Studies on the substrate specificity of the inducible and non-inducible acyl-CoA oxidases from rat kidney peroxisomes

We have studied the substrate specificity of the inducible (acyl-CoA oxidase I) and non-inducible (acyl-CoA oxidase II) oxidases in peroxisome-enriched fractions from rat kidney. The two oxidases were separated by means of ion-exchange chromatography and shown to accept a variety of acyl-CoA esters as substrates, including lignoceroyl-CoA, palmitoyl-CoA, lauroyl-CoA, caproyl-CoA, and trimethyltridecanoyl-CoA. Glutaryl-CoA was found to react exclusively with the inducible enzyme, and pristanoyl-CoA exclusively with the non-inducible enzyme. We conclude that under normal non-induced conditions both acyl-CoA oxidase I and II contribute to the oxidation of the various acyl-CoA esters with the exception of pristanoyl-CoA and glutaryl-CoA, although the extent to which each enzyme contributes to the oxidation was found to differ between the various acyl-CoA esters.     

Evidence for the covalent binding of hydroxyethyl radicals to rat liver microsomal proteins

It is well known that acetaldehyde is capable of covalent binding to liver proteins. However, in experiments using liver microsomes prepared from chronically ethanol-fed rats we have observed that the addition of EDTA-iron complex to the microsomes increases by about 4-5 fold both the spin trapping of hydroxyethyl radicals and the covalent binding of 14C-ethanol to proteins, while it only doubles acetaldehyde formation. Conversely, the presence of GSH strongly decreases the trapping of hydroxyethyl radicals and completely inhibits the covalent binding, without affecting acetaldehyde production. Furthermore, the spin trapping agent 4-pyridyl-N-oxide-t-butyl nitrone (4-POBN), previously employed for the detection of hydroxyethyl radicals, decreases by about 70% the covalent binding of 14C-ethanol to microsomal proteins. 4-POBN does not affect acetaldehyde production by liver microsomes, nor does it interfere with the covalent binding of acetaldehyde produced by ADH-mediated oxidation of ethanol. The results obtained indicate that hydroxyethyl radicals generated during ethanol oxidation by cytochrome P-450 play an important role in the alkylation of microsomal proteins consequent to ethanol metabolism.     

PxF, a prenylated protein of peroxisomes

CAAX farnesyltransferase attaches a farnesyl group to proteins that terminate in the sequence CAAX, where C is cysteine, A is an aliphatic amino acid, and X is typically methionine or serine. A limited number of substrates for the CAAX farnesyltransferase have been identified in cultured cells. These include p21ras proteins and the nuclear lamins A and B. We describe here the use of a CAAX farnesyltransferase inhibitor, together with a hamster cell line that exhibits efficient uptake of [3H]mevalonate, as a means of identifying novel farnesylated proteins. One candidate protein was purified and its attached prenyl group identified as farnesyl. The predicted amino acid sequence of this protein, deduced from a cloned cDNA, terminates with the tetrapeptide Cys-Leu-Ile-Met, which conforms to the consensus sequence for recognition by farnesyltransferase. This farnesylated protein, designated PxF, is localized to the outer surface of peroxisomes as determined by indirect immunofluorescence and electron microscopy.     

Studies on cytosolic 5SrRNA-L5 protein particles (5SRNP) of rat liver

Effects of antiallergic drugs on bradykinin-induced histamine release and intracellular Ca2+ release from peritoneal mast cells were studied in rats. Bradykinin caused a concentration-dependent histamine release as well as Ca2+ release from the intracellular Ca store of peritoneal mast cells. Antiallergic drugs used in this study showed an inhibition of not only histamine release but also Ca2+ release. The Ca2+ release from the intracellular Ca store induced by bradykinin was more sensitive to antiallergic drugs than histamine release from mast cells. Mequitazine and terfenadine caused potent inhibitory effects on both responses, whereas effects of ketotifen and cromolyn sodium were relatively weak. In conclusion, histamine release from mast cells and intracellular C2+ release induced by bradykinin were inhibited by antiallergic drugs similar to those induced by substance P and compound 48/80.     

Inhibition of haem synthesis caused by cobalt in rat liver. Evidence for two different sites of action

Cobalt inhibits liver haem synthesis in vivo by acting at least two different sites in the biosynthetic pathway: (1) synthesis of 5-aminolaevulinate and (2) conversion of 5-amino-laevulinate into haem. The first effect is largely, if not entirely, due to inhibition of the activity of 5-aminolaevulinate synthase, rather than to inhibition of the formation of the enzyme. The second effect results from diversion of 5-aminolaevulinate into an unidentified liver pool with solubility properties similar to those of cobalt protoporphyrin.     

The relevance framework for category-based induction: Evidence from garden-path arguments.

