Site of action of substrates requiring cytochrome b5 for oxidation by cytochrome P450.
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Animals↗
Biomedical subjects
J L Vigne
Publications and source records attributed to J L Vigne.
Truncated forms of DNA-binding estrogen receptors in human breast cancer.
The likelihood a breast cancer will respond to antiestrogen therapy depends on the tumor content of immunoreactive or ligand-binding estrogen receptor (ER). To investigate the failure of many ER-positive breast cancers to respond to antiestrogen therapy, we examined by gel-shift assay the ability of tumor ER to bind its cognate estrogen response element (ERE). Analysis of 38 primary breast cancers showed that some tumors containing abundant immunoreactive ER failed to demonstrate DNA binding ER. In many other ER-positive tumors, the fraction of DNA binding ER was low and consisted primarily of truncated receptor forms, which on Western analysis were revealed to be 50 kD homodimers and 67-50 kD ER heterodimers. The use of protease inhibitors during tumor extraction and the demonstration of nuclear-localizing ER and ERE-binding COUP (chicken ovalbumin upstream promoter) protein in these tumors indicated that the truncated forms of ER were likely present in vivo. The presence of intact DNA binding ER correlated with higher tumor content of immunoreactive sex steroid receptors (ER and/or PR), standard predictors of tumor responsiveness to antiestrogen, suggesting that loss or truncation of DNA binding ER may be an important prognostic parameter accounting for some forms of clinical resistance to antiestrogen therapy.
Base Sequence↗
Physical and chemical characteristics of apolipoprotein A-I-lipid complexes produced by Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene.
Chinese hamster ovary cells transfected with the human apolipoprotein A-I gene linked to the human metallothionein gene promoter region secrete large quantities of apolipoprotein A-I (7.1 +/- 0.4% total secreted protein) in the presence of zinc. Approx. 16% of the secreted apolipoprotein A-I is complexed with lipid and can be isolated ultracentrifugally at d less than or equal to 1.21 g/ml. The latter complexes are composed of discs and vesicles as judged by electron microscopy and can be further separated by column chromatography into three fractions: fraction I, mostly vesicles (60-260 nm) and large discs (18-20 nm diameter); fraction II, discs 14.2 +/- 2.6 nm diameter; and fraction III, nonresolvable by electron microscopy. The latter fraction is extremely lipid-poor (94% protein, 6% phospholipid); in contrast, the protein, phospholipid and unesterified cholesterol content for the other fractions are 43, 33 and 24%, respectively, for fraction I and 53, 33 and 14%, respectively, for fraction II. Fraction II particles contain three and four apolipoprotein A-Is per particle as determined by protein crosslinking while large structures in fraction I contain primarily six to seven apolipoprotein A-Is per particle. Following incubation with purified lecithin: cholesterol acyltransferase, discoidal particles were transformed into apparent spherical particles 12.9 +/- 3.4 nm diameter; this transformation coincided with 19-21% conversion of unesterified cholesterol to esterified cholesterol. The apolipoprotein A-I-lipid complexes isolated from Chinese hamster ovary cell media are similar to nascent HDL found in plasma of lecithin:cholesterol acyltransferase-deficient patients and those secreted by the human hepatoma line, Hep G2. The ability of the Chinese hamster ovary cell nascent HDL-like particles to undergo transformation in the presence of purified lecithin:cholesterol acyltransferase indicates that they are functional particles.
Animals↗
Characterization of recombinant human prorenin and renin.
A cell line that secretes substantial quantities of recombinant human prorenin was prepared by transfecting Chinese hamster ovary cells with a gene encoding preprorenin. The prorenin was purified to homogeneity and was found to have a single amino terminus, reflecting cleavage after a typical 23 amino acid signal sequence. The purified inactive prorenin was not a substrate for active renin and was not capable of self-activation. Prorenin could be converted to renin by addition of exogenous protease, and deglycosylation of the prorenin did not alter the sensitivity to protease activation. The enzymatic activity of deglycosylated renin was kinetically identical to that of the native protein. Multimilligram quantities of recombinant human renin and prorenin were purified, providing suitable material for studies directed toward greater understanding of the function of these proteins and for structural studies such as x-ray diffraction for use in design of renin inhibitors.
Animals↗
Expression and characterization of human apolipoprotein A-I in Chinese hamster ovary cells.
