Hepatic metabolism and secretion of a cholesterol-enriched lipoprotein fraction

A potentially important source of cholesterol secreted in bile is cholesterol-rich lipoproteins. However, the fate of the cholesterol carried in these lipoproteins after hepatic uptake has not been investigated. We harvested an apoE- and cholesterol-rich lipoprotein fraction (d 1.02-1.06 g/ml) from hypercholesterolemic rats and examined the acute effects of these lipoproteins on hepatic cholesterol metabolism, very low density lipoprotein (VLDL) secretion, and biliary lipid secretion. Administration of a lipoprotein bolus (20 mg of cholesterol) to rats resulted in a significant decrease in 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and a significant increase in acyl-coenzyme A:cholesterol acyltransferase activity over controls at 1 hr. Hepatic cholesteryl ester content increased 400% with no change in hepatic free cholesterol content or biliary cholesterol secretion. These cholesterol-rich lipoproteins delivered in the isolated perfused liver effected a fivefold increase in hepatic VLDL secretion with no change in composition. Therefore, cholesterol-rich lipoproteins do not acutely alter biliary cholesterol secretion. Rather, the majority of the cholesterol delivered to the liver in these lipoproteins is either esterified and stored as cholesteryl ester or resecreted as free and esterified cholesterol in hepatic VLDL.     

Blockade of intestinal lipoprotein clearance in rabbits injected with Triton WR 1339-ethyl oleate

Although Triton WR 1339 has been used to block triglyceride or cholesterol removal from plasma, no data are available on the extent to which Triton WR 1339 administered to rabbits blocks clearance of newly absorbed dietary lipids. In the present study, we have measured the efficiency of this blockade during a 24-hr interval. After the Triton WR 1339 administration, plasma Sf greater than 400 and d less than 1.019 g/ml lipoprotein lipid concentrations increased greatly, but the concentration of d greater than 1.019 g/ml lipids decreased. In the rabbits fed 0.5% cholesterol for 1 week, the increase in d less than 1.019 g/ml and the decrease in 1.019 less than d less than 1.063 g/ml lipoprotein fractions 24 hr after the Triton WR 1339 injection were much greater than in the chow-fed Tritonized rabbits. After the Triton treatment, 50% of intravenously injected LDL-125I-labeled apoB disappeared in 24 hr, but little or no apoB appeared in other lipoprotein fractions and no VLDL apoB was converted to LDL. Labeled cholesterol and retinol were fed to rabbits and 24-hr increments in plasma cholesteryl- and retinyl-ester label and mass were measured. In chow-fed Tritonized rabbits about one-half of the absorbed oral doses of both labeled lipids was recovered in plasma, indicating that Triton WR 1339 does not completely inhibit the clearance of intestinal lipoproteins. When rabbits were injected with Triton and an ethyl oleate emulsion, the blockade of dietary lipid removal from plasma was substantially improved and chylomicron cholesterol uptake by extra-hepatic tissues was completely abolished.     

Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat

The lumen of the small intestine in anesthetized rats was recirculated with 50 ml perfusion fluid containing normal salts, 25 mM glucose and low concentrations of hydrophilic solutes ranging in size from creatinine (mol wt 113) to Inulin (mol wt 5500). Ferrocyanide, a nontoxic, quadrupally charged anion was not absorbed; it could therefore be used as an osmotically active solute with reflection coefficient of 1.0 to adjust rates of fluid absorption, Jv, and to measure the coefficient of osmotic flow, Lp. The clearances from the perfusion fluid of all other test solutes were approximately proportional to Jv. From Lp and rates of clearances as a function of Jv and molecular size we estimate (a) the fraction of fluid absorption which passes paracellularly (approx. 50%), (b) coefficients of solvent drag of various solutes within intercellular junctions, (c) the equivalent pore radius of intercellular junctions (50 A) and their cross sectional area per unit path length (4.3 cm per cm length of intestine). Glucose absorption also varied as a function of Jv. From this relationship and the clearances of inert markers we calculate the rate of active transport of glucose, the amount of glucose carried paracellularly by solvent drag or back-diffusion at any given Jv and luminal glucose concentration and the concentration of glucose in the absorbate. The results indicate that solvent drag through paracellular channels is the principal route for intestinal transport of glucose or amino acids at physiological rates of fluid absorption and concentration. In the absence of luminal glucose the rate of fluid absorption and the clearances of all inert hydrophilic solutes were greatly reduced. It is proposed that Na-coupled transport of organic solutes from lumen to intercellular spaces provides the principal osmotic force for fluid absorption and triggers widening of intercellular junctions, thus promoting bulk absorption of nutrients by solvent drag. Further evidence for regulation of channel width is provided in accompanying papers on changes in electrical impedance and ultrastructure of junctions during Na-coupled solute transport.     

