A new relationship between cholesterolemia and cholesterol synthesis determined in rats fed an excess of cystine

The present study deals with an attempt to describe how the plasma cholesterol level is related to input into the plasma of cholesterol synthesized in the liver and in the intestine. It has previously been shown in our laboratory that, for a given absorption of alimentary cholesterol, the rat plasma cholesterol level decreases when internal secretion of cholesterol (cholesterol synthesized in the organs and poured into the plasma) increases. This relationship was established using rats in which the major source of cholesterol synthesis was the intestine. We used rats fed a cystine-enriched diet (5%) which was previously shown to increase cholesterolemia and internal secretion of cholesterol. It was first demonstrated that a significant positive linear correlation exists between individual values of cholesterolemia and those of internal secretion of cholesterol. Secondly, using [14C]acetate as the cholesterol precursor it was shown that ingestion of the cystine-enriched diet increased hepatic but not intestinal cholesterogenesis. Individual values of cholesterolemia were linearly correlated to those of [14C]acetate incorporation into the hepatic sterols. Results obtained by this method were validated by determining the 13C-labeling pattern of cholesterol synthesized de novo by the liver and the intestine after [13C]acetate infusion. Indeed, this labelling indicated that the dilution of exogenous acetyl-CoA in the liver was not changed by cystine feeding, whereas that in the intestine was enhanced. It is concluded that the plasma cholesterol level varies with internal cholesterol secretion, depending on the organ which determines the variations of this secretion: it decreases when intestinal cholesterogenesis increases, whereas it increases when hepatic cholesterogenesis increases. Finally, the use of [14C]acetate coupled with lipoprotein analysis in rats fed the cystine-enriched diet, in control rats and in rats fed a cholesterol-enriched diet, allowed a new linear correlation to be demonstrated: between cholesterol concentration in LDL2 (lipoproteins of density 1.040-1.063 g/ml) and [14C]acetate incorporation into liver sterols. Our results suggest that LDL2 are produced by the liver in relation to cholesterogenesis in this organ.     

Availability to monoclonal antibodies of antigenic sites of the alpha and beta subunits in active, denatured or membrane-bound mitochondrial F1-ATPase

The binding of five monoclonal antibodies to mitochondrial F1-ATPase has been studied. Competition experiments between monoclonal antibodies demonstrate that these antibodies recognize four different antigenic sites and provide information on the proximity of these sites. The accessibility of the epitopes has been compared for F1 integrated in the mitochondrial membrane, for purified beta-subunit and for purified F1 maintained in its active form by the presence of nucleotides or inactivated either by dilution in the absence of ATP or by urea treatment. The three anti-beta monoclonal antibodies bound more easily to the beta-subunit than to active F1, and recognized equally active F1 and F1 integrated in the membrane, indicating that their antigenic sites are partly buried similarly in purified or membrane-bound F1 and better exposed in the isolated beta-subunit. In addition, unfolding F1 by urea strongly increased the binding of one anti-beta monoclonal antibody (14 D5) indicating that this domain is at least partly shielded inside the beta-subunit. One anti-alpha monoclonal antibody (20 D6) bound poorly to F1 integrated in the membrane, while the other (7 B3) had a higher affinity for F1 integrated in the membrane than for soluble F1. Therefore, 20 D6 recognizes an epitope of the alpha-subunit buried inside F1 integrated in the membrane, while 7 B3 binds to a domain of the alpha-subunit well exposed at the surface of the inner face of the mitochondrial membrane.     

Regional assignment of the gene for diphtheria toxin sensitivity using subchromosomal fragments in microcell hybrids

Human x mouse microcell hybrids resistant to G418 were constructed between mouse hepatoma cells and human x mouse whole cell hybrids containing only intact human chromosome 5 and 22 with an integrated neo ^r-gene. Among these, microcell hybrid BG15 produced four subclones, BG15-4, BG15-6, BG15-7 and BG15-9, which contained variously sized complements of human chromosome 5. BG15-6 contained an intact human chromosome 5, BG15-7 a deleted human chromosome 5 (5pter-q22) and BG15-4 and BG15-9 a translocation between parts of human chromosome 5 (pter-qter? and pter-q23, respectively) and a mouse chromosome. Southern DNA blot analysis showed that the human dihydrofolate reductase (DHFR) gene was present in all four subclones, whereas the human homolog of the v-fms gene was present in BG15-4 and 15-6, but absent from BG15-7 and 15-9. BG15-4, 15-6 and 15-9 were sensitive to diphtheria toxin, and only BG15-7 was resistant to the toxin. We used these microcell hybrids to restrict further the regional location of the gene for diphtheria toxin sensitivity to the q23 region of human chromosome 5.     

