Hyperthermostabilization of Bacillus licheniformis{alpha}-amylase and modulation of its stability over a 50{degrees}C temperature range

Bacillus licheniformis     

Genetic identification of Saccharomyces bayanus var. uvarum, a cider-fermenting yeast

Twenty-one Saccharomyces strains isolated from a cider process were analysed in terms of karyotypes, Y' S. cerevisiae sequence occurrence, rDNA structure and cross-fertility with species tester strains. A strong predominance of S. bayanus var. uvarum G. Naumov was found (18 strains vs. three S. cerevisiae). Among the S. bayanus var. uvarum, only three strains proved to contain species-specific Y' S. cerevisiae sequences.     

Sequence analysis of two cosmids from Schizosaccharomyces pombe chromosome III

We report the complete sequence of two cosmids, SPCC895 (38457 bp insert, EMBL Accession No. AL035247) and SPCC1322 (42068 bp insert, EMBL Accession No. AL035259), localized on chromosome III of the Schizosaccharomyces pombe genome. Fourteen Coding DNA sequences (CDSs) were identified in SPCC895 and 17 in SPCC1322. Two known genes were found in each cosmid: map2 and gms1 on SPCC895, encoding the mating type P-factor precursor and an UDP-galactose transporter, respectively, and bub1 and ade6 in SPCC1322, encoding a protein kinase and a phosphoribosylaminoimidazole carboxylase, respectively. The fission yeast K RNA gene has been localized to SPCC895. Three ribosomal proteins have been predicted among these two cosmids. Nine CDSs similar to known proteins were found on SPCC895, and seven on SPCC1322. They include putative genes for an uridylate kinase, a proteasome catalytic component, an ion transporter, a checkpoint protein, a translation initiation protein, a SNARE complex protein, a protein involved in cytoskeletal organization, a spindle pole body-associating protein, pre-mRNA splicing factor RNA helicase, a 3'-5' exonuclease for RNA 3' ss-tail, an UTP-glucose-1-phosphate uridylyltransferase, a leukotriene A(4) hydrolase, a member of the RanBP7-importin beta-Cse1p superfamily, a Ca(++)-calmodulin-dependent serine/threonine protein kinase and a prohibitin antiproliferative protein. One CDS is predicted to be an integral membrane protein. One CDS from SPCC895 is similar to a CDS of unknown function from Saccharomyces cerevisiae and three from SPCC1322 are similar to CDSs of unknown function from Candida albicans, S. cerevisiae and Sz. pombe, respectively. Finally, one CDS of SPCC895 and three of SPCC1322 correspond to orphan genes.     

P2Y11 Agonism Prevents Hypoxia/Reoxygenation- and Angiotensin II-Induced Vascular Dysfunction and Intimal Hyperplasia Development

Vascular dysfunction in cardiovascular diseases includes vasomotor response impairments, endothelial cells (ECs) activation, and smooth muscle cells (SMCs) proliferation and migration to the intima. This results in intimal hyperplasia and vessel failure. We previously reported that activation of the P2Y11 receptor (P2Y11R) in human dendritic cells, cardiofibroblasts and cardiomyocytes was protective against hypoxia/reoxygenation (HR) lesions. In this study, we investigated the role of P2Y11R signaling in vascular dysfunction. P2Y11R activity was modulated using its pharmacological agonist NF546 and antagonist NF340. Rat aortic rings were exposed to angiotensin II (AngII) and evaluated for their vasomotor response. The P2Y11R agonist NF546 reduced AngII-induced vascular dysfunction by promoting EC-dependent vasorelaxation, through an increased nitric oxide (NO) bioavailability and reduced AngII-induced H₂O₂ release; these effects were prevented by the use of the P2Y11R antagonist NF340. Human vascular SMCs and ECs were subjected to AngII or H/R simulation in vitro. P2Y11R agonist modulated vasoactive factors in human ECs, that is, endothelial nitric oxide synthase (eNOS) and endothelin-1, reduced SMC proliferation and prevented the switch towards a synthetic phenotype. H/R and AngII increased ECs secretome-induced SMC proliferation, an effect prevented by P2Y11R activation. Thus, our data suggest that P2Y11R activation may protect blood vessels from HR-/AngII-induced injury and reduce vascular dysfunctions. These results open the way for new vasculoprotective interventions.     

