Amoxicillin-clavulanic acid and ciprofloxacin-treated SPF mice as gnotobiotic model

Applied Microbiology and Biotechnology - The experiment was carried out on 24 SPF BALB/c female mice and lasted for 15 days with a 5-day antibiotic (ATB) treatment and then...     

Adeno-Associated Virus Type 2-Mediated Gene Transfer: Correlation of Tyrosine Phosphorylation of the Cellular Single-Stranded D Sequence-Binding Protein with Transgene Expression in Human Cells In Vitro and Murine Tissues In Vivo

Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single-stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3′ end of the AAV genome and may prevent viral second-strand DNA synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the β-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line, and in CD34⁺ primary human hematopoietic progenitor cells; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1⁺ lin⁻ primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, contained predominantly the dephosphorylated form of the ssD-BP. Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.     

Stereospecific Biohydroxylations of Protected Carboxylic Acids with Cunninghamella blakesleeana

Cunninghamella blakesleeana DSM 1906 was found to be an efficient biocatalyst for the biotransformation of cycloalkylcarboxylic acids into hydroxy and oxo derivatives. When cultivated in submerged culture, the fungus grew in pellets. In comparison with malt extract-glucose-peptone-yeast extract medium (medium E), Czapek-Dox medium was found to reduce pellet size. Cycloalkylcarboxylic acids were protected against microbial degradation by chemical transformation into 2-cycloalkyl-1,3-benzoxazoles. The transformations of protected cyclopentyl-, cyclohexyl-, cycloheptyl-, and cyclooctylcarboxylic acids by C. blakesleeana were investigated. The biotransformations were performed in medium E by using an aerated, stirred-tank bioreactor. The transformation of 2-cyclopentyl-1,3-benzoxazole yielded (1S,3S)-3-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol as the main product. The main by-product was (1R)-3-(benz-1,3-oxazol-2-yl)cyclopentan-1-one, and 2-(benz-1,3-oxazol-2-yl)cyclopentan-1-ol was also obtained in small amounts. During the experiment, the enantiomeric excess of the main product increased up to 64%. 2-Cyclohexyl-1,3-benzoxazole was hydroxylated to 4-(benz-1,3-oxazol-2-yl)cyclohexan-1-ol. 2-Cycloheptyl-1,3-benzoxazole and 2-cyclooctyl-1,3-benzoxazole were transformed into several alcohols and ketones, all in low yields (2 to 19%).     

Closing in on the toxic domain through analysis of a variant Clostridium difficile cytotoxin B

Strain 1470 is the standard typing strain for serogroup F of Clostridium difficile containing both toxin genes, toxA-1470 and toxB-1470 . A polymerase chain reaction (PCR)-based approach to the sequencing of the total toxB-1470 gene identified an open reading frame (ORF) of 7104 nucleotides. In comparison with the previously sequenced toxB of C. difficile VPI10463, the toxB-1470 gene has 16 additional nucleotides, 13 within the 5'-untranslated region and three within the coding region. The M _r of ToxB-1470 is 269 262, with an isoelectric point (IP) of 4.16. The equivalent values for ToxB are M _r 269 709 and IP 4.13. In comparison with ToxB, ToxB-1470 differs primarily in the N-terminal region between positions 1 and 868 where 148 amino acids residues are changed. The C-terminal region between residues 869–2367 is highly conserved with only six amino acid alterations. Dot matrix comparison of ToxB-1470 with ToxA and ToxB reveals the highest homology between ToxB-1470 and ToxB. Thus ToxB-1470 did not originate from recombination between ToxA and ToxB. On cultured endothelial cells, from porcine pulmonary artery, purified ToxB-1470 is less potent than ToxB. The cytopathic effects of ToxB-1470 are indistinguishable from those caused by the lethal toxin (LT) of Clostridium sordellii , but are clearly different from the patterns observed after exposure of endothelial cells to ToxA and ToxB of C. difficile (VPI10463) or α-toxin (Tcnα) of Clostridium novyi . The LT-like action of ToxB-1470 was not due to altered internalization processes, as microinjection and addition to the medium induced identical effects on the cells. Since the differences between ToxB and ToxB-1470 are clustered within the N-terminal third of the respective proteins, we conclude that these domains carry the toxic determinants. A three-domain structure is proposed for the family of large clostridal cytotoxins.     

Detection of confined placental mosaicism in trisomy 18 conceptions using interphase cytogenetic analysis

Fluorescence in situ hybridization provides a rapid and accurate technique for detecting chromosomal aneuploidy. It is an excellent method for identifying mosaicism in placental tissues following prenatal diagnosis. Mosaicism, in the form of confined placental mosaicism, occurs im approximately 1%–2% of viable pregnancies studied by chorionic villus sampling at 9–11 weeks of gestation. It has been detected in pregnancies with both diploid and trisomic fetuses and appears to have an important effect on the intrauterine fetal survival. Using both standard cytogenetic analysis and fluorescence in situ hybridization, we have studied 12 placentas from pregnancies with trisomy 18 for the presence of chromosomal mosaicism. These included 2 that were spontaneously aborted, 5 that were terminated after prenatal diagnosis, and 4 that were delivered as either stillborn or liveborn. Significant levels of mosaicism, confined exclusively to cytotrophoblast, were detected in 7 pregnancies. This study demonstrates the usefulness of interphase cytogenetic analysis of uncultured tissues as an alternative method for the detection of mosaicism.     

An operator binding-negative mutation of Xyl repressor from Bacillus subtilis is trans dominant in Bacillus megaterium

Abstract We have selected a Bacillus subtilis 168-borne xylR Ser to Leu mutation at position 41 of the encoded amino acid sequence showing a constitutive expression phenotype for the xyl operon. When cloned on a multi-copy plasmid in a B. megaterium strain harbouring a single-copy xylA-lacZ fusion it leads to derepressor of β-galactosidase expression. Thus, it is trans dominant over the endogenous xylR , indicating that Xyl repressor functions as a multimer. This result also supports the assumption that the mutation is in a putative α-helix-turn-α-helix operator binding motif of Xyl repressor.