Biliary lipid secretion in the rat. The uncoupling of biliary cholesterol and phospholipid secretion from bile acid secretion by sulfated glycolithocholic acid

Glycolithocholic acid and its sulfated derivative are major metabolites of the secondary bile acid lithocholic acid in man. Both compounds are known to induce cholestasis in experimental animals. We compared the effects of these endogenous hepatotoxins on bile production and biliary lipid composition in rats with chronic biliary drainage. The compounds were administered enterally at relatively low rates (5-50% of the rats' endogenous bile acid secretion in these experiments) to simulate enterohepatic circulation. Both compounds were substantially secreted into bile (more than 90% of dose); sulfated glycolithocholic acid unchanged and glycolithocholic acid after hepatic hydroxylation predominantly in the form of glyco-beta-muricholic acid (cf. Kuipers et al. (1986) Am. J. Physiol. 251, G189-G194). Neither glycolithocholic acid nor its sulfated derivative affected the biliary excretion of endogenous bile acids or bile flow in these experiments. In spite of this, phospholipid and cholesterol secretion were significantly reduced by sulfated glycolithocholic acid but were not altered by glycolithocholic acid. Phospholipid and cholesterol secretion rapidly decreased to 25 and 50% of their initial values, respectively, at biliary output rates of sulfated glycolithocholic acid up to 2 mumol/h, and did not further decrease when this output was increased to 6 mumol/h. Small unilamellar liposomes consisting of cholesterol, [Me-14C]choline-labeled phosphatidylcholine, phosphatidylserine and [3H]cholesteryl oleate in a 5:4:1:0.1 molar ratio were employed to label intrahepatic lipid pools. Administration of sulfated glycolithocholic acid slightly reduced bile acid synthesis from [3H]cholesteryl oleate, but significantly reduced the biliary secretion of [14C]phospholipid. Glycolithocholic acid did not affect the hepatic processing of liposomal lipids. It is concluded that sulfated glycolithocholic acid at low doses causes the uncoupling of biliary lipid secretion from that of bile acids, which might represent in initiating event in sulfated glycolithocholic acid hepatotoxicity.     

Cloning and sequencing of the peroxisomal amine oxidase gene from Hansenula polymorpha

We have cloned the AMO gene, encoding the microbody matrix enzyme amine oxidase (EC 1.4.3.6) from the yeast Hansenula polymorpha. The gene was isolated by differential screening of a cDNA library, immunoselection, and subsequent screening of a H. polymorpha genomic library. The nucleotide sequence of a 3.6 kilobase stretch of DNA containing the amine oxidase (AMO) gene was determined. The AMO gene contains an open reading frame of 692 amino acids, with a relative molecular mass of 77,435. The 5' and 3' ends of the gene were mapped and show that the transcribed region measures 2134 nucleotides. The derived amino-acid sequence was confirmed by sequencing an internal proteolytic fragment of the purified protein. Amine oxidase contains the tripeptide sequence Ser-Arg-Leu, located 9 residues from the carboxy terminus, which may represent the topogenic signal for protein import into microbodies.     

Evidence for reverse cholesterol transport in vivo from liver endothelial cells to parenchymal cells and bile by high-density lipoprotein.

Acetylated low-density lipoprotein (acetyl-LDL), biologically labelled in the cholesterol moiety of cholesteryl oleate, was injected into control and oestrogen-treated rats. The serum clearance, the distribution among the various lipoproteins, the hepatic localization and the biliary secretion of the [3H]cholesterol moiety were determined at various times after injection. In order to monitor the intrahepatic metabolism of the cholesterol esters of acetyl-LDL in vivo, the liver was subdivided into parenchymal, endothelial and Kupffer cells by a low-temperature cell-isolation procedure. In both control and oestrogen-treated rats, acetyl-LDL is rapidly cleared from the circulation, mainly by the liver endothelial cells. Subsequently, the cholesterol esters are hydrolysed, and within 1 h after injection, about 60% of the cell- associated cholesterol is released. The [3H]cholesterol is mainly recovered in the high-density lipoprotein (HDL) range of the serum of control rats, while low levels of radioactivity are detected in serum of oestrogen-treated rats. In control rats cholesterol is transported from endothelial cells to parenchymal cells (reverse cholesterol transport), where it is converted into bile acids and secreted into bile. The data thus provide evidence that HDL can serve as acceptors for cholesterol from endothelial cells in vivo, whereby efficient delivery to the parenchymal cells and bile is assured. In oestrogen-treated rats the radioactivity from the endothelial cells is released with similar kinetics as in control rats. However, only a small percentage of radioactivity is found in the HDL fraction and an increased uptake of radioactivity in Kupffer cells is observed. The secretion of radioactivity into bile is greatly delayed in oestrogen-treated rats. It is concluded that, in the absence of extracellular lipoproteins, endothelial cells can still release cholesterol, although for efficient transport to liver parenchymal cells and bile, HDL is indispensable.     

Regulation and structure of an Escherichia coli gene coding for an outer membrane protein involved in export of K88ab fimbrial subunits.