Morphological parameter variations between sister chromatids by electron microscopy

Five morphological parameters of sister chromatids, viz radius, length, centromeric index, volume and centromere width, were studied by electron microscopy, from 140 human number 1 chromosomes, and 122 human number 2 chromosomes. The degree of variation obtained ranged from 0.277 +/- 0.003% and 0.286 +/- 0.005% for the centromeric index of chromosomes 1 and 2, respectively, to 9.835 +/- 0.933% and 13.472 +/- 1.461% for centromere width.     

Respiratory syncytial virus fusion glycoprotein: further characterization of a major epitope involved in virus neutralization

Competition experiments and biological assays with a panel of 15 monoclonal antibodies confirmed the presence of at least four antigenic sites on the fusion protein of human respiratory syncytial virus, three of which were involved in virus neutralization. One antigenic site, recognized by two strongly neutralizing antibodies, was conserved after reduction and denaturation and shown by immunoblotting to be localized on the F1 fragment of the fusion protein. Cleavage of this protein with staphylococcal protease V8 or papain produced a series of smaller peptides from 11 to 7 kilodaltons that retained this important neutralization determinant. Compared with the other neutralization sites, the epitope defined by monoclonal antibody 7C2 thus appears as the major neutralization epitope. Our peptide mapping results support the hypothesis that this major epitope is composed of a continuous sequence on the viral genome.     

Effects of organic solutes on the Raman spectra of barnacle muscle fibers

The Raman spectra observed from barnacle muscle fibers are quite complex because the cytoplasm of these cells contains several proteins and solutes. An extraction procedure was used to separate organic solutes from the contractile proteins. Glycine, trimethylamine oxide, taurine, and alanine were found to contribute to the Raman spectra of barnacle muscle fibers, while spectra of lobster fibers reveal the presence of betaine in addition. We have observed that the increase in osmolarity of the intracellular fluid caused by the augmentation of the salinity of sea water (density, 1.023-1.030) in which the barnacles were kept, induces a reduction of intensity of the amide I band. To distinguish among the different parameters which are modified by the sea water salinity, observations were made on glycerinated barnacle muscle fibers. The reduction of intensity of the amide I band in the Raman spectra of glycerinated muscle fibers was also observed with the addition of taurine (0.08 M) in the external relaxing solution. Therefore, under these experimental conditions, the Raman scattering intensity in the amide I region assigned to the alpha-helix conformation (1645-1650 cm-1) is increased when the concentration of organic electrolytes is reduced. However, as no significant decrease of the scattering intensity in the 1660-1670 cm-1 region where the amide I bands of either beta-sheet or disordered conformations normally appear was observed, the increase of intensity of the amide I band centered at 1645 cm-1 is assigned to a change of orientation of alpha-helical segments of the myosin molecules. Our results suggest that organic solutes influence the position of the S-2 segments relative to the thick filaments.     

L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide): a novel thromboxane-prostaglandin endoperoxide antagonist

