The role of OAT2 (SLC22A7) in the cyclic nucleotide biokinetics of human erythrocytes

The present study was conducted to characterise the transporter(s) responsible for the uptake of cyclic nucleotides to human erythrocytes. Western blotting showed that hRBC expressed OAT2 (SLC22A7), but detection of OAT1 (SLC22A6), or OAT3 (SLC22A8) was not possible. Intact hRBC were employed to clarify the simultaneous cyclic nucleotide egression and uptake. Both these opposing processes were studied. The K_m‐values for high affinity efflux was 3.5 ± 0.1 and 39.4 ± 5.7 μM for cGMP and cAMP, respectively. The respective values for low affinity efflux were 212 ± 11 and 339 ± 42 μM. The uptake was characterised with apparently low affinity and similar K_m‐values for cGMP (2.2 mM) and cAMP (0.89 mM). Using an iterative approach in order to balance uptake with efflux, the predicted real K_m‐values for uptake were 100–200 μM for cGMP and 50–150 μM for cAMP. The established OAT2‐substrate indomethacin showed a competitive interaction with cyclic nucleotide uptake. Creatinine, also an OAT2 substrate, showed saturable uptake with a K_m of 854 ± 98 μM. Unexpectedly, co‐incubation with cyclic nucleotides showed an uncompetitive inhibition. The observed K_m‐values were 399 ± 44 and 259 ± 30 μM for creatinine, in the presence of cGMP and cAMP, respectively. Finally, the OAT1‐substrate para‐aminohippurate (PAH) showed some uptake (K_m‐value of 2.0 ± 0.4 mM) but did not interact with cyclic nucleotide or indomethacin transport.     

Binding site of ABC transporter homology models confirmed by ABCB1 crystal structure

The human ATP-binding cassette (ABC) transporters ABCB1, ABCC4 and ABCC5 are involved in resistance to chemotherapeutic agents. Here we present molecular models of ABCB1, ABCC4 and ABCC5 by homology based on a wide open inward-facing conformation of Escherichia coli MsbA, which were constructed in order to elucidate differences in the electrostatic and molecular features of their drug recognition conformations. As a quality assurance of the methodology, the ABCB1 model was compared to an ABCB1 X-ray crystal structure, and with published cross-linking and site directed mutagenesis data of ABCB1. Amino acids Ile306 (TMH5), Ile340 (TMH6), Phe343 (TMH6), Phe728 (TMH7), and Val982 (TMH12), form a putative substrate recognition site in the ABCB1 model, which is confirmed by both the ABCB1 X-ray crystal structure and the site-directed mutagenesis studies. The ABCB1, ABCC4 and ABCC5 models display distinct differences in the electrostatic properties of their drug recognition sites.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Automated high-content live animal drug screening using C. elegans expressing the aggregation prone serpin α1-antitrypsin Z

The development of preclinical models amenable to live animal bioactive compound screening is an attractive approach to discovering effective pharmacological therapies for disorders caused by misfolded and aggregation-prone proteins. In general, however, live animal drug screening is labor and resource intensive, and has been hampered by the lack of robust assay designs and high throughput work-flows. Based on their small size, tissue transparency and ease of cultivation, the use of C. elegans should obviate many of the technical impediments associated with live animal drug screening. Moreover, their genetic tractability and accomplished record for providing insights into the molecular and cellular basis of human disease, should make C. elegans an ideal model system for in vivo drug discovery campaigns. The goal of this study was to determine whether C. elegans could be adapted to high-throughput and high-content drug screening strategies analogous to those developed for cell-based systems. Using transgenic animals expressing fluorescently-tagged proteins, we first developed a high-quality, high-throughput work-flow utilizing an automated fluorescence microscopy platform with integrated image acquisition and data analysis modules to qualitatively assess different biological processes including, growth, tissue development, cell viability and autophagy. We next adapted this technology to conduct a small molecule screen and identified compounds that altered the intracellular accumulation of the human aggregation prone mutant that causes liver disease in α1-antitrypsin deficiency. This study provides powerful validation for advancement in preclinical drug discovery campaigns by screening live C. elegans modeling α1-antitrypsin deficiency and other complex disease phenotypes on high-content imaging platforms.     

