Crystallographic analysis of triclosan bound to enoyl reductase

Molecular genetic studies with strains of Escherichia coli resistant to triclosan, an ingredient of many anti-bacterial household goods, have suggested that this compound works by acting as an inhibitor of enoyl reductase (ENR) and thereby blocking lipid biosynthesis. We present structural analyses correlated with inhibition data, on the complexes of E. coli and Brassica napus ENR with triclosan and NAD(+) which reveal how triclosan acts as a site-directed, picomolar inhibitor of the enzyme by mimicking its natural substrate. Elements of both the protein and the nucleotide cofactor play important roles in triclosan recognition, providing an explanation for the factors controlling its tight binding to the enzyme and for the emergence of triclosan resistance.     

Functional identification of AtFao3, a membrane bound long chain alcohol oxidase in Arabidopsis thaliana

The Arabidopsis thalina genome database was searched for homologues of the Candida cloacae fao1 gene which encodes a membrane bound, flavin-containing, hydrogen peroxide generating, long chain alcohol oxidase. This gene has not been isolated from plants or animals. Four putative candidates were found in the database but their function has not been proven. The cDNAs for two of them were cloned by RT-PCR from Arabidopis suspension culture and one of them [AtFAO3] was overexpressed in Escherichia coli and shown to functionally express long chain alcohol oxidase activity. The protein has been solubilised and retains biological activity thereby preparing the way for crystallographic studies. This is the first functional proof identifying a long chain alcohol oxidase in higher plants.     

Proteomic analysis of changes in the extracellular matrix of Arabidopsis cell suspension cultures induced by fungal elicitors

A proteomic approach has been applied to investigate changes in the extracellular matrix of Arabidopsis thaliana cell suspension cultures following treatments with two fungal pathogen elicitors, chitosan and extracts of Fusarium moniliforme. The oxidative burst and induction of glutathione S-transferase were used as markers for induction of the pathogen defence response. Changes in the cell wall and culture filtrate proteome were profiled. Proteins whose abundance changed reproducibly were analysed via matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS). An increase in the level of two classical cell wall proteins (a putative endochitinase and a polygalacturonase inhibiting protein) and two novel proteins (a putative receptor-like protein kinase and a probable apospory-associated protein) were seen at 24 hours following elicitation. The level of an unknown protein and a hypothetical protein, which has some homology to serine carboxypeptidases, were decreased at 24 hours post-elicitation. In the culture filtrate extracts, we identified two pathogen elicitor responsive proteins, a xyloglucan endo-1,4-beta-D glucanases (XEG) and a peroxidase. Using a combination of two-dimensional polyacrylamide gel electrophoresis, immunoblotting with a phosphotyrosine-specific antibody, and MALDI-TOF MS we discovered that spots that represent putative lectin receptor-like kinase, a putative endochitinase and a XEG possess phosphorylated tyrosine residues. The identification of phosphorylated bona fide cell wall proteins and a putative extracellular receptor-like kinase with no transmembrane domain implicate the existence of an extracellular phosphorylation network which could be involved in intercellular communication.     

Phylogenetic relationships and taxonomic position of Chlorella -like isolates from low pH environments (pH < 3.0)

Background  

Little is known about phytoplankton communities inhabiting low pH environments such as volcanic and geothermal sites or acidic waters. Only specialised organisms are able to tolerate such extreme conditions. There is, thus, low species diversity. We have characterised the previously isolated acid tolerant Chlorella -like microalgae Viridiella fridericiana and Chlorella protothecoides var. acidicola by microscopical and biomolecular methods in order to assess their phylogenetic relationships.     