The effects of L-641,953 (R-8-fluoro-dibenzo[b, f]thiepin-3-carboxylic acid-5-oxide) have been studied on pulmonary and other smooth muscle preparations in vitro and in vivo. When studied in vitro on guinea-pig tracheal chains, L-641,933 produced significant shifts in the dose-response curves to the prostaglandin endoperoxide analogues, U-44069 (pA2 7.06) and U-46619 (pA2 7.14), and prostaglandin (PG) F2 alpha (pA2 6.33) had minimal activity against contractions induced by histamine (pA2 4.38), 5-hydroxytryptamine (pA2 4.63), and acetylcholine (pA2 4.56) and slightly enhanced relaxation induced by PGE2. When tested on the guinea-pig gall bladder strip in vitro, L-641,953 antagonized contractions induced by U-44069 (pA2 7.03) but was less active against those induced by PGF2 alpha (pA2 6.03), PGE1 (pA2 5.62), and histamine (pA2 4.84). When tested in vitro on the guinea-pig pulmonary artery, L-651-953 significantly antagonized contractions induced by U-44069 (pA2 7.04), U-46619 (pA2 7.14), and PGF2 alpha (pA2 7.16) but was less effective against contractions induced by histamine (pA2 4.19). Schild analysis indicated that L-641,953 was fully competitive against contractions of either the guinea-pig tracheal chain induced by U-46619 or the guinea-pig pulmonary artery induced by U-44069 and U-46619. When tested on human platelets in vitro L-641,953 inhibited aggregation induced by U-44069 (IC50 1.3 X 10(-6) M) but not ADP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pH and fructose 2,6-bisphosphate on oxidized and reduced spinach chloroplastic fructose-1,6-bisphosphatase

This report describes the effects of pH and fructose 2,6-bisphosphate (an analog of fructose 1,6-bisphosphate) on the activity of oxidized and reduced fructose-1,6-bisphosphatase from spinach chloroplasts. Studies were carried out with either fructose 1,6-bisphosphate, the usual substrate, or sedoheptulose 1,7-bisphosphate, an alternative substrate. The reduction of the oxidized enzyme is achieved by a thiol/disulfide interchange. The pK values relative to each redox form for the same substrate (either fructose 1,6-bisphosphate or sedoheptulose 1,7-bisphosphate) are identical, suggesting the same site for both substrates on the active molecule. The finding that the analog (fructose 2,6-bisphosphate) behaves like a competitive inhibitor for both substrates also favours this hypothesis. The inhibitory effect of this sugar is more important when the enzyme is reduced than when it is oxidized. The shift in the optimum pH observed when [Mg2+] was raised is interpreted as a conformational change of oxidized enzyme demonstrated by a change in fluorescence. The reduced and oxidized forms have the same theoretical rates relative to both substrates, but the reduced form has an observed Vmax which is 60% of the theoretical Vmax while that of the oxidized form is only 37% of the theoretical Vmax. The reduced enzyme appears more efficient than the oxidized one in catalysis.     

Construction of an Unstable Ring-X Chromosome Bearing the Autosomal Dopa Decarboxylase Gene in Drosophila melanogaster and Analysis of Ddc Mosaics

An unstable Ring-X chromosome, Ddc⁺- Ring-X carrying a cloned Dopa decarboxylase (Ddc) encoding segment was constructed. The construction involved a double recombination event between the unstable Ring-X, R(1)w^vC and a Rod-X chromosome which contained a P-element mediated Ddc ⁺ insert. The resulting Ddc⁺-Ring-X chromosome behaves similarly to the parent chromosome with respect to somatic instability. The Ddc⁺-Ring-X chromosome was used to generate Ddc mosaics. Analyses of Ddc mosaics revealed that while there was no absolute requirement for the Ddc ⁺ expression in either the epidermis or the nervous system, very large mutant clones did affect the viability of the mosaic.     

Conditions of formation of the heparin-fibronectin-collagen complex and the effect of plasmin

The formation and composition of the insoluble heparin-fibronectin-collagen complex and its degradation by proteolysis was investigated. At fixed concentrations of the other molecular components of the complex, the maximal rate of complex formation, measured turbidimetrically, was reached at a concentration of 4 microM heparin and 0.9 microM collagen, while the rate of complex formation was linearly related to concentrations of fibronectin as high as 3 microM. Heparin was incorporated into the complex in a saturable manner, and was released in active anticoagulant form by plasmin but not by urokinase. The complex formation was inhibited by 5 mM calcium or 250 mM NaCl as well as by polybrene or spermin. It is suggested that fibronectin binds both heparin and collagen cooperatively to form an insoluble ternary complex of the extracellular matrix.