UMP kinase from the Gram-positive bacterium Bacillus subtilis is strongly dependent on GTP for optimal activity.

The gene encoding Bacillus subtilis UMP kinase (pyrH/smbA) is transcribed in vivo into a functional enzyme, which represents approximately 0.1% of total soluble proteins. The specific activity of the purified enzyme under optimal conditions is 25 units.mg-1 of protein. In the absence of GTP, the activity of B. subtilis enzyme is less than 10% of its maximum activity. Only dGTP and 3'-anthraniloyl-2'-deoxyguanosine-5'-triphosphate (Ant-dGTP) can increase catalysis significantly. Binding of Ant-dGTP to B. subtilis UMP kinase increased the quantum yield of the fluorescent analogue by a factor of more than three. UTP and GTP completely displaced Ant-dGTP, whereas GMP and UMP were ineffective. UTP inhibits UMP kinase of B. subtilis with a lower affinity than that shown towards the Escherichia coli enzyme. Among nucleoside monophosphates, 5-fluoro-UMP (5F-UMP) and 6-aza-UMP were actively phosphorylated by B. subtilis UMP kinase, explaining the cytotoxicity of the corresponding nucleosides towards this bacterium. A structural model of UMP kinase, based on the conservation of the fold of carbamate kinase and N-acetylglutamate kinase (whose crystals were recently resolved), was analysed in the light of physicochemical and kinetic differences between B. subtilis and E. coli enzymes.     

Carbohydrate cycling in micro-organisms: what can (13)C-NMR tell us?

The extension of (13)C-nuclear magnetic resonance (NMR) techniques to study cellular metabolism over recent years has provided valuable data supporting the occurrence, diversity and extent of carbon cycling in the carbohydrate metabolism of micro-organisms. The occurrence of such cycles, resulting from the simultaneous operation of different and sometimes opposite individual steps, is inherently related to the network organisation of cellular metabolism. These cycles are tentatively classified here as 'reversibility', 'metabolic' and 'substrate' cycles on the basis of their balance in carbon and cofactors. Current hypotheses concerning the physiological relevance of carbohydrate cycles are discussed in light of the (13)C-NMR data. They most likely represent system-level mechanisms for coherent and timely partitioning of carbon resources to fit with the various biosynthetic, energetic or redox needs of cells and/or additional strategies in the adaptive capacity of micro-organisms to face variation in environmental conditions.     

Mitochondrial targeting of recombinant RNAs modulates the level of a heteroplasmic mutation in human mitochondrial DNA associated with Kearns Sayre Syndrome

Mitochondrial mutations, an important cause of incurable human neuromuscular diseases, are mostly heteroplasmic: mutated mitochondrial DNA is present in cells simultaneously with wild-type genomes, the pathogenic threshold being generally >70% of mutant mtDNA. We studied whether heteroplasmy level could be decreased by specifically designed oligoribonucleotides, targeted into mitochondria by the pathway delivering RNA molecules in vivo. Using mitochondrially imported RNAs as vectors, we demonstrated that oligoribonucleotides complementary to mutant mtDNA region can specifically reduce the proportion of mtDNA bearing a large deletion associated with the Kearns Sayre Syndrome in cultured transmitochondrial cybrid cells. These findings may be relevant to developing of a new tool for therapy of mtDNA associated diseases.     

A two-component signal-transducing system is involved in competence and penicillin susceptibility in laboratory mutants of Streptococcus pneumoniae

Penicillin resistance in Streptococcus pneumoniae has been attributed so far to the production of penicillin-binding protein (PBP) variants with decreased affinities for β-lactam antibiotics. Cefotaxime-resistant laboratory mutants, selected after several steps on increasing concentrations of this β-lactam, become deficient in transformation as well. A DNA fragment conferring both cefotaxime resistance and transformation deficiency was isolated and cloned from the mutant C306. The cefotaxime resistance associated with this resistance determinant was not accompanied with apparent changes in PBP properties, and it mapped on the chromosome distinct from the known resistance determinants, genes encoding PBP2x, PBP1a or PBP2b. Determination of a 2265 bp DNA sequence of the resistance determinant revealed two open reading frames, claR and claH, whose deduced amino acid sequence identified the corresponding proteins as the response regulator and histidine kinase receptor, respectively (members of the two families of bacterial signal-transducing proteins). Two hydrophobic peptide regions divided the histidine kinase ClaH into two putative domains: an N-terminal extracelluiar sensor part, and an intracelluiar C-terminal domain with the conserved His-226 residue, the presumed phosphorylation site. The single point mutations responsible for cefotaxime-resistance and transformation deficiency of C306 and of another two independently isolated cefotaxime-resistant mutants were each located in the C-terminal half of ClaH. A small extracellular protein, the competence factor, is required for induction of competence. Neither C306 nor the transformants obtained with the mutated claH gene produced competence factor, and exogenous competence factor could not complement the transformation deficiency, indicating that the signal-transducing system cia is involved in early steps of competence regulation.     