Relevance theory (Sperber & Wilson, 1995) suggests that people expend cognitive effort when processing information in proportion to the cognitive effects to be gained from doing so. This theory has been used to explain how people apply their knowledge appropriately when evaluating category-based inductive arguments (Medin, Coley, Storms, & Hayes, 2003). In such arguments, people are told that a property is true of premise categories and are asked to evaluate the likelihood that it is also true of conclusion categories. According to the relevance framework, reasoners generate hypotheses about the relevant relation between the categories in the argument. We reasoned that premises inconsistent with early hypotheses about the relevant relation would have greater effects than consistent premises. We designed three premise garden-path arguments where the same 3rd premise was either consistent or inconsistent with likely hypotheses about the relevant relation. In Experiments 1 and 2, we showed that effort expended processing consistent premises (measured via reading times) was significantly less than effort expended on inconsistent premises. In Experiment 2 and 3, we demonstrated a direct relation between cognitive effect and cognitive effort. For garden-path arguments, belief change given inconsistent 3rd premises was significantly correlated with Premise 3 (Experiment 3) and conclusion (Experiments 2 and 3) reading times. For consistent arguments, the correlation between belief change and reading times did not approach significance. These results support the relevance framework for induction but are difficult to accommodate under other approaches. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Studies of nicotinic acetylcholine receptor protein from rat brain. II. Partial purification

The pharmacological specificity of the binding of 125I-labeled alpha-bungarotoxin to a 1% Emulphogene BC-720 extract of a rat brain particulate fraction has been investigated. The extract contains a component which possesses the binding characteristics of a nicotinic acetylcholine receptor protein. The crude soluble acetylcholine receptor protein was purified by affinity chromatography utilizing the alpha-neurotoxin of Naja naja siamensis as ligand and 1.0 M carbamylcholine chloride as eluant. A single, batch-wise, affinity chromatography procedure yields an average purification of 510-fold. When this purified material is treated a second time by affinity chromatography, purification as high as 12600-fold has been obtained. Binding of 125I-labeled alpha-bungarotoxin to this purified acetylcholine receptor protein is saturable with a Kd of 1 - 10(-8) M. Nicotine and acetylcholine iodide at concentrations of 10(-5) M inhibit 125I-labeled toxin-acetylcholine receptor protein complex formation by 41 and 61% respectively. At 10(-4) M, carbamylcholine chloride and (+)-tubocurarine chloride give respectively 52 and 82% inhibition. Eserine sulfate and atropine sulfate have no effect on complex formation at a concentration of 10(-4) M. These data support the isolation of a partially purified nicotinic acetylcholine receptor protein.     

Purification of rat liver nuclear protein kinase NII

Rat liver nuclear protein kinase activity (NII), which is eluted from DEAE-Sephadex columns, has been purified approximately 1500-fold from solubilized nuclear protein. The method of purification involved chromatography of protein eluted from DEAE-Sephadex successively on phosvitin-Sepharose, mixed histone-Sepharose, and histone H2b-Sepharose followed by gel filtration on Sephadex G-200. Resulting preparations are homogeneous by polyacrylamide gel electrophoresis. The enzyme consists of three polypeptides with molecular weights of 42,000 (alpha), 39,000 (alpha'), and 26,000 (beta) which are present in the ratio 1:1:2 indicating that the enzyme has a minimum tetrameric subunit composition of alphaalpha'beta2. The molecular weight and s20,w of the purified enzyme were 123,000 and 7.0, respectively, as determined by sucrose density gradient centrifugation in 0.4 M NaCl. The enzyme has maximal activity with phosvitin as substrate and is not stimulated by 10(-5) to 10(-4) M cAMP or cGMP using H2b as substrate.     

Evidence of hepatocyte apoptosis in rat liver after the administration of carbon tetrachloride

In acute liver injury induced by the injection of CCl4, cell death has been attributed to the necrosis of hepatocytes in the centrilobular area. In the present study, we re-examined the hepatic injury evoked by CCl4 in rats and explored the possibility that apoptosis may also contribute to its pathogenesis. Apoptotic hepatocytes were identified and quantified by light and electron microscopy, the in situ immunohistochemical labeling of nuclear DNA fragmentation, flow cytometry, and DNA gel electrophoresis. We found that a substantial number of hepatocytes underwent apoptosis. Apoptotic changes were also observed in ballooned hepatocytes. Apoptotic hepatocytes increased in number at 3 hours and peaked at 6 hours after the CCl4 injection. Apoptotic bodies were sequestrated in the adjacent hepatocytes and sinusoidal cells. Double staining of the cells with immunostaining for phagocytes and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining for labeling of DNA fragmentation showed that the majority of apoptotic hepatocytes were phagocytosed by Kupffer cells and macrophages. The results indicated that apoptosis occurs in the ballooned and injured hepatocytes of the centrilobular area. What occurs after CCl4 administration may be important in reducing inflammation, shortening the course of acute hepatic injury, and preventing the development of fibrosis.     

