained intact plasma membrane profiles as judged by the predominance of adenylate cyclase activity and the presence of occluded lactate dehydrogenase which constituted over 70% of the total activity in these fractions. Morphological examination of the gradient fractions revealed that the P2B fraction contains membrane bound structures which resemble synaptosomes prepared from neonatal rat brain. These structures have a granular matrix in which mitochondria and frequently, neurofilaments were observed. Very few synaptic vesicles were present and there was no evidence for post synaptic attachments. The cholinergic markers choline acetyltransferase, acetylcholinesterase and receptor sites defined by quinuclidinyl benzilate and alpha-bungarotoxin binding were enriched in fractions P2 and P2B which contained the bulk of nerve ending particles. This enriched preparation of fetal synaptosomes may be valuable for functional studies on pre-synaptic terminals in developing brain.

Brain↗