We produced human apolipoprotein A-I (apoA-I) in Chinese hamster ovary (CHO) cells. The CHO cells were transfected with an expression plasmid which placed the human apoA-I gene under the direction of the human metallothionein II gene promoter. Isolation of a clonal cell line resulted in high level expression of apoA-I. Greater than 30% of total protein secreted by these CHO cells was apoA-I, which enabled us to purify apoA-I with a single step purification scheme. As a result, large quantities of apoA-I can be produced and isolated without having to rely on plasma sources. Structural characterization of the recombinant apoA-I showed it to be identical to authentic apoA-I from human serum high density lipoprotein. Furthermore, we demonstrated approximately equal to 90% of the apoA-I secreted by CHO cells is processed, mature protein. A portion of the secreted recombinant apoA-I was associated with lipid and floated at a density approximately equal to 1.10 g/ml. Additional analysis identified the presence of five isoforms of apoA-I in the CHO cell conditioned medium. Processing and post-translational modification of the recombinant apoA-I occurred in the CHO cell cultures in the absence of serum components. We conclude that the human apoA-I produced by CHO cells is identical to circulating, mature apoA-I in humans and that recombinant mammalian expression offers an opportunity to investigate apoA-I processing.
Amino Acid Sequence↗
Effect of pectin, wheat bran and cellulose on serum lipids and lipoproteins in rats fed on a low- or high-fat diet.
1. Four groups of adult male Sprague-Dawley rats were fed for 6 weeks on a diet with a low-fat content (50 g/kg) and another four groups were given a diet rich in fat (250 g/kg) and cholesterol (12 g/kg). In both cases, the basal diets were either fibre-depleted or supplemented with cellulose (60 g/kg), wheat bran (100 g/kg) or low-methoxyl pectin (100 g/kg). 2. Low-methoxyl pectin displayed the most hypocholesterolaemic effect and decreased the cholesterol content of the very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL), when the low-fat diet was given. When rats were fed on the high-fat diet, pectin no longer had a hypocholesterolaemic effect but still decreased the VLDL-cholesterol content. Pectin lowered serum triglyceride and VLDL-triglyceride levels only when the low-fat diet was given. 3. Wheat bran exerted no hypocholesterolaemic effect in rats fed on the low- and high-fat diets, but decreased the cholesterol content of VLDL and lowered serum triglycerides and VLDL-triglycerides when the high-fat diet was given. 4. Purified cellulose had no significant effect on plasma lipids. 5. As shown by multivariance analysis, low-methoxyl pectin and wheat bran both beneficially modified the serum triglyceride and cholesterol variables except VLDL-triglycerides. However, the magnitude of the effect of each individual type of fibre was dependent on the fat and cholesterol content of the diet, suggesting the existence of different mechanisms of action.
Animals↗
Effects of dietary fibers and cholestyramine on the activity of pancreatic lipase in vitro.
Most experiments were conducted in the presence of human gallbladder bile; colipase and pancreatic lipase were purified using porcine pancreas. The adsorption of bile salts, phospholipids and cholesterol from the bile, together with that of pancreatic lipase was measured on wheat bran, cellulose, hemicellulose (xylan), slightly methylated pectin (42%) and cholestyramine. In contrast to cholestyramine which intensively binds biliary lipids (61.7-81.7%) and pancreatic lipase (47.5%), the fibers studied only had a low adsorbent power. The direct influence of these fibers and of cholestyramine at concentrations ranging from 0-5% on lipase activity was measured at constant pH, using two conventional assay systems, long chain triglycerides and tributyrin. In the presence of human bile and colipase, a drastic reduction in triglyceride hydrolysis by lipase was observed with cholestyramine (loss of 66-82%) and wheat bran (loss of 77-94%) at 1% concentration. The other fibers did not have any marked effects on enzyme activity. The use of a radio labeled lipase made it possible to demonstrate that the inhibitory effect of bran on enzyme activity was independent of adsorption phenomena on bran. The fraction of bran that can be solubilized in the aqueous phase, in fact, induced this reduction in activity. The presence of protein inhibitor in bran may be responsible for the reduction in pancreatic lipase activity.
Animals↗
Effect of wheat bran, pectin and cellulose on the secretion of bile lipids in rats.