Isolation and characterization of rat liver amphisomes. Evidence for fusion of autophagosomes with both early and late endosomes

Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms. To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation). Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to autophagosomes). Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion. The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes.     

Uptake of hydroxocobalamin by rat liver mitochondria. Binding to a mitochondrial protein

Lysosome-free preparations of rat liver mitochondria take up hydroxo[57Co]cobalamin by a process which is dependent on mitochondrial swelling, rather than on energy or ion fluxes. The uptake system is saturable and unidirectional, leading to inside/outside concentration ratios of 17. The process also exhibits specificity: cyano[57Co]cobalamin is taken up less rapidly and to a lesser extent than hydroxocobalamin; methylcobalamin and adenoslcobalamin inhibit hydroxocobalamin uptake markedly, while cyanocobalamin does not. The [57Co]cobalamin ([57Co]Cbl) taken up is bound to a mitochondrial protein whose apparent molecular weight is 120,000 by Sephadex G-150 chromatography. Double reciprocal plots of bound [57Co]Cbl versus medium [57Co]Cbl concentration yield estimates for bound Cblmax of 29 pmol/mg of protein and for Kd is 8.2 muM. We conclude that mitochondrial uptake of cobalamins occurs via the diffusion of free cobalamins into the mitochondria and their subsequent binding to a high affinity mitochondrial protein(s) which we propose to be the source of the unidirectional character, the saturability, and the specificity of the uptake system.     

Chemical modification of rat liver cytosolic NADP(+)-linked isocitrate dehydrogenase by N-ethylmaleimide. Evidence for essential sulphydryl groups

It is well known that diabetic patients are at an increased risk of the earlier development of significant atherosclerotic disease. We wish to report the case of a young insulin-dependent diabetic with an 18 months history of rapidly progressing 'diabetic' retinopathy who presented with right-sided weakness and was found to have severe carotid artery disease. We suggest that the sudden proliferation in his retinopathy was due to retinal ischaemia secondary to the carotid artery stenosis and we wish to present this as a case of ocular ischaemic syndrome in a young diabetic patient.     

Tissue distribution of liver regulating protein. Evidence for a cell recognition signal common to liver, pancreas, gonads, and hemopoietic tissues

Liver regulating protein (LRP) is an integral plasma membrane protein that plays a critical role in maintaining the differentiated phenotype of adult rat hepatocytes by mediating cell-cell interactions with rat liver epithelial cells. Using a specific monoclonal antibody (MAb L8) capable of inhibiting the interactions between these two cell types, the cellular distribution of LRP was analyzed in the liver. Various cell types, including hepatocytes and several sinusoidal cells, were found to be positive, whereas vascular endothelial cells and bile duct cells were consistently negative. This observation led us to question whether cells of nonhepatic origin would also express LRP. We show that MAb L8 immunoreactive material was detected in only three groups of tissues and corresponded to molecules similar to LRP but with different molecular weights. LRP-like molecules were demonstrated on acinar cells of the exocrine pancreas and on all hemopoietic cells regardless of their localization in the organism. LRP-like molecules were also expressed by germ cells and surrounding feeder cells in the testis and ovary in a stage-dependent manner. These results demonstrate the existence of a family of LRP proteins and strongly suggest a critical role for these molecules in regulating cell-cell communication in specific tissues.     

Studies of nicotinic acetylcholine receptor protein from rat brain

Specific binding of 125I-labeled alpha-bungarotoxin to a 34800 X g pellet of a whole rat brain homogenate has been obtained at levels of 2 pmol toxin per g of whole brain with a Kd of 8-10(-9) M. Binding is reduced 90% by 10(-5) M (+)-tubocurarine chloride and 10(-4) M nicotine, whereas concentrations of 10(-4) M choline chloride, atropine sulfate and eserine sulfate have essentially no effect on toxin binding. These results compare closely with those obtained from binding studies with 125I-labeled alpha-bungarotoxin and soluble acetylcholine receptor protein preparations from Torpedo nobiliana; suggesting that this mammalian receptor protein is nicotinic in character. Extraction of the 34800 X g pellet with 1% Emulphogene yields a soluble fraction with specifically binds 125I-labeled alpha-bungarotoxin with a Kd of 5-10(-9) M. Nicotine and alpha-bungarotoxin at concentrations of 10(-5) M abolish toxin-receptor complex formation and carbachol and (+)-tubocurarine chloride reduce complex formation 35-40% at similar concentrations. Eserine sulfate, atropine sulfate, decamethonium, and pilocarpine had no effect on complex formation at concentrations of 10(-5) M.