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.     

The effect of some environmental factors on the production of L-DOPA by alginate-entrapped cells of Mucuna pruriens

In-vitro-grown cells of Mucuna pruriens, immobilized in calcium-alginate gels, were able to transform the precursor L-tyrosine into L-dihydroxyphenylalanine (L-DOPA). After the immobilization in alginate the plant cells released 90% of the produced L-DOPA into the medium; supplementation of the medium with calcium inhibited both the transformation of L-tyrosine into L-DOPA and the release of L-DOPA into the medium. Continuous illumination of the beads had a slight beneficial effect on the synthesis of L-DOPA. A simple production medium for the transformation of L-tyrosine into L-DOPA was designed. This medium contained only sucrose and sodium chloride as osmotic stabilizers, a low concentration of calcium chloride for stabilization of the alginate beads, and L-tyrosine as the precursor.     

Vergleichende Untersuchungen an HNO2-Inaktivierten und UV-Inaktivierten Bakteriophagen T4

Summary The inactivation of phage T4 by nitrous acid (HNO₂) is essentially an exponential function of time of treatment. HNO₂-inactivated T4 is able to undergo multiplicity reactivation, and genetic markers may be rescued by live phage, however, the extent of both effects is appreciably less than after UV-inactivation. Also, the survival of phenotypic function of the cistronsr II-A andr II-B is lower with HNO₂-treatment than with a UV-irradiation of a corresponding number of hits.The reduced effects are quantitatively accounted for by the assumption of lethal hits blocking early steps of infection. These early-step damages amount to approximately 1/6 of the total hit number; it is still unknown whether they occur in DNA or in protein. Some indication for the occurrence in protein comes from the result that the host-killing efficiency of HNO₂-inactivated phage is reduced at a similar rate as these early-step damages occur. However, at least 5/6 of the lethal hits are due to chemical changes within the DNA, as can be calculated from the results of multiplicity reactivation, marker rescue, and phenotypic survival of therII-cistrons.

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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.     

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.