Dynamic rearrangement of the filamentous actin network occurs during zoosporogenesis and encystment in the oomycete phytophthora cinnamomi

The organization of filamentous actin (F-actin) in living cells of the oomycete Phytophthora cinnamomi was determined during zoosporogenesis and zoospore encystment by microinjecting sporangia with fluorescently labeled phalloidin and observing resultant fluorescence by confocal microscopy. In multinucleate sporangia prior to the induction of cleavage, phalloidin labeling took the form of plaques which occurred mainly in the periphery of the sporangia. After induction of cleavage, phalloidin labeling showed that the plaques disappeared and that F-actin began to accumulate along the developing cleavage planes and around nuclei and water expulsion vacuoles. F-actin labeling was also observed near the plasma membrane in zoospores and young cysts but reverted to the plaque form in older cysts. Localization of F-actin close to the developing cleavage planes is consistent with the idea that actin microfilaments function in the positioning and expansion of the cleavage membranes. Observations of plaques of actin in living sporangia provide evidence that plaques are not aldehyde-induced fixation artifacts. Copyright 1998 Academic Press.     

HIV-particles in spermatozoa of patients with AIDS and their transfer into the oocyte

By immunocytochemistry and in situ hybridization at the electron microscopy level, and by the PCR technique, we have shown that HIV-1 binds and enters normal sperm; that viral particles, their antigens, and nucleic acid are present in sperm from HIV-1 infected men; and that such sperm can transfer HIV-1 like particles to normal human oocytes. We also present evidence that a galactosylceramide-like compound is present on the sperm membrane and could function as an alternative receptor for HIV.     

Unusual pattern of bacterial ice nucleation gene evolution

Bacterial ice nucleation activity (INA+ phenotype) can be traced to the product of a single gene, ina. A remarkably sparse distribution of this phenotype within three bacterial genera indicates that the ina gene may have followed an unusual evolutionary path. Southern blot analyses, coupled with assays for ice-nucleating ability, revealed that within four bacterial species an ina gene is present in some strains but absent from others. Results of hybridization experiments using DNA fragments that flank the ina gene suggested that the genotypic dimorphism of ina may be anomalous. A phylogenetic analysis of 16S ribosomal RNA gene sequences from a total of 14 ina+ and ina- bacterial strains indicated that the ina+ bacteria are not monophyletic but instead phylogenetically interspersed among ina- bacteria. The relationships of ina+ bacteria inferred from ina sequence did not coincide with those inferred from the 16S data. These results suggest the possibility of horizontal transfer in the evolution of bacterial ina genes.      

Relationship between the golgi apparatus, gerl, and secretory granules in acinar cells of the rat exorbital lacrimal gland

The method of secretory granuleformation in the acinar cells of the rat exorbital lacrimal gland was studied by electron microscope morphological and cytochemical techniques. Immature secretory granules at the inner face of the Golgi apparatus were frequently attached to a narrow cisternal structure similar to GERL as described in neurons by Novikoff et al. (Novikoff, P. M., A. B. Novikoff, N. Quintana, and J.-J. Hauw. 1971. J. Cell Bio. 50:859-886). In the lacrimal gland. GERL was located adjacent to the inner Golgi saccule, or separated from it by a variable distance. Portions of GERL were often closely paralleled by modified cisternae of rough endoplasmic reticulum (RER), which lacked ribosomes on the surface adjacent to GERL. Diaminobenzidine reaction product of the secretory enzyme peroxidase was localized in the cisternae of the nuclear envelope, RER, peripheral Golgi vesicles, Golgi saccules, and immature and mature secretory granules. GERL was usually free of peroxidase reaction product or contained only a small amount. Thiamine pyrophosphatase reaction product was present in two to four inner Golgi saccules; occasionally, the innermost saccule was dilated and fenestrated, and contained less reaction product than the next adjacent saccule. Acid phosphatase (AcPase) reaction product was present in GERL, immature granules, and, rarely, in the innermost saccule, but not in the rest of the Golgi saccules. Thick sections of AcPase preparations viewed at 100 kV revealed that GERL consisted of cisternal, and fenestrated or tublular portions. The immature granules were attached to GERL by multiple connections to the tublular portions. These results suggest that, in the rat exorbital lacrimal gland, the Golgi saccules participate in the transport of secretory proteins, and that GERL is involved in the formation of secretory granules.