Isolation and characterization of IS elements repeated in the bacterial chromosome

Shigella sonnei contains repetitive sequences, including an insertion element IS1, which can be isolated as double-stranded DNA fragments by DNA denaturation and renaturation and by treatment with S1 nuclease. In this paper, we describe a method of cloning the IS1 fragments prepared by the S1 nuclease digestion technique into phage M13mp8 RFI DNA. Several clones contained IS1, usually with a few additional bases. We isolated and characterized five other repetitive sequences using this method. One sequence, 1264 base-pairs in length, had terminal inverted repeats and contained two open reading frames. This sequence, called IS600, showed about 44% sequence homology with IS3 and was repeated more than 20 times in the Sh. sonnei chromosome. Another sequence (named IS629, 1310 base-pairs in length), which was repeated six times, was found also to be related to IS3 and thus IS600. Two other sequences (named IS630 and IS640, 1159 and 1092 base-pairs in length, respectively), which were repeated approximately ten times, had characteristic terminal inverted repeats and contained a large open reading frame coding for a protein. The inverted repeat sequences of IS630 were similar to the sequence at one end of IS200, a Salmonella-specific IS element. The fifth sequence, repeated ten times in Sh. sonnei, had about 98% sequence homology with a portion of IS2. The method described here can be applied to the isolation of IS or iso-IS elements present in any other bacterial chromosome.     

Complete primary structure of vertebrate smooth muscle myosin heavy chain deduced from its complementary DNA sequence. Implications on topography and function of myosin

The 1979 amino acid sequence of embryonic chicken gizzard smooth muscle myosin heavy chain (MHC) have been determined by cloning and sequencing its cDNA. Genomic Southern analysis and Northern analysis with the cDNA sequence show that gizzard MHC is encoded by a single-copy gene, and this gene is expressed in the gizzard and aorta. The encoded protein has a calculated Mr of 229 X 10(3), and can be divided into a long alpha-helical rod and a globular head. Only 32 to 33% of the amino acid residues in the rod and 48 to 49% in the head are conserved when compared with nematode or vertebrate sarcomeric MHC sequences. However, the seven residue hydrophobic periodicity, together with the 28 and 196 residue repeat of charge distribution previously described in nematode myosin rod, are all present in the gizzard myosin rod. Two of the trypsin-sensitive sites in gizzard light meromyosin have been mapped by partial peptide sequencing to 99 nm and 60 nm from the tip of the myosin tail, where these sites coincide with the two "hinges" for the 6 S/10 S transition. In the head sequence, several polypeptide segments, including the regions around the putative ATP-binding site and the reactive thiol groups, are highly conserved. These areas presumably reflect conserved structural elements important for the function of myosin. A multi-domain folding model of myosin head is proposed on the basis of the conserved sequences, information on the topography of myosin in the literature, and the predicted secondary structures. In this model, Mg2+ ATP is bound to a pocket between two opposing alpha/beta domains, while actin undergoes electrostatic interactions with lysine-rich surface loops on two other domains. The actin-myosin interactions are thought to be modulated through relative movements of the domains induced by the binding of ATP.     

