Lipase-Catalyzed Regioselective Acylation and Deacylation of Glucose Derivatives

In the development of an efficient synthesis of 1-O-decanoyl-2,3,4,6-tetra-O-acetyl-β-D-glucose (β-2) several lipase-based approaches have been explored. Among five immobilized Upases tested, the lipase from Candida antarctica proved particularly efficient for catalyzing selective hydrolysis in the 1-position of 1,2,3,4,6-penta-O-acetyl-β-D-glucose (β-1). Using triethylamine as catalyst, the hydrolysis product 2,3,4,6-tetra-O-acetyl-D-glucose (3) can be esterified with decanoyl chloride to form β-2 selectively, thereby providing an efficient chemo-enzymatic synthesis starting from readily available raw materials. Attempts to produce β-2 from β-1 by lipase-catalyzed interesterification or to esterify 3 with decanoic acid using a lipase as catalyst were unsuccessful. The latter finding was explained by the hemiacetal OH group of glucose being unable to act as nucleophile in the lysis of the lipase acyl-enzyme intermediate. Furthermore, β-2 was found to bee a too bulky substrate to fit into the active site of any of the lipases tested.     

Equilibrium hormone binding to human estrogen receptors in highly diluted cell extracts is non-cooperative and has a Kd of approximately 10 pM

It is generally accepted that the K_d for hormone binding to estrogen receptors in extracts ranges between 0.1–1 nM and that binding displays positive cooperativity due to formation of homodimers. After carefully optimizing assay procedures, to diminish ligand depletion phenomena and to fully control recoveries, we find a single class of non-interacting high affinity hormone binding sites with a K_d of approx. 10 pM. Ligand depletion was avoided by decreasing receptor concentrations to 5–8 pM. We were therefore obliged to employ radioiodinated estradiol as a probe as the specific radioactivity of tritiated estradiol was too low to maintain the accuracy of the binding assay. Human estrogen receptor extracted from the MCF7 cell line and recombinantly produced (in yeast) wild-type human receptor have identical equilibrium hormone binding characteristics.     

Variation and taxonomy in Hordeum depressum and in the H. brachyantherum complex (Poaceae)

The variation pattern in the perennial Hordeum brachyantherum complex and in the annual H. depressum are described. The diploid form of H. brachyantherum s. lat., endemic to California in USA, previously recognized as a separate species is here treated as a subspecies ( H. brachyantherum ssp. californicum ). Despite its restricted distribution it shows a considerable variation and overlap in morphology with the tetraploid ssp. brachyantherum , and no unambiguous determination based on morphology between the two tax a is possible. The tetraploid cytotype has a large distribution area in western North America and easternmost Asia and a very wide morphological variation. It has also a small disjunct distribution area in easternmost Canada. A single hexaploid population from California is referred to ssp. brachyantherum .     

The distribution and taxonomic value of fatty acids and eicosanoids in the Lipomycetaceae and Dipodascaceae

Using radioimmunoassay, blood platelet aggregation studies and GC-MS the existence of prostaglandins in the endomycetalean yeastDipodascopsis uninucleata was confirmed by our group. These findings triggered the search for similar eicosanoids in the rest of the Endomycetales. We commenced by scanning for the easily detectable precursors of eicosanoids, linoleic- and linolenic acid. We selected two families (i.e. Lipomycetaceae and Dipodascaceae), both producing these precursors, for further investigation.Representative strains of the two families were tested for their ability to grow in the presence of 1mM aspirin, a specific inhibitor of prostaglandin biosynthesis. In contrast to the lipomycetaceous species the dipodascaceous species were insensitive to this drug. These results were verified when representative strains of both families were investigated for their ability to produce eicosanoids from externally fed radio-labeled arachidonic acid along an aspirin sensitive pathway. Thin layer chromatography of culture extracts, followed by autoradiography, showed that while none of the Dipodascaceae produced aspirin sensitive arachidonic acid metabolites, the members of the Lipomycetaceae tested positive for these metabolites. These findings supported the separation of the lipomycetaceous yeastDipodascopsis from the Dipodascaceae. The findings also correlate with the delimitation of these yeasts in two families (i.e. Dipodascaceae and Lipomycetaceae).Further investigation indicated that prostaglandin production by the genusDipodascopsis is mainly associated with ascosporogenesis. Thin layer chromatography of cell extracts fromDipodascopsis tóthii, followed by scintillation counting, indicated the presence of PGF₂ and PGE₂ during ascosporogenesis.     

Microgradients of Microbial Oxygen Consumption in a Barley Rhizosphere Model System

A microelectrode technique was used to map the radial distribution of oxygen concentrations and oxygen consumption rates around single roots of 7-day-old barley seedlings. The seedlings were grown in gel-stabilized medium containing a nutrient solution, a soil extract, and an inert polymer. Oxygen consumption by microbial respiration in the rhizosphere (<5 mm from the root) and in bulk medium (>30 mm from the root) was determined by using Fick's laws of diffusion and an analytical approach with curve fitting to measured microprofiles of oxygen concentration. A marked increase of microbial respiration was observed in the inner 0- to 3-mm-thick, concentric zone around the root (rhizosphere). The volume-specific oxygen consumption rate (specific activity) was thus 30 to 60 times higher in the innermost 0 to 0.01 mm (rhizoplane) than in the bulk medium. The oxygen consumption rate in the root tissue was in turn 10 to 30 times higher than that in the rhizoplane. Both microbial respiration and oxygen uptake by the root varied between different roots. This was probably due to a between-root variation of the exudation rate for easily degradable carbon compounds supporting the microbial oxygen consumption.     

