A molecular basis for polyketide biosynthesis in marine dinoflagellates with emphasis on Karenia brevis

Polyketides derived from dinoflagellates are among the most complex and unique structures identified to date. The carbon framework of all polyketides is assembled by a polyketide synthase (PKS). No studies of the biosynthesis of dinoflagellate derived polyketides at the genomic level have been reported to date. Nine strains representing seven different species of dinoflagellates were screened for the presence of type I and type II polyketide synthases (PKS) by PCR and RT-PCR. Seven of the nine strains yielded products that were homologous with known and putative type I polyketide synthases. In each case, the presence of a PKS gene was correlated with the presence of bacteria in the cultures as identified by amplification of the bacterial 16S rRNA gene. However, residual phylogenetic signals, resistance to methylation sensitive restriction enzymes and the lack of hybridization to bacterial isolates support a dinoflagellate origin for most of these genes. ^ A more detailed analysis of Karenia brevis, a toxic marine dinoflagellate endemic to the Gulf of Mexico, also supports the hypothesis that dinoflagellates have polyketide synthase genes. Blooms of this harmful alga cause fish kills, marine mammal mortalities and neurotoxic shellfish poisonings. These harmful effects are attributed to a suite of polyketide secondary metabolites known as the brevetoxins. PKS encoding genes amplified from K. brevis culture were found to be similar to PKS genes from the closely related protist, Cryptosporidium parvum. This suggested that these genes originate from the dinoflagellate. However, K. brevis has not been grown axenically. The associated bacteria might be the source of the toxins or the PKS genes. This dissertation reports the localization of these PKS encoding genes by a combination of flow cytometry/PCR and fluorescence in situ hybridization (FISH). Two genes localized exclusively to K. brevis cells while a third localized to both K. brevis and associated bacteria. While these genes have not yet been linked to toxin production, the work describes the first definitive evidence of resident PKS genes in any dinoflagellate. ^

Red mangroves emit hydrocarbons

The objective of this study is to investigate hydrocarbon species and amounts released by red mangrove foliage and determine if these quantities warrant future research on atmospheric chemical processing of these compounds. The field investigation took place during July 2001 at Key Largo, Florida Bay, Florida. Foliage still attached to plants was enclosed in cuvettes while air of known flow rates circulated around leaves to study, hydrocarbon emissions. Cuvette air samples underwent gas chromatographic analyses to determine species and amounts of hydrocarbons released by mangrove foliage. Red mangrove foliage emits isoprene and trace amounts of the monoterpenes of alpha-pinene, beta-pinene, camphene, and d-limonene. The mangrove flowers released these latter compounds in amounts ranging from 0.5 to 10 mg (monoterpene) per gram of dry biomass per hour. These fluxes are normalized to, the foliage temperature of 30 degreesC. When normalized to the foliage temperature of 30 degreesC and light levels of 1000 mumol m(-2) s(-1), isoprene emission rates as high as 0.092 +/- 0.109 mug (isoprene) per gram of dry biomass per hour were measured. Compared to terrestrial forest ecosystems, red mangroves are low isoprene emitters. During peak flowering periods in the summertime, however, red mangroves may emit sufficient amounts of monoterpenes to alter ground-level ozone concentrations and contribute to biogenic aerosol formation.

Action vasorelaxante du 17[bêta]-oestradiol, implication du monoxyde d'azote et des récepteurs aux oestrogènes

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Action vasorelaxante du 17[bêta]-oestradiol, implication du monoxyde d'azote et des récepteurs aux oestrogènes

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Structure and lipid interactions of membrane-associated glycosyltransferases : Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes

This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.

Characterization of the glial scar tissue in a murine model of spinal cord compression, with focus on hyaluronan

Spinal cord injury (SCI) is a devastating neurological disorder that affects thousands of people each year. Although in recent decades significant progress has been made in relation to understanding the molecular and cellular events underlying the nervous damage, spinal cord injury is still a highly disabling condition for which there is no curative therapy. People affected by spinal cord injuries manifested dysfunction or loss, temporary or permanent, of motor, sensory and / or autonomic functions depending on the spinal lesion damaged. Currently, the incidence rate of this type of injury is approximately 15-40 cases per million people worldwide. At the origin of these lesions are: road accidents, falls, interpersonal violence and the practice of sports. In this work we placed the hypothesis that HA is one of the component of the scar tissue formed after a compressive SCI, that it is likely synthetised by the perilesional glial cells and that it might support the permeation of the glial scar during the late phase of SCI. Nowadays, much focus is drawn on the recovery of CNS function, made impossible after SCI due to the high content of sulfated proteoglycans in the extracellular matrix. Counterbalancing the ratio between these proteoglycans and hyaluronic acid could be one of the experimental therapy to re-permeate the glial scar tissue formed after SCI, making possible axonal regrowth and functional recovery. Therefore, we established a model of spinal cord compression in mice and studied the glial scar tissue, particularly through the characterization of the expression of enzymes related to the metabolism of HA and the subsequent concentration thereof at different distances of the lesion epicenter. Our results show that the lesion induced in mice shows results similar to those produced in human lesions, in terms of histologic similarities and behavioral results. but these animals demonstrate an impressive spontaneous reorganization mechanism of the spinal cord tissue that occurs after injury and allows for partial recovery of the functions of the CNS. As regards the study of the glial scar, changes were recorded at the level of mRNA expression of enzymes metabolizing HA i.e., after injury there was a decreased expression of HA synthases 1-2 (HAS 1-2) and an increase of the expression HAS3 synthase mRNA, as well as the enzymes responsible for the HA catabolism, HYAL 1-2. But the amount of HA measured through the ELISA test was found unchanged after injury, it is not possible to explain this fact only with the change of expression of enzymes. At two weeks and in response to SCI, we found synthesized HA by reactive astrocytes and probably by others like microglial cells as it was advanced by the HA/GFAP+ and HA/IBA1+ cells co-location.

An Amidinohydrolase Provides the Missing Link in the Biosynthesis of Amino Marginolactone Antibiotics

Desertomycin A is an aminopolyol polyketide containing a macrolactone ring. We have proposed that desertomycin A and similar compounds (marginolactones) are formed by polyketide synthases primed not with gamma-aminobutanoyl-CoA but with 4-guanidinylbutanoyl-CoA, to avoid facile cyclization of the starter unit. This hypothesis requires that there be a final-stage de-amidination of the corresponding guanidino-substituted natural product, but no enzyme for such a process has been described. We have now identified candidate amidinohydrolase genes within the desertomycin and primycin clusters. Deletion of the putative desertomycin amidinohydrolase gene dstH in Streptomyces macronensis led to the accumulation of desertomycin B, the guanidino form of the antibiotic. Also, purified DstH efficiently catalyzed the in vitro conversion of desertomycin B into the A form. Hence this amidinohydrolase furnishes the missing link in this proposed naturally evolved example of protective-group chemistry.