Taxonomic characterisation of Pseudomonas strain L48 and formal proposal of Pseudomonas entomophila sp. nov

An entomopathogenic, Gram-negative bacterium isolated from a female specimen of the fruit fly Drosophila melanogaster was taxonomically characterised. Strain L48(T) was strictly aerobic, non-fermentative, oxidase and catalase positive, rod-shaped, and motile due to a polar inserted flagellum. Phylogenetic analysis of the 16S rRNA gene and three other housekeeping genes placed strain L48 (T) in the Pseudomonas putida phylogenetic group. DNA-DNA hybridisation studies together with phenotypic metabolic tests and MALDI-TOF MS analysis justified the proposal of strain L48(T) as a representative of a novel species, for which the name Pseudomonas entomophila sp. nov. is proposed. The type strain is deposited in culture collections under accession numbers CCUG 61470(T) and CECT 7985(T).     

Thymic alterations in EphA4-deficient mice

In the present work, we have demonstrated in vivo an altered maturation of the thymic epithelium that results in defective T cell development which increases with age, in the thymus of Eph A4-deficient mice. The deficient thymi are hypocellular and show decreased proportions of double-positive (CD4+CD8+) cells which reach minimal numbers in 4-wk-old thymi. The EphA4 (-/-) phenotype correlates with an early block of T cell precursor differentiation that results in accumulation of CD44-CD25+ triple-negative cells and, sometimes, of CD44+CD25- triple-negative thymocytes as well as with increased numbers of apoptotic cells and an important reduction in the numbers of cycling thymocytes. Various approaches support a key role of the thymic epithelial cells in the observed phenotype. Thymic cytoarchitecture undergoes profound changes earlier than those found in the thymocyte maturation. Thymic cortex is extremely reduced and consists of densely packed thymic epithelial cells. Presumably the lack of forward Eph A4 signaling in the Eph A4 -/- epithelial cells affects their development and finally results in altered T cell development.     

Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum

Physical mapping with large-insert clones is becoming an active area of genomics research, and capillary electrophoresis (CE) promises to revolutionize the physical mapping technology. Here, we demonstrate the utility of the CE technology for genome physical mapping with large-insert clones by constructing a robust, binary bacterial artificial chromosome (BIBAC)-based physical map of Penicillium chrysogenum. We fingerprinted 23.1× coverage BIBAC clones with five restriction enzymes and the SNaPshot kit containing four fluorescent-ddNTPs using the CE technology, and explored various strategies to construct quality physical maps. It was shown that the fingerprints labeled with one or two colors, resulting in 40–70 bands per clone, were assembled into much better quality maps than those labeled with three or four colors. The selection of fingerprinting enzymes was crucial to quality map construction. From the dataset labeled with ddTTP–dROX, we assembled a physical map for P.chrysogenum, with 2–3 contigs per chromosome and anchored the map to its chromosomes. This map represents the first physical map constructed using the CE technology, thus providing not only a platform for genomic studies of the penicillin-producing species, but also strategies for efficient use of the CE technology for genome physical mapping of plants, animals and microbes.     

The thermophilic biomass-degrading fungus Thielavia terrestris Co3Bag1 produces a hyperthermophilic and thermostable β-1,4-xylanase with exo- and endo-activity

Extremophiles - A hyperthermophilic and thermostable xylanase of 82 kDa (TtXynA) was purified from the culture supernatant of T. terrestris Co3Bag1, grown on carboxymethyl cellulose (CMC),...     

