Non-motile mini-transposon mutants of Bordetella bronchiseptica exhibit altered abilities to invade and survive in eukaryotic cells

Non-motile mutants of Bordetella bronchiseptica were generated after mini-transposon mutagenesis. One non-motile mutant (designated VMM1) was derived from the bvg-positive strain BB7865 and four mutants (designated AMM1–4) were derived from the isogenic bvg-negative strain BB7866. Southern hybridisation analysis indicated that loss of motility was not due to the disruption of the flagellin subunit gene. Western blot and transmission electron microscopic analysis indicated that three of the five mutants expressed neither the flagellin subunit (40 kDa) nor flagella whereas one mutant expressed intact flagella under all conditions tested. One unique bvg-negative mutant, AMM4, exhibited temperature-dependent repression of flagella biosynthesis and motility at 37°C. The ability of AMM4 to invade and survive in HeLa cells was significantly decreased.     

Comparison of type IV-pilin genes of Pseudomonas aeruginosa of various habitats has uncovered a novel unusual sequence

Abstract All known pilin sequences in Pseudomonas aeruginosa were amplified by a set of consensus primers located in the 5"-conserved region of pilA and the threonine-specific t-RNA following pilA . This also enabled the discovery of a novel pilin gene in a strain pair of clonal variants, which differs from known pilin genes in its increased GC-content. The mature protein has 173 amino acids making it the longest pilin known to date in P. aeruginosa . Different inserted sequences detected between the 3"-end of the pilin gene and the t-RNA in this strain and in strains with group I pilin genes seemed to be specific for each pilin group indicating a horizontal cotransfer of sequences.     

Uptake of Mineral Particles by Biomphalaria glabrata (Say.) (Pulmonata,Mollusca) and its Importance for Growth Rate and Egg Production

The ingestion of mineral particles by Biomphalaria glabrata is proved and the importance of these particles in the diet of the snails is investigated. The number of particles ingested rises with the size of the snails and they are retained in quantity in the alimentary tract when an adequate supply is not provided. The size of grit found in the gut differs widely from the mixture offered. Mineral particles are ingested actively and selectively. Quantitative investigations comparing snails cultured with and without sand showed a slight advantage concerning growth and reproduction for snails cultured with sand.     

Direct gas chromatographic determination of dechloroethylcyclophosphamide following microsomal incubation of cyclophosphamide

A method for the sensitive determination of dechloroethylcyclophosphamide (3-DCl) in microsomal incubation mixtures was developed. 3-DCl, a side-chain oxidation product of cyclophosphamide (CP), was isolated by extraction with acetic acid ethyl ester following solid-phase extraction on C₈ cartridges. Quantification of the metabolite was performed by direct capillary gas chromatography with a nitrogen-phosphorus detector without prior derivatization. The method showed good sensitivity and reproducibility with a detection limit of 1 ng/ml and a limit of quantification of 5 ng/ml. The suitability of the method is shown for the quantification of 3-DCl following incubation of CP with human liver microsomes.     

Fusion of Endosomes Involved in Synaptic Vesicle Recycling

Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid but not by the slow Ca²⁺ chelator EGTA. Endosome fusion was restored by the addition of Ca²⁺ with an optimum at a free Ca²⁺ concentration of 0.3 × 10⁻⁶ M. Other divalent cations did not substitute for Ca²⁺. A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca²⁺. We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca²⁺ from the endosomal interior.     

YB‐1 orchestrates onset and resolution of renal inflammation via IL10 gene regulation

The Y‐box‐binding protein (YB)‐1 plays a non‐redundant role in both systemic and local inflammatory response. We analysed YB‐1‐mediated expression of the immune regulatory cytokine IL‐10 in both LPS and sterile inflammation induced by unilateral renal ischaemia–reperfusion (I/R) and found an important role of YB‐1 not only in the onset but also in the resolution of inflammation in kidneys. Within a decisive cis‐regulatory region of the IL10 gene locus, the fourth intron, we identified and characterized an operative YB‐1 binding site via gel shift experiments and reporter assays in immune and different renal cells. In vivo, YB‐1 phosphorylated at serine 102 localized to the fourth intron, which was paralleled by enhanced IL‐10 mRNA expression in mice following LPS challenge and in I/R. Mice with half‐maximal expression of YB‐1 (Yb1^+/?) had diminished IL‐10 expression upon LPS challenge. In I/R, Yb1^+/? mice exhibited ameliorated kidney injury/inflammation in the early‐phase (days 1 and 5), however showed aggravated long‐term damage (day 21) with increased expression of IL‐10 and other known mediators of renal injury and inflammation. In conclusion, these data support the notion that there are context‐specific decisions concerning YB‐1 function and that a fine‐tuning of YB‐1, for example, via a post‐translational modification regulates its activity and/or localization that is crucial for systemic processes such as inflammation.     