We assayed the lipid content of bile from rats that had been fed either a standard diet (5% fat) or a high fat diet (25% fat, 1.2% cholesterol) in the presence or in the absence of various dietary fibers (namely, wheat bran, pectin and cellulose). The cholesterol concentration in bile from rats fed the high fat diet plus wheat bran or pectin was lower than that of the rats fed the high fat, high cholesterol diet without fiber. Bile phospholipids did not vary significantly from one group to another. In comparison to the standard diet, the high fat, high cholesterol diet led to a greater ratio of primary to secondary bile salts and a higher level of glycoconjugates. The observed differences may be explained by a variation in the metabolism of bile salts brought about by the difference in diet.
Animals↗
Immunohistochemical localization of the apoproteins of the bile lipoprotein complex in the human intestine.
The human bile lipoprotein complex includes an apoprotein fraction of IgA fragments and an acidic polypeptide tightly bound to bile cholesterol and phosphatidylcholines. The fate of the intestinal bile lipoprotein complex was immunohistochemically studied. Its localization in the human duodenum is consistent with selective capture or synthesis. The result of both might be the presence of bile apoprotein in the bloodstream. These two mechanisms may explain the cross-reaction between the lipoprotein complex apoprotein and the high density lipoprotein.
Apolipoproteins↗
Adsorption of pancreatic (pro)phospholipase A2 to various physiological substrates.
The adsorption of pancreatic phospholipase was studied in vitro in the presence of egg yolk lipoprotein emulsion, Intralipid emulsion, and milk fat globules. When the emulsions are incubated with bile salts, the latter dissociate a considerable fraction of the phospholipids initially associated with the emulsions, leading to the coexistence of an emulsified phase and a phase of mixed micelles. After the addition of pancreatic phospholipase A2, gel filtration shows that the enzyme was more than 90% bound to mixed micelles, regardless of the type of emulsion used. Comparable results were obtained by replacing the bile salts with human gallbladder bile. In parallel, pancreatic zymogen was never found to be bound to any of the lipid structures present (emulsion or mixed micelles). When the catalytic site of pancreatic phospholipase A2 was blocked with 4-bromophenacylbromide, there was no fixation on mixed micelles. Fixation was restored by the presence of lysolecithins and fatty acids in the incubation mixtures. The partial transformation of all emulsified substrates to mixed micelles by bile salts in vivo would thus lead to optimum activity of pancreatic phospholipase A2.
Adsorption↗
Comparative immunohistochemical localization of IgA and of the IgA part of the bile lipoprotein complex in dog and rat liver.
In bile, phospholipids and cholesterol were found in close association with an apoprotein fraction which cross-reacted to antiserum anti-IgA. In order to determine the origin of the IgA part of the apo bile lipoprotein complex and to establish its relationship with IgA, th localizations of these two components in rat and in dog livers were compared. Two immunohistochemical methods were used. Whereas IgA was found on the sinusoidal surface of the hepatocytes, in cytoplasmic particles and in bile canaliculi, the IgA part of the apo bile lipoprotein complex was only detected in the bile canaliculi, and in particles which presented the same pattern of location as lysosomes. These results suggest that the IgA part of the apo bile lipoprotein complex could originate from the transit of a part of the internalized IgA via the lysosomal compartment of the hepatocytes.
Animals↗
Properties of lipoproteins in blood plasma and liver perfusates of rats with cholestasis.
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Animals↗
Implications of a dynamic theory on the behavior of the bile salt-lecithin association in water.
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Bile Acids and Salts↗
Interaction of immunoglobulins and lipids in human gallbladder bile.
The lipoprotein complex from human gallbladder bile was challenged with anti-IgA and anti-IgG antisera in order to determine whether the apolipoprotein complex isolated from the detergent-free form of bile lipoprotein contains IgA and IgG. The apolipoprotein complex indeed crossreacts with anti-IgA and anti-IgG. In addition, the interactions of IgA and IgG with lipids were studied by ultracentrifugation and gel chromatography to determine whether these interactions occur in human bile.
Bile↗
Metabolism of apolipoprotein A-I of chylomicrons in rats and humans.