The nucleotide sequence of the faeD gene of Escherichia coli and the amino acid sequence of its product is presented. The faeD product is an outer membrane protein required for transport of K88ab fimbrial subunits across the outer membrane. The protein is synthesized as a precursor containing a signal peptide, and the tentative mature protein comprises 777 amino acid residues. The distribution of amino acids in the faeD protein is similar to that of other outer membrane proteins; showing a fairly even distribution of charged residues and the absence of extensive hydrophobic stretches. Secondary structure predictions revealed a region of 250 amino acid residues which might be embedded in the outer membrane. The 5'-end of faeD is located within a region showing dyad symmetry. This region serves to couple translation of faeD to the translation of the gene preceding it (faeC). The 3'-end of faeD shows an overlap of 5 bases with the next gene (faeE).     

Relationship between exercise-induced muscle damage and enzyme release in rats.

The relationship between the amount of exercise-induced muscle damage and the release of creatine kinase (CK), aspartate aminotransferase (AST), and lactate dehydrogenase (LD) was studied. Gender differences in enzyme release and histological damage were also studied. Serial pre- and postexercise blood samples were drawn from untrained male and female catheterized Wistar rats that ran 1.5 or 2.5 h on a treadmill (incline 10 degrees). Three days postexercise, muscle damage was quantified morphometrically in five different hindlimb and forearm muscles. The 1.5 and 2.5 h of exercise elicited histological damage only in the soleus muscle. Significant plasma CK, AST, and LD elevations were found immediately postexercise both in male and female rats. However, the enzyme release was significantly greater in males than in females. Part of this could be explained by differences in clearance rates between males and females. No gender difference in amount of histological damage was found. The actual volume of histological muscle damage was significantly less than the calculated muscle damage based on enzyme release. An increase in the exercise duration from 1.5 to 2.5 h resulted in a disproportional increase in both histological muscle damage and muscle enzyme release. From the present study it is concluded that muscle enzyme release is not clearly reflected in histological muscle damage.     

Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Cyclooxygenase and 5-lipoxygenase inhibitory activity of 2,6 di-t-butylphenols linked by a sulfur atom to 1,3,4-thiadiazoles and 1,3,4-oxadiazoles

The preparation of a series of 1,3,4-thiadiazoles and 1,3,4-oxadizoles linked by a thioether to 2,6-di-t-butylphenol and the inhibition of cyclooxygenase (CO) and 5-lipoxygenase (5-LO) by these compounds is dicussed.     

Identification of a locus involved in meningococcal lipopolysaccharide biosynthesis by deletion mutagenesis

A novel method for insertion/deletion mutagenesis in meningococci was devised. This consisted of ligating a digest of total chromosomal DNA to a 1.1 kb restriction fragment containing an erythromycin-resistance marker ( ermC ), and subsequent transformation of the ligation mixture into the homologous meningococcal strain H44/76. Southern blotting of a number of the resulting erythromycin-resistant transformants demonstrated that all carried the ermC gene inserted at different positions in the chromosome. Mutants with a specific phenotype were identified by screening with the anti-lipopolysaccharide (LPS) monoclonal antibody MN4A8B2, which is specific for immunotype L3. In this way, two independent L3-negative mutant strains were isolated. In transformation experiments with chromosomal DNA from these mutants, erythromycin-resistance and lack of MN4A8B2 reactivity were always linked, showing that the insertion/deletion was in a locus involved in LPS biosynthesis. On SDS–PAGE, the mutant LPS displayed an electrophoretic mobility intermediate between that produced by the previously isolated galE and rfaF mutant strains. Chemical analysis of the mutant LPS revealed that the structure was probably lipid A–(KDO)₂–(Hep)₂. Chromosomal DNA flanking the ermC insertion in these two mutant strains was cloned, and used as probe for the isolation of the corresponding region of the wild-type strain. From hybridization and polymerase chain reaction (PCR) analysis, it could be concluded that both mutations map to the same locus. The affected gene probably encodes the glycosyltransferase necessary for adding N -acetylglucosamine to heptose.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

The genus Archidendron (Leguminosae – Mimosoideae)

An account is given of the morphology and the taxonomy of the Asian, Australian and Pacific genus Archidendron (Leguminosae – Mimosoideae). A new infrageneric classification based on morphological data is presented, the genus being subdivided in 8 series. The phylogeny of the genus is discussed, the base of discussion being all available morphological, palynological and wood–anatomical characters. The presence/absence of stipules, the length of the staminal tube compared with that of the corolla–tube, the sessile/stipitate ovary(–ies), the morphology of the pods and the wood–anatomy have been particularly useful in determining the evolutionary trends within the genus. Analyses of the geographical range of selected character states are presented. The data suggest a Central – W. Malesian origin of the genus. The series endemic to the E. Malesian – Australian area have probably evolved more recently. The pluricarpellate condition of the flowers in several species endemic to the E. Malesian and Australian area is considered to be a derived character state. The following new taxa are proposed: Ser. Calycinae Nielsen, ser. Ptenopae Nielsen, ser. Bellae Nielsen, Archidendron falcatum Nielsen, A. cockburnii Nielsen, A. sabahense Nielsen, A. fagifolium (Bl. ex Miq.) Nielsen var. borneense Nielsen, A. kunsrteri (Prain) Nielsen subsp. ashtonii Nielsen, A. ellipticum (Bl.) Nielsen subsp. cordifoliolatum Nielsen. New combinations are proposed in the Malesian species formerly referred to Abarema, Zygia and Morolobium by Kostermans. Keys to and an enumeration of the species are presented.