Rational approaches towards reversible inhibition of type B monoamine oxidase. Design and evaluation of a novel 5H-Indeno[1,2-c]pyridazin-5-one derivative

The stereoelectronic properties of several potent reversible monoamine oxidase B (MAO-B) inhibitors were studied with a view to develop a pharmacophore model for reversible MAO-B inhibition. This study suggested that important specific H-bond and hydrophobic interactions are required for potent and selective MAO-B inhibition. These requirements were applied in the design and synthesis of a novel reversible and selective MAO-B inhibitor, 3-methyl-8-(4,4,4-trifluoro-butoxy)indeno[1,2-c]pyridazin-5-one, that is ca. 7000 times more selective as an inhibitor for MAO-B than for MAO-A, with K(i(MAO-B)) in the low nanomolar range.     

Substituted azoloquinolines and -quinazolines as new potent farnesyl protein transferase inhibitors

A series of (4-chlorophenyl)--(1-methyl-1H-imidazol-5-yl)azoloquinolines and -quinazolines was prepared. These compounds displayed potent Farnesyl Protein Transferase inhibitory activity and tetrazolo[1,5-a]quinazolines are promising agents for oral in vivo inhibition.     

Crystal structure of the C67A mutant of isopentenyl diphosphate isomerase complexed with a mechanism-based irreversible inhibitor

Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase is a key enzyme in the biosynthesis of isoprenoids. The mechanism of the isomerization reaction involves protonation of the unactivated carbon-carbon double bond in the substrate. Analysis of the 1.97 A crystal structure of the inactive C67A mutant of E. coli isopentenyl diphosphate:dimethylallyl diphosphate isomerase complexed with the mechanism-based inactivator 3,4-epoxy-3-methyl-1-butyl diphosphate is in agreement with an isomerization mechanism involving Glu 116, Tyr 104, and Cys 67. In particular, the results are consistent with a mechanism where Glu116 is involved in the protonation step and Cys67 in the elimination step.     

New 3-deoxyanthocyanidins from leaves of Arrabidaea chica

Two new 3-deoxyanthocyanidins, 6,7,3',4'-tetrahydroxy-5-methoxyflavylium and 6,7,4'-trihydroxy-5-methoxyflavylium, and the pigment carajurin, which has been previously identified, were isolated from dried leaves of Arrabidaea chica, a creeper native to the American tropics. The structures of the components were elucidated by 1H- and 13C-NMR spectroscopy and HPLC-MS, including X-ray crystallographic analysis for carajurin.     

Multiple deletions of the osmolyte transporters BetL,Gbu, and OpuC of Listeria monocytogenes affect virulence and growth at high osmolarity

The success of Listeria monocytogenes as a food-borne pathogen owes much to its ability to survive a variety of stresses, both in the food environment and, after ingestion, within the animal host. Growth at high salt concentrations is attributed mainly to the accumulation of organic solutes such as glycine betaine and carnitine. We characterized L. monocytogenes LO28 strains with single, double, and triple deletions in the osmolyte transport systems BetL, Gbu, and OpuC. When single deletion mutants were tested, Gbu was found to have the most drastic effect on the rate of growth in brain heart infusion (BHI) broth with 6% added NaCl. The highest reduction in growth rate was found for the triple mutant LO28BCG (DeltabetL DeltaopuC Deltagbu), although the mutant was still capable of growth under these adverse conditions. In addition, we analyzed the growth and survival of this triple mutant in an animal (murine) model. LO28BCG showed a significant reduction in its ability to cause systemic infection following peroral coinoculation with the wild-type parent. Altering OpuC alone resulted in similar effects (R. D. Sleator, J. Wouters, C. G. M. Gahan, T. Abee, and C. Hill, Appl. Environ. Microbiol. 67:2692-2698, 2001), leading to the assumption that OpuC may play an important role in listerial pathogenesis. Analysis of the accumulation of osmolytes revealed that betaine is accumulated up to 300 micro mol/g (dry weight) when grown in BHI broth plus 6% NaCl whereas no carnitine accumulation could be detected. Radiolabeled-betaine uptake studies revealed an inability of BGSOE (DeltabetL Deltagbu) and LO28BCG to transport betaine. Indeed, for LO28BCG, no accumulated betaine was found, but carnitine was accumulated in this strain up to 600 micro mol/g (dry weight) of cells, indicating the presence of a possible fourth osmolyte transporter.     

Effects of High Pressure on Inactivation Kinetics and Events Related to Proton Efflux in Lactobacillus plantarum

Knowledge of the mechanism of pressure-induced inactivation of microorganisms could be helpful in defining an effective, relatively mild pressure treatment as a means of decontamination, especially in combination with other physical treatments or antimicrobial agents. We have studied the effect of high pressure on Lactobacillus plantarum grown at pH 5.0 and 7.0. The classical inactivation kinetics were compared with a number of events related to the acid-base physiology of the cell, i.e., activity of F(0)F(1) ATPase, intracellular pH, acid efflux, and intracellular ATP pool. Cells grown at pH 5.0 were more resistant to pressures of 250 MPa than were cells grown at pH 7.0. This difference in resistance may be explained by a higher F(0)F(1) ATPase activity, better ability to maintain a DeltapH, or a higher acid efflux of the cells grown at pH 5.0. After pressure treatment at 250 MPa, the F(0)F(1) ATPase activity was decreased, the ability to maintain a DeltapH was reduced, and the acid efflux was impaired. The ATP pool increased initially after mild pressure treatment and finally decreased after prolonged treatment. The observations on acid efflux and the ATP pool suggest that the glycolysis is affected by high pressure later than is the F(0)F(1) ATPase activity. Although functions related to the membrane-bound ATPase activity were impaired, no morphological changes of the membrane could be observed.     

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Distribution of calcium in a subset of chronic hibernating myocardium in man

Summary The structural correlates of chronic hibernating myocardium in man consist of myocardial cells which transformed from a functional state (rich in contractile material) to a surviving state (poor in contractile material, rich in glycogen). Since the calcium-handling organelles such as SR, sarcolemma and mitochondria underwent structural changes in cells so affected, the distribution of calcium was investigated in biopsies obtained from hibernating areas. The material was processed for microscopic localization of total calcium (laser microprobe mass analysis, LAMMA) and of exchangeable calcium (phosphate-pyroantimonate precipitation method, PPA). Subcellular distribution of total calcium as assessed by LAMMA revealed that in the structurally affected cells the areas in which sarcomeres were replaced by glycogen contained significantly more calcium than all other areas probed such as mitochondria, remaining sarcomeres at the cell periphery and subcellular areas of normally structured cells. Calcium precipitate, obtained after PPA assessment, was localized at the sarcolemma but was virtually absent in the mitochondria of affected cells. The high calcium content in the myolytic areas of affected cells most probably belongs to a pool of bound calcium. The observations that calcium is retained at the sarcolemma and that mitochondria are devoid of precipitate favour the hypothesis that cells structurally affected as such are not ischaemic and are still able to regulate their calcium homeostasis.     

Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic relationships that may be important for the understanding of pathogenicity in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phylogeny of Hartmannella vermiformis, a free-living amoeba that can harbor the etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide sequences were determined for nuclear srDNA from three strains of H. vermiformis isolated from the United Kingdom, Germany, and the United States. These sequences then were compared with a sequence previously obtained for a North American isolate by J. H. Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an average GC content of 49.6%. Sequence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 monotypic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA secondary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent with a separation between European and North American strains of Hartmannella, but results of other tests of this relationship were statistically inconclusive.