Immobilization of Burkholderia cepacia in polyurethane-based foams: embedding efficiency and effect on bacterial activity

Immobilization of the trichloroethylene-degrading bacterium Burkholderia cepacia was evaluated using hydrophilic polyurethane foam. The influence of several foam formulation parameters upon cell retention was examined. Surfactant type was a major determinant of retention; a lecithin-based compound retained more cells than pluronic- or silicone-based surfactants. Excessive amounts of surfactant led to increased washout of bacteria. Increasing the biomass concentration in the foam from 4.8 to 10.5% dry weight per wet weight of foam resulted in fewer cells being washed out. Embedding at reduced temperature did not significantly affect retention, while the use of a silane binding agent gave inconsistent results. The optimal formulation retained all but 0.2% of total embedded cells during passage of 2 L of water through columns containing 2 g of foam. All foam formulations tested reduced the culturability of embedded cells by several orders of magnitude, but O₂ consumption and CO₂ evolution rates of embedded cells were never less than 50% of those of free cells. Nutrient amendments stimulated an increase in cell volume and ribosomal activity in immobilized cells as indicated by hybridization studies using fluorescently labeled ribosomal probes. These results indicate that, although immobilized cells were mostly nonculturable, they were metabolically active and thus could be used for biodegradation of toxic compounds. Received 23 December 1996/ Accepted in revised form 13 March 1997     

The critical size bony defect in a small animal for bone healing studies (I): Comparative anatomical study on rats' femur.

Laboratory rats are small animal models which are often used for scientific investigations in medicine. So far there are only few scientific data about the meaning of these small animal models for in vivo bone healing studies available in literature. Although the rat's femur with its cyclic loadings during gait is an appropriate model for investigations in the field of experimental orthopaedics and traumatology there is a lack of morphometric information with respect to its anatomy. The aim of this study is to evaluate the anatomy of rat femurs in two species, which are often performed in animal experimental medicine. These morphometric data should contribute to develope an appropriated osseous fragment fixation system in the rat's femur. The femurs of Wistar (WR) and Sprague Dawley (SDR) cadavers were prepared and analysed by x-rays in two standard planes. The results were compared with the corresponding data for humans by literature. It could be demonstrated that SDR showed a higher caput-collum-diaphyseal and antetorsion angle, but a lower transcondylar femur valgus angle compared to WR. Cortical thickness, bone marrow cavity diameter and femur length were higher in WR. Wistar rat's femur anatomy shows more similarities to human anatomy than Sprague Dawley rats.     

Restriction-map variation associated with the G6PD polymorphism in natural populations of Drosophila melanogaster

Restriction-map variation was studied in 126 copies of the G6pd region in X chromosome lines of Drosophila melanogaster from North America, Europe, and Africa. Special attention was focused on the distribution of variation relative to the geographically variable polymorphism for two electrophoretic variants. Nucleotide heterozygosity as determined by eight six-cutter restriction enzymes for the 13-kb region is estimated, on the basis of the worldwide sample, to be 0.065%, which is the lowest value reported for any comparable region in the D. melanogaster genome. Significant linkage disequilibrium between electrophoretic alleles and restriction-site variation is observed for several sites. In contrast to published studies of other genetic regions, there are large insertions that reach significant frequencies and are found across considerable geographic distances. There is a clustering of this variation inside the first large intervening sequence of the G6PD gene.      

A novel mitotic spindle pole component that originates from the cytoplasm during prophase

Several unique aspects of mitotic spindle formation have been revealed by investigation of an autoantibody present in the serum of a patient with the CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, schlerodacytyly, and telangiectasias) syndrome. This antibody was previously shown to label at the spindle poles of metaphase and anaphase cells and to be absent from interphase cells. We show here that the serum stained discrete cytoplasmic foci in early prophase cells and only later localized to the spindle poles. The cytoplasmic distribution of the antigen was also seen in nocodazole-arrested cells and prophase cells in populations treated with taxol. In normal and taxol-treated cells, the microtubules appeared to emanate from the cytoplasmic foci and polar stain, and in cells released from nocodazole block, microtubules regrew from antigen-containing centers. This characteristic distribution suggests that the antigen is part of a microtubule organizing center. Thus, we propose that a prophase originating polar antigen functions in spindle pole organization as a coalescing microtubule organizing center that is present only during mitosis. Characterization of the serum showed reactions with multiple proteins at 115, 110, 50, 36, 30, and 28 kD. However, affinity-eluted antibody from the 115/110-kD bands was shown to specifically label the spindle pole and cytosolic foci in prophase cells.