UCP2 is a mitochondrial transporter with an unusual very short half-life

This study focused on the stability of UCP2 (uncoupling protein 2), a mitochondrial carrier located in the inner membrane of mitochondrion. UCP2 is very unstable, with a half-life close to 30min, compared to 30h for its homologue UCP1, a difference that may highlight different physiological functions. Heat production by UCP1 in brown adipocytes is generally a long and adaptive phenomenon, whereas control of mitochondrial ROS by UCP2 needs more subtle regulation. We show that a mutation in UCP2 shown to modify its activity, actually decreases its stability.     

Genome sequencing in the sea urchin embryo: what is new concerning the cell cycle?

Sea urchin is a classical research model system in developmental biology; moreover, the external fertilization and growth of embryos, their rapid division cycle, their transparency and the accessibility of these embryos to molecular visualization methods, made them good specimens to analyze the regulatory mechanisms of cell division. These features as well as the phylogenetic position of sea urchin, close to vertebrates but in an outgroup within the deuterostomes, led scientists working on this model to sequence the genome of the species S. purpuratus. The genome contains a full repertoire of cell cycle control genes. A comparison of this toolkit with those from vertebrates, nematodes, drosophila, as well as tunicates, provides new insight into the evolution of cell cycle control. While some gene subtypes have undergone lineage-specific expansions in vertebrates (i.e. cyclins, mitotic kinases,...), others seem to be lost in vertebrates, for instance the novel cyclin B identified in S. purpuratus. On the other hand, some genes which were previously thought to be vertebrate innovations, are also found in sea urchins (i.e. MCM9). To note is also the absence of cell cycle inhibitors of the INK type, which are apparently confined to vertebrates. The uncovered genomic repertoire of cell-cycle regulators will thus provide molecular tools that should further enhance future research on cell cycle control and developmental regulation in this model.     

The two authentic methionine aminopeptidase genes are differentially expressed in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Background  

Two putative methionine aminopeptidase genes,map(essential) andyflG(non-essential), were identified in the genome sequence ofBacillus subtilis. We investigated whether they can function as methionine aminopeptidases and further explored possible reasons for their essentiality or dispensability inB. subtilis.     

Therapeutic vaccination against autologous cancer stem cells with mRNA-transfected dendritic cells in patients with glioblastoma

Background

The growth and recurrence of several cancers appear to be driven by a population of cancer stem cells (CSCs). Glioblastoma, the most common primary brain tumor, is invariably fatal, with a median survival of approximately 1 year. Although experimental data have suggested the importance of CSCs, few data exist regarding the potential relevance and importance of these cells in a clinical setting.

Methods

We here present the first seven patients treated with a dendritic cell (DC)-based vaccine targeting CSCs in a solid tumor. Brain tumor biopsies were dissociated into single-cell suspensions, and autologous CSCs were expanded in vitro as tumorspheres. From these, CSC-mRNA was amplified and transfected into monocyte-derived autologous DCs. The DCs were aliquoted to 9–18 vaccines containing 10⁷ cells each. These vaccines were injected intradermally at specified intervals after the patients had received a standard 6-week course of post-operative radio-chemotherapy. The study was registered with the ClinicalTrials.gov identifier NCT00846456.

Results

Autologous CSC cultures were established from ten out of eleven tumors. High-quality RNA was isolated, and mRNA was amplified in all cases. Seven patients were able to be weaned from corticosteroids to receive DC immunotherapy. An immune response induced by vaccination was identified in all seven patients. No patients developed adverse autoimmune events or other side effects. Compared to matched controls, progression-free survival was 2.9 times longer in vaccinated patients (median 694 vs. 236 days, p = 0.0018, log-rank test).

Conclusion

These findings suggest that vaccination against glioblastoma stem cells is safe, well-tolerated, and may prolong progression-free survival.     

Genetic Structure and Internal Rearrangements of Stable Merodiploids from BACILLUS SUBTILIS Strains Carrying the trpE26 Mutation

Transformation and transduction to tryptophan independence of strains of Bacillus subtilis carrying the "trpE26" chromosomal aberrations (a translocation and an inversion) with a "normal" 168 type strain as donor induce a tandem duplication of the thrA-ilvA region of the chromosome. The clones possessing this unstable duplication segregate besides the Trp^- some stable Trp⁺ cells which retain only part of the duplication (the trpE-ilvA region) in nontandem configuration. Such clones may also be produced directly during the crosses. The genetic map of these clones (designated as class I stable merodiploids) was constructed: they possess the tranlocation and the inversion of the trpE26 parental strain. Another type of stable Trp⁺ clones (class II) also appears, although more rarely, in similar crosses. Studies on their genetic structure revealed that they are haploid for the trpE-ilvA region and carry a nontandem duplication of the thrA-trpE region. In these clones the cysB-tre region has the orientation of the 168 type strain. The duplications in both classes are stable, that of class I being more stable than that of class II where loss of one copy of the thrA-trpE region leads to about 1% haploid cells. Detailed genetic studies on heterozygous clones from both classes have shown exchange of alleles between copies of the nontandem duplications. Models are proposed for the formation of each class of merodiploids and for recombination events taking place in them. These models imply recombination at sequences of intrachromosomal homology and (or) introduction of heterologous juncions ("novel joints") by transformation or transduction.