After intravenous injection of a large amount of small chylomicrons into intact rats, the concentration of apolipoprotein (apo) A-I was increased by about 40% and remained at this elevated level as most of the chylomicron triglycerides were removed from plasma during the ensuing hour. This apo A-I rapidly left the chylomicrons and was transferred to lipoproteins of higher density. Such transfer of apo A-I did not occur when chylomicrons were incubated at comparably high concentrations with rat serum. In normolipidemic humans, the concentration of apo A-I and apo A-II, as well as phospholipids, increased in the light subfraction of high density lipoproteins (HDL2) 4 to 7 h after ingestion of a meal containing 1.5 g cream fat per kilogram body weight. The concentration of these components increased in the heavy subfraction of high density lipoproteins (HDL3) after 12 to 24 h. The concentration of apo E in plasma was unaffected by fat ingestion, but the concentration of this protein increased in lipoproteins of density less than 1.006 g . mL-1 and fell in lipoproteins of higher density. It is concluded that apo A-I in rat chylomicrons is transferred quantitatively to HDL as chylomicron remnants are formed. Chylomicron apo A-I and apo A-II appear to be transferred similarly to HDL in humans, whereas apo E is transferred from HDL to chylomicrons after chylomicrons enter the blood.
Adult↗
Pancreatic phospholipase A2 hydrolysis of phosphatidylcholines in various physicochemical states.
Bile salts-phosphatidylcholines-cholesterol mixed micelles, native bile, egg yolk and intralipid emulsions were used as pancreatic phospholipase A2 (EC 3.1.1.4) substrates. The enzyme activity depends on the bile salt/phosphatidylcholine molar ratio. The enzyme had a low specific activity on bile phosphatidylcholines, because of the existence in native bile of a high bile salt/phosphatidylcholine molar ratio generating unfavorable conditions of hydrolysis, as demonstrated with mixed micelles. However when the bile salt/phosphatidylcholine molar ratio from bile was decreased to 2 : 1, enzyme activity increases up to an optimum. This optimal activity was about one third that observed when the substrate was mixed micelles. Under these optimal conditions a simultaneous hydrolysis of intralipid and bile phosphatidylcholine mixture shows comparable initial hydrolysis rates. During an extended incubation, however, nearly all intralipid phosphatidylcholines and only half the bile phosphatidylcholines were hydrolyzed by pancreatic phospholipase A2. Bile salts mixture or native bile desorb a portion of the phosphatidylcholines from the intralipid emulsion in optimally hydrolysable bile salts phosphatidylcholines mixed micelles. These micelles bind about 85% of the enzyme indicating that hydrolysis occurs primarily in the micellar phase. These results are discussed in terms of fat lipolysis in vivo.
Animals↗
Effect of bile lipids on the adsorption and activity of pancreatic lipase on triacylglycerol emulsions.
Emulsions of natural triacylglycerols obtained with different shear forces were used to study lipase adsorption and lipolysis. The influence of the bile lipoprotein complex on these two processes was determined. Optimal lipase activity was observed to occur with a given phospholipid : triacylglycerol ratio. This ratio depended on the degree of triacylglycerol emulsification and was accompanied by maximal adsorption of the bile lipoprotein complex. These results support our previous model for pancreatic lipolysis under physiological conditions, according to which colipase controls lipase binding to the bile lipoprotein complex and the resulting association directs enzyme adsorption to the acylglycerol particle (Lairon, D., Nalbone, G., Lafont, H., Léonardi, J., Domingo, N., Hauton, J.C. and Verger, R. (1978) Biochemistry 17, 5263--5269).
Adsorption↗
The effect of a high cholesterol and saturated fat diet on serum high-density lipoprotein-cholesterol, apoprotein A-I, and apoprotein E levels in normolipidemic humans.
The effects of a high cholesterol, high saturated fat diet on serum high density lipoprotein cholesterol, apo A-I, and apo E levels were studied in six normolipidemic subjects. The study was done on an outpatient basis and mixed natural foods normally consumed by humans were used. When compared with a low cholesterol (98 mg/day) high polyunsaturated fat (P/S ratio 1.6) diet, the high cholesterol (1021 mg/day), high saturated fat (P/S ratio 0.4) diet increased serum cholesterol (23%) by raising the cholesterol concentration in very low-density lipoproteins (59%), low-density lipoproteins (15%), and high-density lipoproteins (30%). The low-density lipoprotein-cholesterol/high-density lipoprotein-cholesterol ratio fell significantly from 1.78 to 1.58. The increased high-density lipoprotein-cholesterol was associated with an elevation of serum apo A-I but not apo E. Serum triglycerides did not change significantly.
Adult↗