Glucose oxidation and PQQ-dependent dehydrogenases in Gluconobacter oxydans

Gluconobacter oxydans is famous for its rapid and incomplete oxidation of a wide range of sugars and sugar alcohols. The organism is known for its efficient oxidation of D-glucose to D-gluconate, which can be further oxidized to two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, as well as 2,5-di-keto-D-gluconate. For this oxidation chain and for further oxidation reactions, G. oxydans possesses a high number of membrane-bound dehydrogenases. In this review, we focus on the dehydrogenases involved in D-glucose oxidation and the products formed during this process. As some of the involved dehydrogenases contain pyrroloquinoline quinone (PQQ) as a cofactor, also PQQ synthesis is reviewed. Finally, we will give an overview of further PQQ-dependent dehydrogenases and discuss their functions in G. oxydans ATCC 621H (DSM 2343).     

Gestational diabetes mellitus modulates cholesterol homeostasis in human fetoplacental endothelium

Gestational diabetes mellitus (GDM) is associated with excessive oxidative stress which may affect placental vascular function. Cholesterol homeostasis is crucial for maintaining fetoplacental endothelial function. We aimed to investigate whether and how GDM affects cholesterol metabolism in human fetoplacental endothelial cells (HPEC). HPEC were isolated from fetal term placental arterial vessels of GDM or control subjects. Cellular reactive oxygen species (ROS) were detected by H₂DCFDA fluorescent dye. Oxysterols were quantified by gas chromatography–mass spectrometry analysis. Genes and proteins involved in cholesterol homeostasis were detected by real-time PCR and immunoblotting, respectively. Cholesterol efflux was determined from [³H]-cholesterol labeled HPEC and [¹⁴C]-acetate was used as cholesterol precursor to measure cholesterol biosynthesis and esterification. We detected enhanced formation of ROS and of specific, ROS-derived oxysterols in HPEC isolated from GDM versus control pregnancies. ROS-generated oxysterols were simultaneously elevated in cord blood of GDM neonates. Liver-X receptor activation in control HPEC by synthetic agonist TO901319, 7-ketocholesterol, or 7β-hydroxycholesterol upregulated ATP-binding cassette transporters (ABC)A1 and ABCG1 expression, accompanied by increased cellular cholesterol efflux. Upregulation of ABCA1 and ABCG1 and increased cholesterol release to apoA-I and HDL₃ (78?±?17%, 40?±?9%, respectively) were also observed in GDM versus control HPEC. The LXR antagonist GGPP reversed ABCA1 and ABCG1 upregulation and reduced the increased cholesterol efflux in GDM HPEC. Similar total cellular cholesterol levels were detected in control and GDM HPEC, while GDM enhanced cholesterol biosynthesis along with upregulated 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and sterol O-acyltransferase 1 (SOAT1) mRNA and protein levels. Our results suggest that in GDM cellular cholesterol homeostasis in the fetoplacental endothelium is modulated via LXR activation and helps to maintain its proper functionality.     

<Emphasis Type="Italic">LTRharvest</Emphasis>, an efficient and flexible software for <Emphasis Type="Italic">de novo</Emphasis> detection of LTR retrotransposons

Background  

Transposable elements are abundant in eukaryotic genomes and it is believed that they have a significant impact on the evolution of gene and chromosome structure. While there are several completed eukaryotic genome projects, there are only few high quality genome wide annotations of transposable elements. Therefore, there is a considerable demand for computational identification of transposable elements. LTR retrotransposons, an important subclass of transposable elements, are well suited for computational identification, as they contain long terminal repeats (LTRs).