Interleukin-8 activates microtubule-associated protein 2 kinase (ERK1) in human neutrophils

The signal transduction initiated by the human cytokine interleukin-8 (IL-8), the main chemotactic cytokine for neutrophils, was investigated and found to encompass the stimulation of protein kinases. More specifically, IL-8 caused a transient, dose and time dependent activation of a Ser/Thr kinase activity towards myelin basic protein (MBP) and the MBP-derived peptide APRTPGGRR patterned after the specific concensus sequence in MBP for ERK enzymes. The activated MBP kinase was furthermore identified as an extracellular signal regulated kinase (ERK1) based on several criteria such as substrate specificity, molecular weight, activation-dependent mobility shift, and recognition by anti-ERK antibodies. For comparison, the chemotactic response of neutrophils to a stimulus of bacterial origin (fMet-Leu-Phe or fMLP) was also examined and found to involve the activation of a similar ERK enzyme. The present data clearly indicate that in terminally differentiated, non-proliferating human cells, the MBP kinase/ERK activity can serve other purposes than mitogenic signaling, and that processes such as chemotaxis, induced by bacterial peptides as well as by human cytokines like IL-8, involve the regulation of ERK enzyme.Abbreviations IL-8 interleukin-8 - fMLP fMet-Leu-Phe - MBP myelin basic protein - ERK extracellular signal regulated kinase - MAP2 microtubule-associated protein 2 - PK-A cAMP dependent protein kinase - PKI protein kinase inhibitor - PMSF phenyl-methanesulfonyl fluoride - PVDF poly-vinylidene difluoride - HBSF Hank's buffered salt solution - DAB 3,3-diaminobenzidine tetrahydrochloride - PNPP p-nitrophenyl-phosphate - HSA human serum albumin - EGTA [ethylenebis (oxyethylenenitrilo)]tetraacetic acid - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

A Fos-Jun element in the first intron of an α2u-globulin gene

The hepatic expression of the _2u gene family is controlled by a variety of hormones including steroids, growth hormone and insulin. The mechanisms by which these hormones affect -globulin expression are only partially understood. Recently we isolated and characterized clone RAP 01, an _2u-globulin gene expressed in the liver. In preliminary experiments we noted that partial hepatectomy, a procedure which results in a sharp rise in the level of the oncoproteins c-Fos and c-Jun, also causes a transient induction of the messenger RNA corresponding to clone RAP 01. Using the DNAseI footprinting technique we were able to show that this clone contains a TPA (phorbol 12-myristate 13-acetate)-responsive element (TRE) in its first intron. This element (denoted as element X) is identical to the consensus AP-1 binding site (TGACTCAG) and is protected by rat liver nuclear extracts as well as by purified c-Jun. Gel retardation experiments show that an oligonucleotide containing the TRE consensus sequence competes for binding of liver nuclear proteins to element X and that antibodies directed against the M₂ peptide of the mouse Fos protein or the PEP-2 peptide of Jun prevent the formation of specific complexes with the same element. Moreover, element X functions as a TRE in transfected BWTG₃ hepatoma cells treated with TPA. Co-transfection withfos andjun expression vectors mimics the effects of TPA suggesting that AP-1 is in fact the mediator of the observed response. It is concluded that the first intron of RAP 01 contains a functional Fos-Jun element.     

Microtubule-associated distribution of specific granules and secretion of atrial natriuretic factor in primary cultures of rat cardiomyocytes

A close spatial relationship between specific granules containing atrial natriuretic factor (ANF) and microtubules was demonstrated in primary cultures of neonatal rat cardiac myocytes. For the detection of specific granules and microtubules, the myocytes were double immunolabelled with antibodies against -ANF and -tubulin and examined by conventional fluorescence or laser scanning confocal microscopy. In addition, the ultrastructural distribution of specific granules was demonstrated by electron microscopy. In the atrial myocytes, ANF was stored in numerous specific granules that were mainly localized in the perinuclear sarcoplasm. In the ventricular myocytes, however, a minority of the cells (10%) exhibited limited ANF immunoreactivity after 4 days in culture. Microtubules were present throughout the sarcoplasm of the myocytes. They were most densely packed in the perinuclear regions. Depolymerization of the microtubules with nocodazole was followed by dispersal of ANF immunostaining both in the atrial myocytes and in the ventricular myocytes exhibiting ANF immunoreactivity. When the microtubules were allowed to recover, the perinuclear distribution of specific granules, as seen in non-treated myocytes, reappeared. Measurements of secreted immunoreactive ANF by radioimmunoassay revealed that the secretion of ANF from atrial myocytes into the medium was significantly reduced following nocodazole treatment, whereas a similar decrease in secretion from ventricular myocytes was not observed. These findings indicate that ANF-containing specific granules are closely associated with microtubules within the myocytes. It is suggested that secretion of ANF from the atrial myocytes, in contrast to the ventricular myocytes, is microtubule-dependent.