Structure of dihydropyrimidinase from Sinorhizobium meliloti CECT4114: New features in an amidohydrolase family member

The recombinant dihydropyrimidinase from Sinorhizobium meliloti CECT4114 (SmelDhp) has been characterised and its crystal structure elucidated at 1.85 Å. The global architecture of the protein is reminiscent of that of the amidohydrolase superfamily, consisting of two domains; an (α/β)₈ TIM-like barrel domain, where the catalytic centre is located, and a smaller β-sheet sandwich domain of unknown function. The c-terminal tails of each subunit extend toward another monomer in a swapping-like manner, creating a hydrogen bond network which suggests its implication in protein oligomerisation. Mutational and structural evidence suggest the involvement of a conserved tyrosine in the reaction mechanism of the enzyme. SmelDhp presents both hydantoinase and dihydropyrimidinase activities, with higher affinity for the natural six-membered ring substrates. For the five-membered ring substrates, affinity was greater for those with aliphatic and apolar groups in the 5th carbon atom, with the highest rates of hydrolysis for d-5-methyl and d-5-ethyl hydantoin (k_cat/K_m = 2736 ± 380 and 944 ± 52 M^?1 s^?1, respectively). The optimal conditions for the enzyme activity were found to be 60 °C of temperature at pH 8.0. SmelDhp retains 95% of its activity after 6-hour preincubation at 60 °C. This is the first dihydropyrimidinase used for the hydrolytic opening of non-natural 6-monosubstituted dihydrouracils, which may be exploited for the production of β-amino acids.     

3-Hydroxy-3-methylglutaryl-CoA reductase, mevalonate-5-pyrophosphate decarboxylase and acyl-CoA: cholesterol acyl-transferase from mucosa scrapings and isolated enterocytes from chick duodenum, jejunum and ileum

3-Hydroxy-3-methylglutaryl-CoA reductase, mevalonate-5-pyrophosphate decarboxylase and acyl-CoA: cholesterol acyltransferase activities were assayed in mucosal scrapings and isolated enterocytes from chick duodenum, jejunum and ileum. Maximal reductase and decarboxylase specific activities were found in ileum and jejunum, while ileum exhibited the minimal acyltransferase specific activity. The isolated epithelial cells showed levels of reductase and acyltransferase specific activities higher than those found in mucosa scrapings, probably due to the contact of these microsomal proteins with proteolytic enzymes during homogenization of the mucosa. However, no protecting effect of the trypsin inhibitor (2mg/ml) could be observed on reductase activity in mucosa scrapings. The cytosolic location of decarboxylase may account for the similar levels of specific activities found in mucosa scrapings and isolated enterocytes.     

Varying additive effects of bromocriptine with two somatostatin analogs in cultures of GH-secreting adenomas.

In this study, we have investigated the effect of combined treatment using two somatostatin analogs, lanreotide or octreotide, with bromocriptine on GH release in cultures of GH-secreting pituitary tumors. Sixteen acromegalic patients were included in the study. All patients had been treated with lanreotide prior to the surgery. Five patients (31.2 %) reached GH levels below 2.0 microg/l and normal IGF-I levels according to age and sex after lanreotide treatment. A positive correlation was observed between the lanreotide-induced inhibition of GH release in vitro and serum GH decrease after lanreotide treatment (r = 0.52; p = 0.03). Combined treatment significantly inhibited GH release in vitro in 8 of the 16 tumors (50 %). However, only 5 (31.2 %) of the respective patients had been resistant to presurgical treatment with lanreotide. Three of these 5 patients (18.7 %) responded to a BC concentration similar to that achieved with therapeutic doses, and in 2 patients only when a pharmacological dose of BC was used in the combined treatment. The additive effect was observed with the combination of lanreotide and BC in 6 tumors and with octreotide and BC in 3. Only one tumor showed simultaneous response to both types of combination. These results suggest that the additive effect under the combined treatment might be found between 18 and 30 % of patients who are resistant to these drugs, and that different combinations of somatostatin analogs and dopamine agonists should be tested in resistant patients.     

Salmonella intracellular proliferation: where, when and how?

Salmonella species proliferate within membrane-bound vacuoles of eukaryotic cells. Recent work has shown that macrophages are the main cell type supporting bacterial growth in vivo. In contrast, tissue culture models have traditionally described epithelial cells as the most permissive cells for bacterial growth. Unfortunately, no mechanism used by Salmonella to initiate growth within a vacuole has been characterised. Recently, it has been shown that Salmonella is capable of attenuating intracellular proliferation. This finding suggests that both the host and the pathogen contribute to a fine adjustment of the intracellular growth rate.