Temperature Dependence of Drug Interaction with the Platelet 5-Hydroxytryptamine Transporter: A Clue to the Imipramine Selectivity Paradox

Although [3H]imipramine is a selective radioligand for the 5-hydroxytryptamine (5-HT) transporter in human platelets, its affinity for binding to the 5-HT transporter complex at 0 degrees C (0.6 nM) is significantly higher than its potency for inhibition of [3H]5-HT uptake at the physiological temperature of 37 degrees C (Ki = 29 nM). As this apparent discrepancy could be related to the assay temperature, we studied the thermodynamics of drug interaction with the 5-HT transporter at assay temperatures between 0 degrees C and 37 degrees C, using as radioligands [3H]imipramine (0 degrees C and 20 degrees C) and [3H]paroxetine (20 degrees C and 37 degrees C), a newly available probe for the 5-HT transporter. At 20 degrees C, Ki values of 14 tricyclic and nontricyclic drugs for inhibition of [3H]imipramine and [3H]paroxetine binding to human platelet membranes were highly significantly correlated (r = 0.98, p less than 0.001), validating the use of these two radioligands to study the 5-HT transporter over a temperature range larger than was previously possible with [3H]imipramine alone. The affinity of imipramine for the 5-HT transporter is progressively enhanced with decreasing incubation temperature, thus favoring the selectivity of [3H]imipramine for the 5-HT transporter at 0 degrees C. At 37 degrees C, the Ki of imipramine for inhibition of [3H]paroxetine binding is 32 nM, and equals its Ki value for inhibition of 5-HT uptake into human platelets. With the exception of chlorimipramine, other tricyclic 5-HT uptake inhibitors showed a temperature sensitivity in their interaction with the 5-HT transporter similar to that of imipramine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.     

Lectin histochemical study on the infraorbital gland of the Japanese serow (Capricornis crispus)

Histology and lectin histochemistry were performed in the infraorbital gland of the Japanese serow. The gland is composed of glandular tissues and a pouch filled with the secretion. The tissues consist of an inner layer of sebaceous glands and an outer layer of apocrine glands. The male sebaceous layer is made up of the ordinary type, whereas the female's layer consists of the ordinary and modified types. In the apocrine gland stained with Arachis hypogaea (PNA), nine different patterns of glandular tubules were distinguished on the basis of staining of the cytoplasm, the Golgi area of secretory cells and secretion. Secretory modes of apocrine secretion and exocytosis were included in these stainings. Myoepithelial cells stained constantly with Glycine max (SBA) except when only the Golgi area of secretory cells was positive. The modified sebaceous gland was stained with PNA, SBA, Ricinus communis I (RCA), Triticum vulgaris (WGA), Canavalia ensiformis (Con A) and Ulex europaeus I (UEA), while the ordinary type was positive in PNA, RCA, SBA, WGA and Con A. The secretion in the pouch was stained with PNA, RCA, SBA, Dolichos biflorus (DBA), WGA and Con A. These findings suggest that the modified sebaceous gland contains large amounts of glycoconjugates and the apocrine gland shows a cyclic secretory process of apocrine secretion and exocytosis.     

Retrospective evaluation of gynecologic cytodiagnosis. II. Interlaboratory reproducibility as shown in rescreening large consecutive samples of reported cases

Two laboratories exchanged and rescreened a large sample of cases with cervicovaginal smears they had consecutively accessioned to examine the reproducibility of gynecologic cytodiagnosis under optimum conditions. At least a "working agreement" (diagnoses within +/- 1 category on a ten-category scale) was achieved in diagnoses of normal, benign reaction and squamous abnormality (from minimal dysplasia though invasive cancer) in 18,859 cases (96.8%), of endometrial abnormality in 21 cases (42%) and of "unsatisfactory" in 99 cases (20.7%). Larger differences occurred in greater than or equal to 30% of cases except in the categories of "normal" and "benign reaction," reaching a maximum of 82% for moderate dysplasia. Reexamining 382 cases decreased disagreement by category to the 20% to 65% range only in the five categories of dysplasia plus carcinoma in situ. Agreement was not predicated on the presence of endocervical cells or squamous metaplasia; the basis for "unsatisfactory" calls was not uniform. Comparison of the laboratories' diagnoses with referee diagnoses or, on 178 cases, with tissue diagnoses also demonstrated differences in diagnostic criteria.