Identification of an UDP-Glc:glycoprotein glucosyltransferase in the yeast Yarrowia lipolytica

The UDP-Glc:glycoprotein glucosyltransferase (UGT) is a soluble protein of the endoplasmic reticulum (ER) that plays a determining part in the mechanism by which unfolded, partially folded or misfolded glycoproteins are retained into the ER. We have identified an UGT in the yeast Yarrowia lipolytica. This protein, of a predicted molecular weight of 165.7 kDa, is encoded by a 5054 bp coding sequence containing a 643 bp intron at position 682-1323. The N-terminal part of the protein displays a signal sequence whereas its C-terminal part carries an ER retrieval signal HDEL. An interruption of the gene that removes the 1075 last nucleotides of its sequence did not lead to any evident phenotype except for a slight increased sensitivity to tunicamycin. YlUGT1 mRNA levels respond to tunicamycin treatment by an induction factor of 2-4, which indicates that the gene product participates in the quality control mechanism in this yeast. Finally, an immunofluorescence study of the protein localization, shows that the protein distribution is different from that of previously studied ER resident proteins. This could indicate that UGT distribution in the secretory pathway is not confined to the ER.     

Interaction of Kar2p and Sls1p is required for efficient co-translational translocation of secreted proteins in the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is a model organism for in vivo study of the signal recognition particle-dependent targeting pathway. In this report, we defined solubilization conditions and set up a fractionation procedure of Y. lipolytica microsomes to determine the amounts of Sec61p-containing translocation pores linked to ribosomes. In contrast to Saccharomyces cerevisiae, from 70 to 80% of Sec61p associates with ribosomes in this yeast. The chaperone protein Kar2p and the Sls1p product, a resident protein of the endoplasmic reticulum lumen, partially fractionate with this Sec61p population. Moreover, Sls1p can be co-immunoprecipitated with Kar2p, and the two polypeptides are shown to directly interact in the yeast two-hybrid system. A site-directed mutagenesis was performed on the SLS1 coding sequence that allowed us to define a functional domain in Sls1p. Indeed, co-translational translocation of a reporter protein is affected when one of these mutant proteins is expressed. Moreover, this protein has lost its capacity to interact with Kar2p, and the two lumenal polypeptides might thus cooperate to promote secretory protein co-translational translocation.     

Carbon nanotubes in macrophages: imaging and chemical analysis by X-ray fluorescence microscopy

X-ray fluorescence microscopy (microXRF) is applied for the first time to study macrophages exposed to unpurified and purified single-walled (SW) and multiwalled (MW) carbon nanotubes (CNT). Investigating chemical elemental distributions allows one to (i) image nanotube localization within a cell and (ii) detect chemical modification of the cell after CNT internalization. An excess of calcium is detected for cells exposed to unpurified SWCNT and MWCNT and related toxicological assays are discussed.     

Discovery of new hexagonal supramolecular nanostructures formed by squalenoylation of an anticancer nucleoside analogue

In this study, the dynamically folded conformation of squalene (SQ) is taken advantage of to link this natural compound to the anticancer nucleoside analogue gemcitabine (gem) in order to achieve the spontaneous formation of nanoassemblies (SQgem) in water. Cryogenic transmission electron microscopy examination reveals particles (104 nm) with a hexagonal or multifaceted shape that display an internal structure made of reticular planes, each particle being surrounded by an external shell. X-ray diffraction evidences the hexagonal molecular packing of SQgem, resulting from the stacking of direct or inverse cylinders. The respective volumes of the gem and SQ molecules as well as molecular modeling of SQgem suggest the stacking of inverse hexagonal phases, in which the central aqueous core, consisting of water and gem molecules, is surrounded by SQ moieties. These SQgem nanoassemblies also exhibit impressively greater anticancer activity than gem against a solid subcutaneously grafted tumor, following intravenous administration. To our knowledge, this is the first demonstration of hexagonal phase organization with a SQ derivative.     

A family of laboratory strains of Saccharomyces cerevisiae carry rearrangements involving chromosomes I and III

In order to study meiotic segregation of chromosome length polymorphism in yeast, we analysed the progeny of a cross involving two laboratory strains FL100trp and YNN295. Analysis of the parental strains led us to detect an important length polymorphism of chromosomes I and III in FL100trp. A reciprocal translocation involving 80 kb of the left arm of chromosome III and 45 kb of the right arm of chromosome I was shown to be the cause for the observed polymorphism in this strain. The characterization of the translocation breakpoints revealed the existence of a transposition hot-spot on chromosome I: the sequence of the translocation joints on chromosomes I and III suggests that the mechanism very likely involved homologous recombination between Ty2 transposable elements on each chromosome. Analysis of FL100, FL200 and FL100trp ura, which are related to FL100trp, shows that this reciprocal translocation is present in some of the strains of the FL series, whereas the parental strain FL100 does not carry the same rearrangement. We evidenced instead the duplication of 80 kb of chromosome III on chromosome I and a deletion of 45 kb of the right arm of chromosome I in this strain, indicating that secondary events might have taken place and that the strain currently named FL100 is not the common ancestor of the FL series. © 1998 John Wiley & Sons, Ltd.